Chemical Composition and Protein Antigenicity â•fi Almond ...

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Electronic Theses, Treatises and Dissertations The Graduate School

2008

Chemical Composition and ProteinAntigenicity Almond (Prunus Dulcis) andMacadamia Nut (Macadamia Integrifolia)SeedsErin Kelly Monaghan

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FLORIDA STATE UNIVERSITY

COLLEGE OF HUMAN SCIENCES

CHEMICAL COMPOSITION AND PROTEIN ANTIGENICITY – ALMOND (Prunus

dulcis) AND MACADAMIA NUT (Macadamia integrifolia) SEEDS

By

Erin Kelly Monaghan

A Dissertation submitted to the Department of Nutrition, Food and Exercise Sciences

in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

Degree Awarded: Summer Semester, 2008

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The members of the Committee approve the dissertation of Erin Kelly Monaghan defended on June 24, 2008.

________________________________ Bahram Arjmandi Professor Directing Dissertation

________________________________ Kenneth H. Roux Outside Committee Member

________________________________ Yun-Hwa Peggy Hsieh Committee Member

Approved: ________________________________________________________________ Bahram Arjmandi (Chair, Department of Nutrition, Food, and Exercise Sciences) ________________________________________________________________ Billie Collier (Dean, College of Human Sciences) The Office of Graduate Studies has verified and approved the above named Committee members.

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ACKNOWLEDGMENTS

I would like to express my deepest appreciation to my committee members, Dr. Bahram Arjmandi, Dr. Kenneth Roux, and Dr. Peggy Hsieh for their unwavering support and encouragement throughout my graduate program. Without their wisdom and guidance this dissertation would never have come to fruition.

I owe a special thanks to my major advisor Dr. Bahram Arjmandi for his willingness to

“adopt” me and help me reach the finish line. His infectious enthusiasm and dedication to his students defines a true and trustworthy mentor.

A special thanks to Dr. Mary Ann Moore, a wise and caring advisor, who gave me much-

needed reassurance and guidance. I would like to offer my heartfelt gratitude to Dr. Jason Robotham and Dr. Kenneth Roux.

I am indebted to them for their assistance and guidance. They are extraordinary individuals who I admire tremendously.

I would like to thank all faculty and staff from the Department of Nutrition, Food, and

Exercise Science for their assistance, guidance, and encouragement over the years.

A special thanks to Margaret Seavy for her continuous support, compassion, and understanding.

I am thankful for all my past and present lab mates for making everyday life in the

laboratory enjoyable. It has been a true pleasure to work with each and every one of you and I wish you all the best.

I have been very blessed in my life, particularly in my friendships. In all of the ups and

downs that came my way during the “dissertation years”, I knew I always had the support and love of friends. In particular, I am grateful to Terry Love, April Hambrecht, Jennifer Hura, Debbie Iarossi, Gina Pitisci, Molly Carey, Jenny Hansen, Brent Stoney, David Krum, Mary Susan Gerber, and Elizabeth Ibbotson for always being there when I needed support. A very special thanks to Grant Cotner for being incredibly supportive, patient, and constantly bringing a smile to my face.

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I would like to thank my grandparents, aunts, uncles, cousins, and close family friends for their enthusiasm and support throughout my graduate education.

I am forever grateful to Mallary and Rayleigh, my loyal fans and my greatest comforts, who have blessed my life with their presence and brought it so much joy and pleasure.

I am extremely thankful to Ian Monaghan, who is not only my big brother but also my

oldest and closest friend. His support and humor has enriched my life more than he will ever know.

Finally, I dedicate this dissertation to my parents, Patrick and Mary Monaghan. Words

alone cannot express the thanks I owe to my parents who have guided me, inspired me, and encouraged me throughout my entire life. Their unwavering faith and confidence in me has shaped me into the woman I am today. I am eternally grateful and blessed to have two of the greatest people I have even known as my parents.

"I believe that everything happens for a reason. People change so that you can learn to

let go, things go wrong so that you learn to appreciate them when they're right. You believe lies

so you eventually learn to trust no one but yourself...and sometimes good things fall apart so

better things can fall together." ~ Marilyn Monroe ~

“Everyone who has even done a kind deed for us, or spoken one word of encouragement

to us, has entered into the make-up of our character and of our thoughts, as well as our success.” ~ George Burton Adams ~

“The future belongs to those who believe in the beauty of their dreams.”

~ Eleanor Roosevelt ~ “People grow through experience if they meet life honestly and courageously. This is how

character is built.” ~ Eleanor Roosevelt ~

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TABLE OF CONTENTS

List of Tables vii List of Figures ix Abbreviations xii Abstract xiv

INTRODUCTION 1 Background 1 Statement of the Problem 4 Research Hypothesis 8 Significance of the Study 9 Limitations of the Study 9

REVIEW OF LITERATURE 10 Almond Cultivars 10 Macadamia Nut Cultivars 10 Physical Seed Characteristics 13 Chemical Composition 14 Tree Nut Allergies 23 Detection Methods for Tree Nut Allergens 25 Almond and Macadamia Nut Allergens 25 Cross-Reactivity 26

Allergen Stability 27

MATERIALS AND METHODS 29 Materials 29 Methods 30

Physical Seed Characteristics 30 Individual seed weight 30 Seed dimension 30

Preparation of Nut Seed Flours 30 Chemical Composition 30 Moisture 30 Lipid 30 Protein 31 Amino Acid Composition 31 Total amino acids 31 Free amino acids 32 Ash 33 Total soluble sugars 33 Tannins 33 Soluble Protein Estimation 34

SDS-PAGE 34 Polyclonal Antibody Production and Characterization 35

Monoclonal Antibody 4C10 Production and Characterization 35 Protein G Purification of Rabbit anti-Macadamia Nut Protein IgG 36

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Competitive Inhibition ELISA Development 36 Almond Protein Detection 36 Macadamia Nut Protein Detection 36 Sandwich ELISA Development 38 Western Blot Analysis 38 Dot Blot Analysis 39

Densitometric Quantification 40 Cross-Reactivity Studies 40

Thermal Processing of Almond and Macadamia Nut Seeds 41 Autoclaving 41 Blanching 41 Dry Roasting 41 Frying 41 Microwave Heating 41 pH Treatments 42 Spiking Studies 42 Data and Statistical Analysis Procedures 42

RESULTS AND DISCUSSION 49 Environmental Conditions 49

Physical Seed Characteristics 49 Individual seed weight 49 Seed dimension 50 Chemical Composition 51 Moisture 51 Lipid 52 Protein 53 Ash 54 Total soluble sugars 54 Tannins 55 Amino Acid Composition 60 Total amino acids 60 Free amino acids 61 Electrophoretic Analysis 82 ELISA 82 Western and Dot Blotting 92 Cross-Reactivity Studies 92 Stability Studies 96 Spiking Studies 96

CONCLUSION 97 APPENDIX A 105 ANOVA Tables 105 APPENDIX B 142 Animal Research Approval 142 REFERENCES 145 BIOGRAPHICAL SKETCH 164

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LIST OF TABLES

Table 1. Proximate composition of almond and macadamia nut seeds 18

Table 2. Lipid composition of almond and macadamia nut seeds 19 Table 3. Mineral and vitamin content of almond and macadamia nut seeds 20

Table 4. Amino acid composition of almond and macadamia nut seeds 21

Table 5. Immunoassays for tree nut protein detection 28

Table 6. Cross-reactivity testing matrices 40 Table 7. Almond cultivars and history of origin 44

Table 8. Average climatic conditions in select California counties during 46

2003/2004 and 2005/2006 almond seasons

Table 9. Cultivar, geographic location, and harvest year variation on the 56 physical characteristics and chemical composition of select almond seeds

Table 10. Interaction of cultivar and geographic location on the physical 57

characteristics and chemical composition of select almond seeds

Table 11. Physical characteristics and chemical composition of select 59 macadamia nut seeds

Table 12. Cultivar, geographic location, and harvest year variation on the 63

essential amino acid composition of select almond seeds

Table 13. Cultivar, geographic location, and harvest year variation on the 64 non-essential amino acid composition of select almond seeds

Table 14. Interaction of cultivar and geographic location on the essential 65

amino acid composition of select almond seeds

Table 15. Interaction of cultivar and geographic location on the non-essential 67 amino acid composition of select almond seeds

Table 16. Amino acid composition of select macadamia nut seeds 69

Table 17. Correlation coefficients among various physical and chemical 70

parameters of select almond seeds

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Table 18. Correlation coefficients among various physical and chemical 71 parameters of select macadamia nut seeds

Table 19. Cultivar, geographic location, and harvest year variation on the 75

free (essential) amino acid composition of select almond seeds

Table 20. Cultivar, geographic location, and harvest year variation on the 76 free (non-essential) amino acid composition of select almond seeds

Table 21. Interaction of cultivar and geographic location on the free 77

(essential) amino acid composition of select almond seeds

Table 22. Interaction of cultivar and geographic location on the free 79 (non-essential) amino acid composition of select almond seeds

Table 23. Correlation coefficients among free amino acids of select almond 81

seeds Table 24. Effect of geographic location on the immunoreactivity of select 86

almond cultivars assessed by ELISA

Table 25. Interaction of cultivar and geographic location on the 87 immunoreactivity of select almond cultivars assessed by ELISA

Table 26. IgG purified macadamia nut pAb titer optimization 90

Table 27. Cross-reactivity of select foods/ingredients with Protein G purified 95

rabbit anti-macadamia nut IgG assessed by inhibition ELISA

Table 28. Detection of AMP in 100 mg of food matrices spiked with 102 almond seed protein extract or defatted almond seed flour

Table 29. Detection of macadamia nut protein in 100 mg of food matrices spiked 103

with macadamia nut protein extract or defatted macadamia nut flour Table 30. Detection of almond and macadamia nut in commercially prepared 104

foods using ELISA

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LIST OF FIGURES

Figure 1. 45

Figure 2. 47 Figure 3. 48 Figure 4. 72 Figure 5. 73 Figure 6. 74

Figure 7. 84 Figure 8. 85

Location of almond seed samples included in the current investigation. Estimated timeline for California almond seed production. ** Season varies according to region, variety and weather conditions. Climatic conditions in Fresno (A), Kern (B), and Stanislaus (C) counties during the 2003/2004 and 2005/2006 almond season. Daily weather measurements recorded by the California Irrigation Management Information System (CIMIS) and the National Climatic Data Center (NCDC) of the National Oceanic and Atmospheric Administration (NOAA) were used to calculate county averages. Weather stations: CIMIS #2 and #80 for Fresno County, NCDC #0442 for Kern County, and NCDC #6168 and #5738 for Stanislaus

County. Tmax = maximum temperature and Tmin = minimum temperature. Relationship between (A) seed weight and seed length, and (B) seed weight and seed width in 58 almond seed samples. Relationship between (C) seed weight and seed diameter in 7 macadamia nut seed samples. Data are expressed in grams (seed weight) and millimeters (length, width, and diameter). Relationship between (A) lipid and protein, (B) protein and total soluble sugars, and (C) lipid and total soluble sugars in 58 almond seed samples. Relationship between (D) protein and total soluble sugars in 7 macadamia nut seed samples. Data are expressed in gram per 100 gram edible portion. Relationship between (A) glutamic acid and aspartic acid, (B) glutamic acid and arginine, and (C) arginine and lysine in 58 almond seed samples. Relationship between (D) arginine and lysine in 7 macadamia nut seed samples. Data are expressed in gram per 100 gram protein. Polypeptide profile and AMP antigenicity of select almond cultivars assessed by (A) SDS-PAGE stained with Coomassie stain; protein load = 30�g, (B) Western blot probed with mAb 4C10; protein load = 30�g, (C) Dot blot probed with mAb 4C10; protein load = 500 ng. SDS-PAGE analysis of macadamia nut cultivars. A. Coomassie stain; protein load = 30 �g. B. Silver stain; protein load = 10 �g. C. Glycoprotein stain; protein load = 200 �g. D. Western blot; protein load = 30 �g. Primary Ab = Protein G purified rabbit anti- macadamia nut IgG (1:16,000 v/v). Secondary Ab = HRP-labeled goat anti-rabbit pAb (1:40,000 v/v).

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Figure 9. 89

Figure 10. 90

Figure 11. 91 Figure 12. 91 Figure 13. 93

Figure 14. 94


Checkerboard titration for optimization of coating buffer in non-competitive ELISA. Protein G purified rabbit anti-macadamia nut IgG dilution was 1:4,000 v/v. Data are expressed as mean ± SEM (n = 4). Checkerboard titration using PBS coating buffer in non-competitive ELISA to optimize macadamia nut protein (antigen) coating concentration. Protein G purified rabbit anti-macadamia nut IgG dilutions are indicated in figure legend (X = 1,000). Data are expressed as mean ± SEM (n = 4). Representative competitive inhibition ELISA standard curve for macadamia nut. The IC50 (mean ± SEM) for soluble macadamia nut protein was 21.75 ± 0.94 ng/ml (n = 70). Immunoreactivity (%) of macadamia nut varieties assessed by inhibition ELISA (n= 3). * = significantly different compared to control (Blue Diamond, p � 0.05, LSD = 23.18) Cross-reactivity of mAb 4C10 with select foods/ingredients assessed by (A) Western blot; protein load = 20 �g, (B) Dot blot; protein load = 500 ng for almond and 2 �g for test samples, and (C) sandwich ELISA; values are % cross-reactivity and are expressed as mean ± SEM (n = 3). Cross-reactivity of select foods/ingredients with Protein G purified rabbit anti-macadamia nut IgG (1:16,000 v/v) assessed by Western blotting. Protein loads: 10 �g macadamia and 30 �g for all test samples. Effect of thermal processing on antigenicity of AMP assessed by (A) SDS-PAGE stained with Ponceu S stain; protein load = 30�g, (B) Western blot probed with mAb 4C10; protein load = 30�g, (C) mAb 4C10 sandwich ELISA; values are % immunoreactivity [(IC50 of processed sample/IC50 of unprocessed sample) x 100] and are expressed as mean ± SEM (n = 3, p � 0.05). Effect of pH on the antigenicity of AMP assessed by (A) SDS-PAGE stained with Ponceu S stain and (D) Coomassie stain; protein load = 20 �g, (B and E) Western blot probed with mAb 4C10; protein load = 20 �g, and (C and F) mAb 4C10 sandwich ELISA; values are % immunoreactivity [(IC50 of processed sample/IC50 of unprocessed sample) x 100] and are expressed as mean ± SEM (n = 3, p � 0.05). A,

B, C: Defatted almond seed flour exposed to desired pH value and neutralized prior to analysis. D, E, F: Defatted almond seed flour exposed to desired pH value and analyzed directly (not neutralized).

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Effect of thermal processing on immunoreactivity of macadamia nut proteins assessed by ELISA and Dot blot, (A) SDS-PAGE stained with Coomassie stain; protein load = 30 �g, and (B) Western blot probed with Protein G purified rabbit anti-macadamia nut IgG; protein load = 30 �g. ELISA values are % immunoreactivity [(IC50 of processed sample/IC50 of unprocessed sample) x 100] and are expressed as mean ± SEM (n = 3, p � 0.05). Effect of pH on immunoreactivity of macadamia nut proteins assessed by (A and D) SDS-PAGE stained with Coomassie stain; protein load = 20 �g, (B and E) Western blot probed with Protein G purified rabbit anti-macadamia nut IgG; protein load = 20 �g, and (C and F) inhibition ELISA; values are % immunoreactivity [(IC50 of processed sample/IC50 of unprocessed sample) x 100] and are expressed as mean ± SEM (n = 3, p � 0.05). A, B, C: Defatted macadamia nut flour exposed to desired pH value and analyzed directly (not neutralized). D,

E, F: Defatted macadamia nut flour exposed to desired pH value and neutralized prior to analysis.

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ABBREVATIONS

ABC Almond Board of California ADD Amino acid distribution AMP Almond major protein or amandin ANOVA Analysis of variance AP Alkaline phosphatase �-ME �-mercaptoethanol BSA Bovine serum albumin BSB Borate saline buffer (0.1 M H3BO3, 0.025 M Na2B4O7, 0.075 M NaCl, pH 8.45) CIMIS California Irrigation Management Information System CBBR Coomassie brilliant blue R DI water Distilled, deionized water DMSO Dimethyl sulfoxide ELISA Enzyme-linked immunosorbent assay E/T Essential-to-total amino acid ratio FALCPA Food Allergen Labeling and Consumer Protection Act GC Gas chromatography HAES Hawaii Agricultural Experiment Station HCN Hydrogen cyanide HDL High density lipoproteins HPLC High performance liquid chromatography HRP Horseradish peroxidase IgE Immunoglobulin E IgG Immunoglobulin G IU International unit kDa Kilo Dalton kGy Kilo Gray LDL Low density lipoproteins LEAA Limiting essential amino acid LSD Fisher’s least significant difference mAb Monoclonal antibodies MUFA Monounsaturated fatty acid MW Molecular weight NC Nitrocellulose NCDC National Climatic Data Center ND Not determined NFDM Nonfat dry milk NOAA National Oceanic and Atmospheric Administration pAb Polyclonal antibodies PBS Phosphate buffered saline (0.1 M sodium phosphate buffer containing 0.85% w/v

NaCl, pH 7.2) PCR Polymerase chain reaction pI Isoelectric precipitation PITC Phenyl isothiocyanate

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PNPP p-nitrophenyl phosphate ppm parts per million PUFA Polyunsaturated fatty acid RAE Retinol equivalent RT Room temperature, ~25 °C SEM Standard error of mean SFA Saturated fatty acid SPT Skin prick test TBS-T Tris buffered saline (10 mM Tris, 0.9% w/v NaCl, 0.05% v/v, 0.2% Tween 20,

pH 7.2) TEA Triethylamine 1° Ab Primary antibody 2° Ab Secondary antibody

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ABSTRACT

Almond and macadamia nut cultivars were analyzed for physical seed characteristics,

chemical composition, and protein antigenicity. The effect of environmental conditions

(geographic location and harvest year) on almond seed composition was also investigated.

Almond samples ranged from 0.78-1.44 g in individual seed weight, 18.3-27.8 mm in length,

9.8-13.8 mm in width, and 6.9-10.2 mm in thickness. Macadamia nut samples ranged from 2.41-

3.36 g in individual seed weight, 18.7-20.1 mm in diameter, and 14.4-15.9 mm in thickness.

Moisture, lipid, protein, ash, soluble sugars, and tannins ranged from 2.9-5.6%, 50.9-66.7%,

15.7-26.7%, 2.6-3.5%, 3.0-6.5%, and 0.07-0.36% in almond seeds and 1.7-2.6%, 60.0-66.2%,

5.6-7.1%, 1.0-1.4%, 5.3-8.7%, and 0.03-0.04% in macadamia nut seeds, respectively. The acidic

amino acids (glutamic acid and aspartic acid) were the dominate amino acids in almond and

macadamia nut proteins, accounting for 24.4-43.3% and 35.1-40.8% of total amino acids.

Almond and macadamia nut seed proteins have essential-to-total amino acid ratio ranges of 25.4-

33.8% and 22.5-26.1% and are also rich sources of arginine with ranges of 7.3-11.9% and 9.4-

11.5%, respectively. Total free amino acids in almond seeds ranged from 172.3-723.2 mg/100 g

dry weight, with free asparagine accounting for 14.9-44.9% of total free amino acids. A

monoclonal-based sandwich ELISA and a polyclonal-based inhibition ELISA were developed

for detecting and evaluating the antigenicity of almond and macadamia nut proteins,

respectively. ELISAs, in combination with Western and Dot blot analyses, were used to

investigate the antigenic stability of almond and macadamia nut proteins to various processing

(thermal and pH) treatments and to quantify these proteins in complex food matrices. The

ELISAs were able to detect almond major protein (AMP) and macadamia nut proteins at levels

as low as 19.2 ± 1.3 ng/ml and 21.8 ± 0.9 ng/ml and were able to detect AMP and macadamia

nut proteins at or below 10 ppm in majority of tested food matrices.

INTRODUCTION

Background Almond Seeds

Almond seeds are included in the family Rosaceae in addition to Pomoideae (apples,

pears), Prunoideae (apricot, cherry, peach, and plum) and Rosoideae (blackberry, strawberry)

fruits. Almond seeds (Prunus amygdalus) are of 2 types: 1) sweet almonds (Prunus amygdalus

‘dulcis’) used mainly for culinary purposes and 2) bitter almonds (Prunus amygdalus ‘amara’)

used mainly in the making of oils and flavorings. The bitterness of the latter type is based on the

presence of cyanogenic glycosides which can be degraded by �-glycosidases (present in the seed

or produced by microorganism in the digestive tract of mammals) to generate hydrogen cyanide

(HCN) which may potentially cause cyanide poisoning (1). Prior to their use in the

manufacturing of oils and flavorings, bitter almond seeds are processed by grinding to release the

generated HCN making them safe for consumption (1). Almond seeds, believed to be native to

the Middle East, were domesticated as early as 3000 BC and perhaps much earlier as wild

almond seeds were unearthed in Greek archeological sites dating back to 8000 BC. Spanish

missionaries (specifically Franciscan Padres) are credited for bringing the almond seed to

California in the mid-1700s. Research and cross-breeding of almond seeds began in the 1870’s,

where several of the almond seed cultivars used today were originally developed and selected

(2). The California almond industry was established in the Central Valley of California

(Sacramento and San Joaquin counties) by the turn of the 20th century (2) and grew at a moderate

pace and around 1960 California became the world’s leader in almond seed production. In

2006/2007, worldwide almond seed production totaled 600,884 metric tons (shelled-basis), with

the U.S. (primarily California) producing 82.7%, followed by Spain (11.3%), Greece (2.5%),

Turkey (2.3%), Italy (1.0%), and India (0.2%) (3). California produced 1,117.3 million pounds

of almond seeds in 2006/2007 with the largest production in Kern (22.2%), Fresno (20.8%),

Stanislaus (14.6%) and Merced (11.2%) counties (4). Worldwide (2006/2007), exports of

almond seeds totaled 445,800 metric tons (shelled-basis) with the U.S. (84.1%), Spain (13.7%),

Greece (10.8%), Italy (10.1%), and Turkey (0.1%) the chief exporters (3). Major importers

worldwide (2006/2007) were Spain (35.0%), Italy (22.3%), India (21.8%), the U.S. (11.8%),

Greece (8.0%), and Turkey (1.2%) with imports totaling 125,710 metric tons (shelled-basis) (3).

2

Almond seeds are valued for their sweet taste and crunchy texture. The Almond Board of

California (ABC) report the majority (� 50%) of consumed almond seeds are used as an

ingredient in manufactured goods such as candy, cereal, ice cream, granola bars, and cookies.

The remainder are purchased at retail for consumer snacking, in-home baking and cooking

(~25%) or consumed at the food service level (~25%) (5). Almond seeds are a common

ingredient in nougat, marzipan, cookies (e.g. macaroons, biscotti), ice cream, butters, amaretto (a

sweet liquor made from a base of apricot and/or almond pits which provide bitterness), snacks

(mixed nuts, roasted and/or salted) and as a topping for desserts, salads, and vegetables.

Almond seeds contain approximately 51% lipid, 21% protein, 20% carbohydrate and

12% fiber (Table 1). The majority of lipids are monounsaturated (~67%) and polyunsaturated

(~25%) fatty acids (MUFA and PUFA, respectively) (Table 2). Previous studies indicate the

MUFAs from almond seeds may reduce total cholesterol and low-density lipoproteins (LDL,

“bad cholesterol”) while maintaining healthy high-density lipoproteins (HDL, “good

cholesterol”) levels (6-15). Almond seed proteins typically contain a large proportion of acidic

amino acids (aspartic and glutamic acids), have an essential-to-total amino acid ratio (E/T) of

~29%, and are typically deficient in the sulfur amino acids (methionine and cysteine) (Table 4).

Almond seeds are a good source of phosphorus, calcium, potassium, magnesium, manganese,

copper, zinc, and iron (Table 3). Almond seeds, primarily the skin (hull), contain tannins which

are astringent bitter-tasting polyphenols that act as antioxidants (16-20). Antioxidants inhibit

and/or minimize the production of destructive free radicals which play a key role in the

development and progression of cancer, atherosclerosis, and inflammation (21-23). Another

antioxidant, vitamin E, is also present in almond seeds with a one-ounce serving (~24 seeds)

providing 50% the RDA for vitamin E (15 mg/day for both male and female adults) (24).

Macadamia Nut Seeds

Macadamia nut seeds, native to Australia, are a genus of eight species of flowering

plants within the family Proteaceae. The genus includes 7 Australian species (Macadamia

claudiensis, M. grandis, M. integrifolia, M. jansenii, M. ternifolia, M. tetraphylla, M.

whelaniiseven) and 1 Indonesia Sulawesi specie (M. hildebrandii) (25, 26). Kermond and

Baumgardt (27) also include M. prealta, M heyana, M. roussellii, M. vieillardii, M. francii, M.

alticola, M. augustifolia, M. leptophylla, M. neuophylla, M. verticillata, and M. youngiana as

additional species recognized in the genus Macadamia. Common names include Macadamia,

3

Macadamia nut, Queensland nut, Bush nut, Bauple nut, and Maroochi nut (27). Only two

species, M. integrifolia (smooth-shell) and M. tetraphylla (rough-shell) are of commercial

importance (25). The remaining species in the genus produce poisonous and/or inedible nuts

(28). M. whelanii and M. ternifolia, like bitter almond nut seeds, contain cyanogenic glycosides

and in order to use these species the seeds must be processed by grinding and steeping, followed

by cooking to remove these glycosides (1). Macadamia nut seeds were believed to be a diet

staple of Aboriginal Australians who have lived on the continent of Australia for over 40,000

years (27). Charles Staff, in 1887, is credited for planting the earlier commercial macadamia nut

seed orchard at Rous Mill in Northern New South Wales (27). Around World War II, the Angus

family (believed to be connected to the Mac Farms operation in Australia) first organized the

commercial purchasing and processing of macadamia nut seeds in Australia (27). The first large

scale Australian macadamia nut seed producers and processors were Commonwealth Sugar

Refinery (C.S.R.) and Macadamia Plantations of Australia (M.P.A.) (27). Macadamia nut seeds

are the only plant source native to Australia that are produced and exported in significant

quantity (27). The macadamia nut seed (M. intergrifolia) was introduced to Hawaii from

Australia in 1892 (29), with William Herbert Purvis (1892-1895) introducing a seed from the Mt.

Bauple region, north of Queensland and the Jordan brothers (Captain Robert A. Jordan and E.W.

Jordan) (1892-1893) introducing a seed from Pimpama, south of Brisbane (26, 30). The

Pimpama trees were originally planted in Hawaii for ornamental purposes and reforestation (26).

The Hawaiian macadamia nut seed industry began commercial development in the 1920’s and

1930’s with Castle & Cook and Royal Hawaiian Macadamia Nut the first commercial companies

(27). Worldwide (2005/2006) production of macadamia nut seeds totaled 104,551 metric tons

(in-shell basis), with Australia (37.8%), the U.S. (primarily Hawaii, 26%), Republic of South

Africa (18.7%), Kenya (10.5%), and Guatemala (7.0%) the highest producers (3). Exports of

macadamia nut seeds worldwide (2005/2006) totaled 62,479 metric tons (in-shell basis), with

Australia (47.2%), Republic of South Africa (23.8%), Guatemala (11.5%), Kenya (10.2%), and

the U.S. (7.3%) the chief exporters (3). Total imports of macadamia nut seeds worldwide

(2005/2006) totaled 38,759 metric tons (in-shell basis), with the U.S. (91.0%) and Republic of

South Africa (9.0%) the major importers (3).

Macadamia nut seeds are valued for their sweet taste and creamy texture. A 2006

Australian market usage report (31) show the majority (46%) of macadamia nut seed are

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consumed in snack packs (roasted and salted seeds) and the remainder used in confectionary

(chocolate covered macadamias, 35%), bakery (macadamia nut cookies and biscuits, 15%) and

ice cream (4%) products.

Macadamia nut seeds contain approximately 76% lipid, 14% carbohydrate, 9% fiber, and

8% protein (Table 1). Considered gourmet oil, macadamia nut seed oil is used in cooking, baking

and a wide variety of cosmetic products, especially skin creams, moisturizers, soaps, lotions and

massage oils (32). The oil is high in MUFAs (~81.3%), mainly oleic (60.4%) and palmitoleic

acids (~17.9%), make it an excellent olive oil substitute (Table 2). Similar to almond seeds,

several clinical studies suggest incorporating macadamia nut seeds into a healthy diet may

protect against coronary artery disease (6, 9, 33-36). When compared to a typical American diet

[37% total fat (16% SFA, 7% PUFA, and 14% MUFA)] over 30 days, a macadamia nut-based

diet [37% total fat (9% SFA, 7% PUFA, and 21% MUFA)] significantly lowered total

cholesterol by 4.8% (p < 0.01), LDL cholesterol by 4.5% (p < 0.05), and total triglycerides by

9.2% (p < 0.05) in 30 healthy men and women (36). However, a significant decrease in HDL

cholesterol by 4.2% (p < 0.01) was also observed (dietary cholesterol was the same for both test

diets, 300 mg/day). Another study where 17 hypercholesterolemic men consumed 40-90 g/day of

macadamia nut seeds over a 4 week period had a significant reduction in total (3.2%, p < 0.05)

and LDL cholesterol (6.0%, p < 0.05) (35). Interestingly unlike the previous study, a significant

increase in HDL cholesterol (6.3%, p < 0.05) was observed (no significant difference in

triglyceride levels were observed). A recent study under similar conditions (17

hypercholesterolemic men consumed 40-90 g/day of macadamia nut seeds over a 4 week period)

reported a significant reduction in leukotriene (23.9%, p = 0.024) and 8-isoprostane (22.5%, p =

0.032), both are plasma markers of inflammation and oxidative stress (33).

Macadamia nut seed proteins, like almond seed proteins, also contain a large amount of

acidic amino acids, have an essential-to-total amino acid (E/T) ratio of ~27%, and are typically

deficient in lysine and the sulfur amino acids (Table 4). Additionally, macadamia nut seeds are

low in sodium and are a good source of thiamin, manganese, calcium, and iron (Table 3).

STATEMENT OF THE PROBLEM

Worldwide, the U.S. is a major producer of almond and macadamia nut seeds with

California and Hawaii leading the production, respectively (3). Almond cultivars, although

5

produced in different geographic regions of California, have limited published literature on the

genetic, geographic, and annual variations on seed composition. Published data to date are also

limited on the compositional differences of macadamia nut seed cultivars. As published data on

these topics are lacking the current investigation on compositional differences in almond and

macadamia nut seeds as affected by various parameters (cultivar, geography, and/or harvest year)

will therefore be useful to almond and macadamia nut seed producers, food manufacturers and

consumers. The current study analyzed and compared the physical characteristics (seed weight

and seed size) and chemical composition (moisture, lipid, proteins, ash, total soluble sugars, and

tannins) of select nut seed samples. The study also evaluated the protein amino acid composition

of these select seed samples to assess the nutritional quality and the free amino acid composition

of select almond seed samples mainly to assess the free asparagine content. Analyzing almond

seeds for free asparagine is important as almonds are typically enjoyed roasted and are reported

to contain high levels of free asparagine (37-39), two factors believed to contribute to acrylamide

formation (40-43). Therefore, evaluating the free asparagine content is of importance as

acrylamide is a known carcinogen (37).

Recent reports suggesting the health and nutritional benefits of almond (8, 10-15, 44) and

macadamia nut (33-36) seeds have lead to their increased consumption and use in processed

foods. However, there is a cause for concern as tree nuts are one of the “big 8” most allergenic

foods and the prevalence of food allergies in industrialized counties has risen (45). Their

increased use in the food industry may result in greater risk of accidental exposure (e.g. improper

labeling or cross-contamination) and pose a danger of severe allergic reactions in individuals

sensitive to almond and macadamia nut seed proteins. The risk of accidental exposure has

resulted in several large food recalls due to improper labeling and cross-contamination of known

food allergens (45). Several commercial kits are available for almond seed protein detection (e.g.

Neogen Veratox ® for almond, Tepnel BioKits Rapid 3-D™ Almond test kit, and R-Biopharm

RIDASCREEN®FAST Mandel / Almond) detection limits of 1.0-1.7 ppm, however most are

cross-reactive with non-almond seed proteins which may increase the possibility of improper

detection and labeling. For instance, the R-Biopharm website report significant cross-reactivity

of the RIDASCREEN®FAST Mandel/Almond detection kit with apricot stone (> 100%) and

lower levels of cross-reactivity with hazelnut, pecan, sunflower, sesame, and lima bean (�

0.073%). A monoclonal antibody (mAb)-based immunoassay (46) for peanut seed protein (Ara h

6

1) is reported to have a higher sensitivity range (0.015-0.02 ppm) and limited cross-reactivity.

Although both polyclonal (pAbs) and monoclonal (mAbs) antibodies are useful in developing

immunoassays for protein detection, several differences such as production, consistency,

specificity, and stability should be considered when choosing the appropriate antibody.

Producing pAbs is typically less expensive, more rapid (several months) and requires less

technical skill than mAb production which is more costly, time-consuming (typically a year or

longer), and demanding (47). However, once a hybridoma is generated a mAb can be produced

repeatedly with the same specificity and affinity (47, 48). Whereas, pAb production is dependent

on the size and life span of the chosen animal model. In addition, the concentration and

specificity of the generated pAbs vary from one animal to another (47, 48). The ability of mAbs

to identify a specific epitope (either linear or conformational) on a protein is desirable for

detecting targeted proteins in a complex food mixture. However, changes in the configuration of

a protein due to processing may significantly alter the function of the mAbs by destroying the

epitope, hiding the epitope, exposing a hidden epitope, or creating a new epitope. On the other

hand, pAbs recognize numerous epitopes on a protein(s) and can be useful when trying to

identify several antigenic proteins or a single protein under different conditions and/or in an

altered state (47). Typically, pAbs remain stable over a wide range of pH and salt concentrations

whereas mAbs are less stable (47). The mAb 4C10 produced and described previously by Sathe

et al. (49) is specific to the 63 kDa polypeptide of almond major protein (AMP) and is not cross-

reactive with several tested seed proteins. Therefore, mAb 4C10 was used to develop sensitive

mAb-based immunoassays for the specific detection of AMP. To date, no commercial

macadamia nut seed detection kits are available. For that reason, producing anti-macadamia nut

seed protein rabbit pAbs and utilizing these pAbs to develop sensitive detection immunoassays

would prove useful for food manufacturers and macadamia nut sensitive consumers.

Finally, almond and macadamia nut seeds typically endure some degree of food

processing and/or are incorporated into numerous food ingredients forming complex food

matrices that may alter the detection of these antigenic proteins (by either blocking or exposing

allergenic epitopes). Therefore, examining the stability of these nut seed proteins to various food

processing treatments and their interaction with other food components is important for the

detection of these antigenic proteins in processed or combined food matrices using the developed

immunoassays.

7

The following are the specific experiments performed in the current study:

I. Analysis of Physical Characteristics and Chemical Composition

1. Determined the physical characteristics (seed weight and seed size) of select

almond and macadamia nut seeds.

2. Determined the chemical composition (moisture, lipid, protein, ash, total soluble

sugars, and tannins) of select almond and macadamia nut seeds.

3. Evaluated polypeptide profiles of select almond and macadamia nut seeds using

gel electrophoresis.

4. Determined the amino acid composition of defatted select almond and macadamia

nut seed flours.

5. Determined the free amino acid composition, in particular free asparagine, of

defatted select almond seed flours.

II. Antigenic Protein Detection and Stability

1. Produced rabbit pAbs against total soluble macadamia nut seed proteins.

2. Investigated the effect of cultivar, geography, and harvest year on AMP detection

using a sandwich ELISA where rabbit pAb was used for capture and mAb 4C10

for AMP detection.

3. Investigated different variables (e.g. coating buffer, coating concentration, pAb

concentration) for the development of a sensitive pAb-based competitive

inhibition ELISA for macadamia nut seed protein detection in foods.

4. Examined the specificity of mAb 4C10 and anti-macadamia nut seed protein

pAbs by evaluating their cross-reactivity with select tree nut, legume, cereal, and

other food proteins.

5. Assessed the effects of the following on AMP and macadamia nut seed protein

detection.

a. Thermal processing: subjected whole, raw almond and macadamia nut

seeds to autoclaving, blanching, dry roasting, frying, and microwaving.

b. pH treatment: exposed defatted almond and macadamia nut flours to

distilled, deionzed water (DI water) and then adjusted to pH 1, 3, 5, 7, 9,

11, and 13.

8

c. Food matrices: spiked select commercial products with soluble almond

and macadamia nut proteins and defatted flours. Ingredients and processed

foods that commonly contain almond and macadamia nut seeds were

selected. These foods included baked goods and confectionary products.

RESEARCH HYPOTHESIS

I. Analysis of Physical Characteristics and Chemical Composition

1. Physical characteristics and chemical composition of select almond seeds will be

affected by cultivar, geographic and annual variations.

2. Cultivar variations will affect the physical characteristics and chemical

composition of select macadamia nut seeds.

3. Almond and macadamia nut seed proteins will exhibit variations in the

electrophoretic profile.

4. The amino acid composition of almond and macadamia nut seed proteins will

contain a high amount of acidic amino acids.

II. Antigenic Protein Detection and Stability

1. Rabbit pAbs (anti-total almond seed proteins, -AMP, and -macadamia nut seed

proteins) and mouse mAb 4C10 will recognize antigenic proteins in tested

immunoassay formats.

2. Rabbit anti-total soluble macadamia nut seed protein pAbs will be cross-reactive

with other plant seed proteins, while earlier studies determined the mAb 4C10

was AMP specific.

3. Almond and macadamia nut seed proteins will retain antigenicity upon thermal

processing.

4. Exposure of almond and macadamia nut seed flours to various pH environments

will significantly affect the extraction and stability of antigenic proteins.

5. The antigenicity of almond and macadamia nut seed proteins will be affected by

food matrices.

9

SIGNIFICANCE OF THE STUDY

1. The compositional analysis of select almond seeds will contribute to the existing

database on almond seed composition in addition to supplying new information on

compositional variation as affected by cultivar, geography, and harvest year.

2. Exploring cultivar variations within different geographic regions of California may be

useful in the selection of cultivars for regional breeding purposes.

3. The compositional analysis of macadamia nut seed varieties will add new information to

the sparsely existing macadamia nut seed composition database, in addition

immunoassay results will add new information.

4. Amino acid composition of almond and macadamia nut seed proteins will be useful in

evaluating their nutritional quality.

5. Free amino acid composition, particularly free asparagine, of almond seed flours will be

useful in evaluating the risk associated with roasting and acrylamide formation.

6. Application of immunoassays for detection of trace amounts of almond and macadamia

nut proteins will be useful for the food industry.

LIMITATIONS OF THE STUDY

1. Only select almond seed cultivars and select geographic regions in California were

studied, thus comparisons were not made between all known almond cultivars and

geographic growth regions outside California.

2. Only select macadamia nut varieties, grown in Hawaii or purchased commercially,

were studied.

3. Cross-reactivity and recovery studies were limited to select tree nuts, oilseeds, legumes,

cereals, spices, and processed foods.

4. The pAbs used in the study exhibited cross-reactivity with other food proteins.

5. In vitro immunoassays using IgG antibodies from rabbit and mouse may not be

clinically relevant.

10

REVIEW OF THE LITERATURE

Almond Cultivars

Almond cultivars Nonpareil, Carmel, Butte, and Monterey account for roughly 72% of

the total California almond production (4). Cultivar Nonpareil is valued commercially for it’s

thin outer shell and smooth kernel which allows for blemish-free processing and is ideal for

blanching and cutting. Cultivar Mission has a thick shell and wrinkled kernel making it

unsuitable for blanching, however, the wrinkled kernel is ideal for adhering seasonings and other

foods making it perfect for use in snack mixes and ice creams. Cultivar Mission with a deep

brownish-red skin is darker than cultivar Nonpareil, and the kernel is typically wider and has a

stronger flavor. Cultivar California, which includes a number of varieties, has a medium thick

shell, darker skin (compared to Nonpareil), and is suitable for blanching. Cultivar Carmel has a

soft shell and is used for blanching and roasting (2).

Macadamia Nut Cultivars

Two species of edible macadamia nut (M. intergrifolia and M. tetraphylla) and their

hybrids (containing both species) are cultivated. M. intergrifolia nut seeds dominate the

worldwide industry, as they are more resistant to water stress and have a lower sugar content (26,

27). The higher sugar content of M. tetraphylla nut seeds lead to excess browning when roasted

(27). In addition, the rough shell types (M. tetraphylla) produce lower grade nuts, bloom slowly,

and their production is unreliable and inconsistent in Hawaii (50). Cultivation conditions in

California and Australia are better suited for the rough shell and hybrid cultivars (25). Steiger et

al. (25) found the two commercially important species share high genetic similarities, 83.9%

within M. integrifolia (18 cultivars), 87.9% within M. tetraphylla (2 cultivars) and 72.6%

between M. integrifolia and M. tetraphylla. Peace et al. (26) using DNA typing, evaluated

roughly 80 macadamia nut cultivars from a variety of origins and developed a cultivar family

tree which defines seven cultivar gene-pools. Gene pools 1 and 2, with the least genetic diversity,

consists of almost all the Hawaiian cultivars, some Israeli cultivars (believed to be derived from

a Hawaiian seed), and several Australian cultivars. Gene pools 3 and 4 are mainly the species M.

intergrifolia and contain most of the Australian cultivars. Gene pools 5 and 6 consist primarily of

Australian hybrid cultivars, and gene pool 7 consists of a mix of the M. tetraphyllas and hybrid

cultivars from Australia and South Africa. Most of the M. intergrifolia cultivars, including all of

the Hawaiian and Australian cultivars, are believed to have come from the Mt. Bauple and

11

Amamoor/Imbil regions (26). The Hawaii Agricultural Experiment Station (HAES) named and

introduced several promising selections of the smooth shell type M. integrifolia (25) in 1948,

which led to the modern macadamia industry in Hawaii. The breeding work was initiated in the

1950’s by Beaumont, Moltzou, and Storey and later pursued by Hamilton (1960’s to 1970’s) and

Ito (1970’s and 1980’s) (27). Macadamia nut seed varieties perform differently in various

climates, therefore it is ideal to first examine the climate of an orchard site in order to determine

the suitability of a specific variety (27). The current study evaluated 5 Hawaiian cultivars

obtained from the University of Hawaii, Manoa. The cultivars and their specific details are as

follows:

Keauhou also known as HAES 246, is from the species M. integrifolia and part of gene

pool 1 (26). The oldest Hawaiian cultivar, originating from the Keauhou Orchard of Hawaii

Macadamia Nut Company at Kona, was selected in 1935 and named in 1948 (25, 29, 51, 52).

The tree is broadly spreading and therefore requires wider spacing in the orchard than narrower,

more upright cultivars (27). Harvest season is relatively short, with most of the crop maturing

within about 3 months. The nut is medium-size, averaging about 63 nuts per pound and has a

diameter of about 1 inch (29, 51). The nut represents 37-40% of the total kernel weight and 85%

of the kernels are grade 1 quality (determined by flotation testing in water, meaning 85% of the

kernels float because they have a specific gravity of less than 1.0) (51). The nut has a medium to

thick shell with a slightly pebbled surface (29). Cultivar Keauhou typically produces hard nut

kernels, which is considered a desirable trait (27). The tree yields well and is extremely resistant

to anthracnose (a fungal disease in which dark-colored lesions develop on the leaves, stems, and

fruit) but the kernel quality has proven rather marginal and inconsistent in different growth

locations (28, 29, 53).

Purvis also known as HAES 294, is from the species M. integrifolia and is part of gene

pool 1 (26). The novel cultivar was selected in 1981 at the Nutridge Orchard of the Hawaii

Macadamia Nut Company, Honolulu, Oahu (25, 54, 55). In a recent study, cultivar Purvis

performed very well (89% grade 1 kernels) at 2,000 feet at Captain Cook Experimental Station in

Kona, HI compared to other cultivars (54). At that elevation, cultivar Purvis had an average

kernel weight of 40.2% (averages 2.9 g/kernel) and was medium-sized, averaging about 63 nuts

per pound (54).

12

Kakea also known as HAES 508, is from the species M. integrifolia and belongs to gene

pool 1 (25). The cultivar was selected in 1936 and named in 1948 after the hill, Puu Kakea Oahu,

on which the Nutridge Orchard of the Hawaii Macadamia Nut Company is situated (25, 29, 51,

52). The tree is reasonably robust, producing kernels of excellent commercial quality and has

been a consistently productive, long-lived variety in Poamoho, Waikea and Kona experimental

farms (29, 51). The tree has a more upright growth habit than cultivar Keauhou and the younger

trees often need to be topped. Nurserymen consider cultivar Kakea harder to graft than other

varieties but skilled propagators are able to get a high percentage of takes. It has a kernel weight

of 36% (averages 2.5 g/kernel) and 90% of the kernels are of grade 1 quality (51). The nut is

medium-sized, averaging about 65 nuts per pound (51). Cultivar Kakea was originally believed

to be one of the best and most reliable varieties for commercial planting in Hawaii (29), however

is no longer recommended for commercial use (56).

Keaau also known as HAES 660, is from the species M. integrifolia and is part of gene

pool 2 (26). The cultivar originated at the Deschwanden Orchard, Lawai Valley on Kauai and

was selected in 1948 and named in 1966 (25, 29, 51, 57). The tree has an upright growth habit

permitting somewhat closer planting than most other cultivars without undue crowding (27).

Cultivar Keaau performs best at elevations between 300 to 1,000 feet where there is adequate

rainfall (54) and harvest season is typically August to November. Cultivar Keaau has a kernel

weight of 42-46% (averages 2.8 g/kernel) and more than 95% of the kernels are of grade 1

quality. Cultivar Keaau has lost popularity among growers as it produces nuts that are small in

size, averaging about 80 nuts per pound, however these nuts are ideal for the confectionery

industry (27). The nut has a light cream color flesh and a medium brown, thin, smooth shell. The

nuts are excellent for processing and the trees have performed well and have been very

productive during the limited period that this variety has been tested (57).

Mauka also known as HAES 741, is from the species M. integrifolia and is part of gene

pool 2 (26). The cultivar was selected in 1957 at the Glaisyer Orchard, Lawai Valley on Kauai

(25) and named in 1977 (54). Steiger et al. (25) found cultivars Mauka and Keaau share the

highest genetic similarity (98.5%) of the 26 macadamia nut seeds tested. An earlier study also

reported high (> 98%) genetic similarity between cultivars Mauka and Keaau when evaluating

45 Macadamia species (30). Like cultivar Keaau, cultivar Mauka has an upright growing habit,

and therefore is more suited for closer planting (27). Cultivar Mauka has a kernel weight of 43%

13

(average 2.8 g/kernel) and 98% of the kernels are of grade 1 quality (51). The nut is medium-

sized, averaging about 70 nuts per pound (51). Although sharing a high genetic similarity and

similar nut and kernel characteristics with cultivar Keaau, cultivar Mauka was found superior to

other cultivars in the ability to withstand and thrive at higher elevations (1,800-2,000 feet) (25,

58). However, in a recent Hawaiian study at the Captain Cook Experiment Station in Kona,

cultivar Mauka did not perform as well as other cultivars at 2,000 feet (54). Cultivar Mauka is

also prone to formation of a watermark around its base, however the mark disappears with

roasting (27).

The current commercial macadamia nut varieties were selected from commercial seedling

orchards established in Hawaii between 1920 and 1930. Researchers at the University of Hawaii

organized the selection, evaluation and breeding of improved strains to develop the seven

commercial varieties of trees planted today. These macadamia nut varieties were selected for

their outstanding nut quality and productivity in a tropical climate.

Physical Seed Characteristics

Individual seed weight. Spanish grown almond cultivars Guara and Pons have reported

weights of 0.89-1.11 g and 1.44-1.51 g, respectively (59, 60). Several studies report ranges of

0.95-1.92 g for 52 cultivars grown in Apulia, Italy (61), 0.88-0.94 g for Nonpareil and 2

advanced breeding cultivars (23.5-16 and 23-122) (62), 0.50-1.34 g for 26 genotypes grown in

Turkey (63), 2.64 g for cultivar Ta�badem grown in Turkey (64), 1.00-1.38 g for cultivars

Ferragnes and Texas grown in Greece (65), 0.45 g for Nigerian grown almond seeds (66), 1.01-

2.08 g for cultivars Nonpareil and Glucan 101-23 (67), 1.02-1.67 g for 14 Portuguese cultivars

(68), and 0.62-1.29 g for California grown cultivars Mission, Neplus, Peerless, Carmel, and

Nonpareil (69). Individual seed weights for 8 Hawaiian and 40 Australian grown macadamia nut

cultivars ranged from 2.3-3.3 g (28) and 2.4-2.5 g (70), respectively. Seeds between 2 and 3 g are

desired by the macadamia nut industry, as seeds weighing less than 2 g are prone to over-

roasting and seeds weighing more than 3 g are prone to under-roasting (70).

Seed dimensions. Nonpareil and advanced breeding almond cultivars (23.5-16 and 23-

122) ranged from 21.2-21.7 mm in length, 11.7-11.9 mm in width, and 7.3-7.6 mm in thickness

(62). Almond cultivar Ta�badem grown in Konya, Turkey has a reported length of 25.5 mm,

width of 17.0 mm, and thickness of 18.13 mm (64). Almond cultivars Nonpareil and Gulcan

101-23 from Turkey range from 29.0-35.6 mm in length, 17.0-18.7 mm in width, and 9.9-11.1

14

mm in thickness (67). The length, width and thickness of 14 Portuguese almond varieties range

from 20.1-28.7 mm, 11.6-17.0 mm, and 6.6-9.6 mm, respectively (68). Macadamia nut cultivars

have reported diameters of 15/16 inch for cultivars Pahau and Nuuanu, 7/8 inch for cultivar

Kohala, and ~1 inch for cultivar Keauhou (29).

Chemical Composition

Moisture. Typically, almond and macadamia nut seeds have a low moisture content

which assists in extending their shelf life by decreasing the risk of microbial growth and

germination. Almond seeds have a reported moisture content of 5.3% (Table 1), 9.5% for

cultivar Nonpareil (71), 3.1-4.3% for California grown cultivars (Carmel, Mission, and

Nonpareil) (72), 4.4-5.9% for California grown cultivars (Carmel, Mission, Neplus, Peerless and

Nonpareil) (69), 5.1-5.7% for Italian grown cultivars (Ferragnes, Stelliette, Tuono, and

Supernova) (73), 5.6-6.5% for Greece grown cultivars Ferragnes and Texas (65), 5.0% for

Spanish grown cultivar Pons (60), 6.9% for Spanish grown cultivars Pons, Canaleta, and

Marcona (74), 3.4-4.9% for almond seeds purchased commercially in Austria and Greece (75),

and 5.0-6.8% for 14 Portuguese almond varieties (68). The reported moisture content of

macadamia nut seeds are 1.4% (Table 1), 1.4-2.2% for commercially available varieties in

Austria and Greece (75), 2.1% for U.S. purchased varieties (71), 2.9-5.1% for Australian

varieties (M. integrifolia) (27) and 3.0-6.0% for 4 New Zealand grown cultivars (M. tetraphylla)

(76).

Lipid. The lipid fraction of almond and macadamia nut seeds is dominated by MUFAs

and PUFAs (Table 2). Typically, the lipid content of almond seeds is lower than macadamia nut

seeds. García-López et al. (77) and Cordeiro et al. (68) report ranges of 53.1-61.7% in 19 almond

cultivars grown in Spain, Italy, Australia, and the U.S. and 49.0-58.9% total lipid in 14

Portuguese almond varieties (68). California grown cultivars range from 43.4% (71), 43.3-47.5%

for Carmel, Mission, and Nonpareil (72) and 53.6-56.1% total lipid for Mission, Neplus,

Peerless, Carmel, and Nonpareil (69). Cultivars Ferragnes and Texas grown in Greece and

cultivars Ferragnes, Stelliette, Tuono, and Supernova grown in Italy contain 55.6-61.6% and

52.5-57.0% total lipid, respectively (65, 73). Almond seeds commercially available in Ireland,

South Africa, Austria, Greece, Canada and the U.S. have a reported range of 40.8% (78), 47.0%

(79), 52.1-60.4% (75), 51.2-53.5% (80) and 50.6% (Table 1) total lipid, respectively. A large

variation (25.2-60.8%) was reported for 26 almond genotypes grown in Turkey (63) and a

15

significantly lower lipid content (21.8%) was reported for Nigeria grown almond seeds (66).

Macadamia nut seeds range from 75.8% (Table 1), 66.2% for U.S. purchased seeds (71), and

73.9-77.6% total lipid for seeds commercially available in Austria and Greece (75). Kaijser et al.

(76) found 4 macadamia nut cultivars grown in 7 different locations in the North Island of New

Zealand contain 69.1-78.4% total lipid. Kermond and Baumgardt (27) report a range of 71.4-

75.4% for M. integrifolia varieties grown in Queensland, Australia.

Protein. Protein content of almond seeds is generally higher than macadamia nut seeds

(Table 1). Almond seeds have a reported range of 21.3% (Table 1), 20.5% for cultivars Pons,

Canaleta, and Marcona grown in Mallorca, Spain (74), 18.5-20.0% for cultivars Ferragnes,

Stelliette, Tuono, and Supernova grown in Italy (73), 11.5% for seeds grown in Nigeria (66),

20.6-23.3% for California cultivars Carmel, Mission and Nonpareil (72), 16.4-22.2% for

California cultivars Mission, Neplus, Peerless, Carmel, and Nonpareil (69), 19.5% for seeds

purchased in the U.S. (71), 20.0% for seeds purchased in South Africa (79), 16.1-31.5% for 26

almond genotypes grown in Turkey (63), and 22.5-31.3% protein for 14 Portuguese almond

varieties (68). Macadamia nut seeds have a reported range of 7.9% (Table 1), 13.0% for seeds

purchased in South Africa (79) and 8.4% protein for seeds purchased in the U.S. (71).

Ash. Almond seeds range from 3.1% (Table 1), 2.5% and 5.0% for seeds purchased in

the U.S. (71) and South Africa (79), 3.1% for Spanish grown cultivars (Pons, Canaleta, Marcona)

(74), 2.7-2.9% for California grown almond cultivars (Mission, Ne Plus, Peerless, Carmel,

Nonpareil) (69), 2.3-3.7% for Italian grown cultivars (Ferragnes, Stelliette, Tuono, and

Supernova) (73), and 3.4-3.9% ash for 14 Portuguese varieties (68). Higher ash content (6.8%) is

reported for almond seeds grown in Nigeria (66). Macadamia nut seeds are reported to have ash

contents of 1.1% (Table 1), 1.4-1.9% for M. integrifolia varieties grown in Queensland, Australia

(27), and 1.2% and 4.0% for seeds purchased in the U.S. (71) and South Africa (79).

Total soluble sugars. A wide range is reported for total soluble sugars in almond and

macadamia nut seeds. Soluble sugars range from 3.2-5.0% for almond cultivars Burbank,

Peerless, Ne Plus Ultra, Ai, and Davey (81), 2.8-4.3% for cultivars Ferragnes and Texas grown

in Greece (65), 5.4-7.5% for California cultivars Carmel, Mission, Nonpareil (72), 2.7-5.5% for

Italian cultivars Ferragnes, Stelliette, Tuono, and Supernova (73), 5.0-5.5% for Spanish cultivars

Pons, Canaleta, and Marcona (74, 82), and 5.0-7.1% for 14 Portuguese almond varieties (68).

Almond seeds purchased in the U.S. contain 4.8% (Table 1) and 2.1% soluble sugars (71).

16

Macadamia nut seeds grown in the U.S. and Australia contain 1.4% (71), 4.6% (Table 1), and

3.7-6.5% total soluble sugars (27), respectively. Macadamia nut cultivars, Kau, Keaau, Keauhou,

Kakea, and Nelmar, have a reported range of 2.9-5.6% soluble sugars (81, 83).

Tannins. Limited data is available on the tannin content of almond and macadamia nut

seeds. Venkatachalam and Sathe (71) report 0.29% and 0.01% tannins in almond cultivar

Nonpareil and macadamia nut seed, respectively. Ahrens et al. (72) reported a range of 0.12-

0.18% tannins in almond cultivars Carmel, Mission and Nonpareil. A significantly higher tannin

content (1.82%) is reported for Nigeria grown almond seeds (66). Kornsteiner et al. (75) report

0.13-0.46% and 0.05% tannins for almond and macadamia nut seeds purchased commercially in

Austria and Greece. The latter study found a higher content of total phenolics (11-35% more) in

almond seeds with skin (0.13-0.46%) compared to without skin (0.05%). Milbury et al. (84) also

report 0.13-0.24% total phenolics in California almond cultivars (Butte, Carmel, Fritz, Mission,

Monterey, Nonpareil, Padre, and Price) with the majority (47.5-72.7%) concentrated in the

almond skin. These findings support earlier studies reporting a high amount of phenolics in

almond skins (16-20).

Amino Acid Composition

Total amino acids. Almond and macadamia nut seed proteins are dominated by

hydrophobic and acidic amino acids, accounting for 65.8-79.4% and 69.5-69.7%, respectively

(60, 71, 72, 74, 85). When compared with the FAO/WHO recommended essential amino acid

amounts for pre-school children (2-5 years), the sulfur amino acids (methionine and cysteine)

were the first limiting essential amino acids in almond seeds, while lysine was the first limiting

amino acid in macadamia nut seeds (Table 4). Earlier studies report lysine as the first limiting

amino acid in Spanish and Italian grown almond seeds (60, 73). Almond and macadamia nut

seeds are also a rich source of arginine, containing 11.2 g and 14.56 g per 100 g protein,

respectively (Table 4). The essential-to-total (E/T) amino acid ratio for almond and macadamia

nut seeds range from 28.9-31.0% and 27.2-29.5%, respectively (60, 71, 72, 74, 85).

Free amino acids. Spanish and French almond cultivars contain 98.1 to 1,507.0 mg total

free amino acids per 100 g dry weight (39, 60). Aspartic acid, glutamic acid, and asparagine

dominate (� 60%) the total free amino acid composition of almond seeds (39, 86). The free

asparagine content of raw almond seeds is of importance as free asparagine is reported to be a

precursor for acrylamide formation (37, 42). Acrylamide is formed during the Maillard reaction

17

by the reaction of free asparagine and reactive carbonyls (e.g. reducing sugars) at temperatures

above 120 °C (38, 40, 41). In fact, studies show the backbone of the formed acrylamide

originates directly from asparagine (38, 40, 43). Acrylamide formation in foods is a public health

concern as acrylamide has neurotoxic and carcinogenic properties (37, 38, 42, 87). However,

several human studies found no significant evidence that dietary acrylamide increases the risk of

developing colorectal (88), renal (89), breast (90), pharynx, larynx, esophagus, ovary, and

prostate cancers (87, 91). Asparagine was the major free amino acid in almonds of U.S. and

European origin, ranging from 500-2,760 mg/kg (38). Surprisingly, European almonds contained

significantly less (2.7 times) free asparagine and formed significantly less acrylamide (3.0 times)

during roasting than U.S. almonds (38), indicating the possible role of environmental influences

on free asparagine content. Almond seeds with high levels of free asparagine and natural sugars

may pose a health concern as they are typically enjoyed roasted (38). The acrylamide content of

roasted almonds is reported to range from 260-2,000 �g/kg (38, 92). Researchers proposed

several ways to decrease acrylamide formation in almond seeds, which include selecting almond

cultivars low in free asparagine and reducing roasting temperatures (37, 38, 42). Commercially,

almonds are typically roasted at 145 °C for up to 20 min and 165 °C for up to 15 min (92). Lukac

et al. (92) report acrylamide formation in roasted almonds began when the kernel temperature

reached 134 °C and 153 °C at roasting temperature of 145 °C and 165 °C, respectively. The

latter study also report a positive correlation (R2 = 0.982) between development of roast color

(measured by the redness) and acrylamide formation in roasted almonds (92). Reducing the

roasting temperature of almonds may decrease acrylamide formation, however the roast color

and over-all sensory quality of almonds may be negatively affected.

18

Nutrient Almond Macadamia

Moisture 4.70 1.36

Energy 575.00 718.00

Protein 21.22 7.91

Lipid 49.42 75.77

Ash 2.99 1.14

Carbohydrate, by difference 21.67 13.82Fiber, total dietary 12.20 8.60

Sugars, total 4.80 4.57

Sucrose 3.89 4.43

Glucose 0.12 0.07

Fructose 0.09 0.07

Lactose 0.00 0.00

Maltose 0.04 0.00Starch 0.74 1.05

Table 1. Proximate composition of almond and macadamia nut seedsa

aCompiled from USDA National Nutrient Database for Standard Reference, Release 20 (85). Nut seeds are unprocessed and values are expressed as gram per 100 gram edible portion with the exception of energy values which is expressed as kilocalories per 100 gram edible portion.

19

Lipids Units Almond Macadamia

Total saturated g 3.73 12.06

4:0 g 0.00 0.00

6:0 g 0.00 0.00

8:0 g 0.00 0.00

10:0 g 0.00 0.00

12:0 g 0.00 0.08

14:0 g 0.01 0.66

15:0 g 0.00 0.00

16:0 g 3.04 6.04

17:0 g 0.01 0.12

18:0 g 0.66 2.33

20:0 g 0.01 1.94

22:0 g 0.00 0.62

24:0 g 0.00 0.28

Total monounsaturated g 30.89 58.88

14:1 g 0.00 0.00

16:1 undifferentiated g 0.24 12.98

17:1 g 0.03 0.00


20:1 g 0.01 1.89


24:1 c g 0.00 0.02

Total polyunsaturated g 12.07 1.50



18:4 g 0.00 0.00

20:2 n-6 c,c g 0.00 0.00



20:5 n-3 g 0.00 0.00

22:5 n-3 g 0.00 0.00

22:6 n-3 g 0.00 0.00

Cholesterol mg 0.00 0.00

Phytosterols mg 141.00 116.00

Stigmasterol mg 4.00 0.00

Campesterol mg 5.00 8.00

Beta-sitosterol mg 132.00 108.00

Table 2. Lipid composition of almond and macadamia nut seedsa

aComplied from USDA National Nutrient Database for Standard Reference, Release 20 (85). Nut seeds are unprocessed and values are expressed per 100 gram edible portion. C = ‘cis’ isomer, n = omega position, undifferentiated = c or n not known.

20

Minerals Units Almond Macadamia

Calcium, Ca mg 264.00 85.00

Iron, Fe mg 3.72 3.69

Magnesium, Mg mg 268.00 130.00

Phosphorus, P mg 484.00 188.00

Potassium, K mg 705.00 368.00

Sodium, Na mg 1.00 5.00

Zinc, Zn mg 3.08 1.30

Copper, Cu mg 1.00 0.76

Manganese, Mn mg 2.29 4.13

Selenium, Se �g 2.50 3.60

Vitamins Units Almond Macadamia

Vitamin C mg 0.00 1.20

Thiamin mg 0.21 1.20

Riboflavin mg 1.01 0.16

Niacin mg 3.39 2.47

Pantothenic acid mg 0.47 0.76

Vitamin B-6 mg 0.14 0.28

Folate, total �g 50.00 11.00

Choline, total mg 52.10 0.00

Betaine mg 0.50 0.00

Vitamin B-12 �g 0.00 0.00

Vitamin A, IU IU 1.00 0.00

Vitamin A, RAE �g_RAE 0.00 0.00

Retinol �g 0.00 0.00

Tocopherol, � mg 26.22 0.54




Table 3. Mineral and vitamin content of almond and macadamia nut seedsa

aComplied from USDA National Nutrient Database for Standard Reference, Release 20 (85). Nut seeds are unprocessed and values are expressed per 100 gram edible portion. IU = International Unit, RAE = Retinol Equivalent.

21

Amino Acid Almond Macadamia

Tryptophan 0.91 0.70

Threonine 2.54 3.84

Isoleucine 2.99 3.26

Leucine 6.33 6.25

Lysine 2.47 0.19

Methionine 0.64 0.24

Cystine 0.80 0.06

Phenylalanine 4.76 6.90

Tyrosine 1.92 5.31

Valine 3.47 3.77

Arginine 10.40 14.56

Histidine 2.37 2.02

Alanine 4.37 4.03

Aspartic acid 12.38 11.41

Glutamic acid 28.97 23.54

Glycine 6.25 4.71

Proline 4.39 4.86

Serine 4.03 4.35

ADD (%)b

Almond Macadamia

Hydrophobic 34.11 34.72

Hydrophilic 9.29 13.56

Acidic 41.35 34.95

Basic 15.24 16.77

LEAAc

Almond Macadamia

first Lys Lys

second Met/Cys Met/Cys

third Trp Trp

LEAAd

Almond Macadamia

first Met/Cys Lys

second Met/Cys

thirdE/T (%) 26.48 27.17

Table 4. Amino acid composition of almond and macadamia nut seedsa

aAll amino acid values are expressed as gram per 100 gram protein. Data Source: USDA National Nutrient Database for Standard Reference, Release 20 (85). bADD (amino acid distribution). LEAA (limiting essential amino acid) for cpre-school child (2-5 years) and dadult recommended by the report of a joint WHO/FAO expert consultation (93). E/T (%) represents essential-to-total amino acid ratio.

22

Climactic, Geographic and Genetic Effects on Composition

Physical seed characteristics and chemical composition of tree nuts can be affected by

environmental conditions, harvest year, geographic location as well as genetic variations.

Evaluating these variations are important as superior cultivars with high production yields and

desired quality can be selected for commercial production.

Almond cultivars (52 total) from the Apulia region in Southern Italy differed significantly

in kernel yield (kg of almonds per tree), shelling percentage (shell weight representing total

weight of almond nut), number of double kernels (presence of 2 kernels in a single shell), kernel

weight, almond nut weight, total lipid content, and �-tocopherol content (61). Kodad and Socias i

Company (94) also found bloom density, fruit set, fruit density and productivity were highly

dependent on genotype and harvest year when studying 9 almond genotypes over 2 consecutive

years (2003 and 2004). Pereira-Lorenzo et al. (95) report 47 chestnut cultivars grown in 6

Spanish regions differed significantly in chemical composition. Cultivar and geographic location

significantly affected moisture, starch, total sugars, ash, phosphorus, and magnesium content but

had no affect on crude fiber, sodium, zinc, copper, and iron content. Crude protein, fat,

manganese and calcium only varied by cultivar but not by geographic location and potassium

only varied by geographic location but not by cultivar. Cultivar variation significantly influenced

the proximate composition of 24 pecan cultivars (96). Additionally, the proximate composition

of 2 pecan cultivars (Desirable and Choctaw) was affected by growth location (Georgia or

Texas) (96). Harvest year and cultivar had no significant influence on lipid, fiber, vitamin C,

fatty acid, mineral, and protein content of Australian grown pecan cultivars Wichita and Western

Schley from 1995-1997 (97). However, sugar content was significantly affected by harvest year

with pecans harvested in 1997 containing 44-56% less sugar than pecans harvested in 1995 and

1996 (97). Severe weather conditions (e.g. flooding) during 1997 were believed to be a

contributing factor to the reported variation in sugar content (97). Growth location was reported

to significantly affect the protein content of pecans, with U.S. grown pecans containing more

protein (~42%) than Australian grown pecans (97). Effect of irrigation regime (non-irrigated or

irrigated soil), fertilizer treatment (inorganic or organic), and harvest year (2002 and 2003) on

the physical properties of almond cultivar Guara were reported (59). Non-irrigated soil produced

almond seeds of greater mass, width, and geometric mean diameter, while irrigated soil produced

longer and more spherical seeds. The physical properties of almond seeds were significantly

23

affected by harvest year, but not by fertilizer treatments. Elevation significantly influenced shell

thickness and ash content of hazelnut cultivar Tombul grown at 4 elevations (0-50, 100-150,

200-250, and 300-350 m) in Persembe, Turkey (98). Cultivar and harvest year but not

geographic location significantly affected the lipid content of 17 chestnut (Castanea sativa Mill)

cultivars grown in 3 regions of Portugal (Terra Frai, Padrela and Soutos da Lapa) over 2 crop

years (2001 and 2002) (99). In a recent study, Borges et al. (100) also report cultivar and

geographic location significantly affected moisture, starch, crude protein, reducing sugars, crude

fat, ash, phosphorus, potassium, calcium, magnesium, copper, iron, manganese, and zinc content

of 17 chestnut cultivars grown in 3 regions of Portugal. Cultivar variation (Uzun, Kirmizi, Halebi

and Sirrt) was not found to significantly affect the total fat and protein content of pistachio nuts

(101). In another study, significant differences were reported in moisture, protein, ash,

potassium, magnesium, and sodium content of 5 pistachio cultivars (Uzun, Kirmizi, Siirt, Ohadi,

and Halebi), while no difference was observed in total fat and copper content (102). Soil

composition was found to influence total protein in 15 Spanish chestnuts, with significantly

higher protein in chestnuts grown in schists-based [consisting largely of micas, chlorite, talc,

hornblende (calcium-rich), graphite, and other minerals] soil than those from granite-based soil

(103). The same Spanish chestnuts also varied significantly in moisture, sucrose, glucose,

fructose, fiber, protein, lipids, and mineral content but not in starch content. Another study report

cultivar, [‘Emka’, ‘Kora’ (both from Poland), ‘Pyra (Czech Republic), and ‘Krupinka (Ukraine)],

geographic location (�eské Bud�jovice and Humpolec in the Czech Republic) and harvest year

(1999 and 2000) had a significant influence on the crude protein content of buckwheat flour

(104). The authors report soil in Humpolec contained a higher quantity of mineral nitrogen than

soil in �eské Bud�jovice, which partly explained the significant impact on crude protein content.

Soil with a higher supply of plant available nitrogen is reported to increase the crude protein

content of rapeseed (105), lupine seed (106), pearl millet (107), and rice (108). Cultivar and

geographic location influenced the free amino acid composition of almond seeds with serine,

asparagine, and glutamic acid varying by cultivar and arginine and glycine varying by

geographic location (39, 109).

Tree Nut Allergies

Tree nuts are one of the “big 8” common food allergens, in addition to eggs, shellfish,

fish, wheat, soy, milk, and peanuts (110, 111). Proteins present in these foods are responsible for

24

over 90% of all food-related allergic reactions (112, 113). An estimated 6% of children and 3.7%

of adults in the U.S. have food allergies, of which tree nut allergies affect 0.2% of the children

and 0.5% of the adults (111). Allergies to peanuts and tree nuts tend to be more severe, causing

life-threatening and sometimes fatal reactions (111, 114). In fact Bock et al. (114) found of the

32 food induced anaphylaxis fatalities evaluated in the U.S., peanuts (62.5%) and tree nuts

(31.3%) were responsible for 94% (111). A more recent report by Bock et al. (115) found 80.6%

of the 31 food induced anaphylaxis fatalities evaluated in the U.S. from 2001-2006 were also

caused by peanuts (54.8%) and tree nuts (25.8%). Allergic reactions to tree nuts and peanuts are

classified as type I reaction (often referred to as acute or immediate hypersensitivity reactions),

in which symptoms are mediated by allergen-specific immunoglobulin E (IgE) antibodies (Abs).

Mast cells and basophils in tissue have a high affinity for the Fc region of IgE Abs, and become

sensitized and triggered when bound with IgE. Upon subsequent exposure to the antigen/allergen

IgE-bound mast cells or basophils will cross-link and release (referred to as ‘mast cell

degranulation’) histamine and other inflammatory mediators. Symptoms generally appear within

minutes up to 2 h following ingestion of the antigen and depend on the location of these

degranulated cells, in the skin (hives, urticaria), respiratory (rhinitis, asthma), GI tract (diarrhea),

or combined systems (anaphylactic shock). An estimated 1-2% of the U.S. population is allergic

to peanuts, tree nuts, or both (116, 117). Over a 5 year period (1997-2002), Sicherer et al. (117)

reported a 100% increase in the rate of nut allergies. The most frequently reported tree nut

allergies are to walnut (34% of respondents), followed by cashew (20%), almond (15%), pecan

(9%), pistachio (7%), and other tree nuts (less than 5% each) (116). In the U.S. and the U.K.,

fatal cases of anaphylaxis to food are caused primarily by tree nuts and peanuts with tree nuts

accounting for 15-30% of fatalities and peanuts accounting for 50-62% of fatalities (114, 118-

121). Until recently, it was believed that children did not out grow allergies to tree nuts and

suffered a life-long challenge. However, Fleisher et al. (122) report 9% of children do out grow

nut allergies. The new Food Allergen Labeling and Consumer Protection Act of 2004 (FALCPA)

which took effect January 1, 2006, mandates that foods containing the “big 8” common food

allergens must declare the food in plain language on the ingredient list (123, 124). The Act was

passed to protect atopic consumers from the potential risk of a severe allergic reaction from

offending foods that may contain a “hidden” or undeclared allergen (124). The presence of

undeclared allergens in food has lead to several published reports of serious anaphylactic

25

reactions (125-131) as well as several large scale food recalls (45, 127). The occurrence of these

undeclared allergens in food that lead to food recalls are typically caused by inaccurate labeling

through ingredient omissions and/or errors and cross-contamination of manufacturing equipment

(127). In order for the food industry to fully comply with the new law, there must be accurate,

sensitive, and robust detection methods to test for trace amounts of these allergens in processed

foods.

Detection Methods for Tree Nut Allergens

Currently, immunochemical techniques, such as ELISA, have been developed and are

available commercially to detect potential allergenic proteins (132-134). Molecular biological

techniques, such as polymerase chain reaction (PCR), have also been introduced as an alternative

and suitable detection method for hidden allergens (135-138). Immunoassays and their level of

sensitivity are listed in Table 5. However, immunochemical detection methods are lacking for

several tree nuts and several of the previously developed methods are not sufficiently sensitive

(1-100 ppm or mg allergenic protein/kg food) to detect trace amounts of these allergens (137,

165, 166).

Almond and Macadamia Nut Allergens

Almond major protein (AMP), a 14S globulin, also referred to as amandin is the major

allergen recognized by � 50% of the almond-sensitive patient sera IgE tested (49, 139, 167, 168).

AMP, with an estimated MW of 379-475 kDa, represents 65-70% of the aqueous solvent

extractable proteins in almond seed (49). AMP is composed of 2 major types of polypeptides, an

acidic (42-64 kDa range) and basic (20-27 kDa range) subunit, linked by disulfide bonds (49,

169, 170) and also contains several additional polypeptides (49). Corresponding Western blots of

AMP probed with almond sensitive human sera show strong reactivity with the 42-44 kDa

polypeptide bands (139, 167). Bargman et al. (171) had earlier reported IgE from almond

sensitive patients to react with two polypeptides, 70 kDa and 45-50 kDa. Other IgE reactive

proteins from almond seed (45, 37, 30 and 12 kDa) have also been identified (172, 173). N-

terminal amino acid sequence analysis indicated the 45 and 30 kDa bands had 60% homology to

the conglutin-γ heavy chain from lupine seed (Lupinus albus) and the basic 7S globulin from

soybean (Glycine max), while the 12 kDa band showed good homology to the 2S albumin from

English walnut (Jug r 1) (172). Tawde et al. (174) reported nearly half of the almond sensitive

26

patient sera reacted to a profilin (designated Pru du 4) protein with a molecular weight of 12-16

kDa.

Allergic reactions to macadamia nut, although not as common as other tree nuts have

been reported (121, 169, 175-181). However, no allergens from macadamia nut have been fully

characterized to date. Sutherland et al. (177) report IgE from a macadamia sensitive patient

reacted strongly with a 17.4 kDa polypeptide, with also a weaker reactivity to several higher

molecular weight polypeptides (kDa not given) in raw and roasted macadamia nuts. In addition,

macadamia nut oil was reported to contain IgE reactive proteins when probed with macadamia

sensitive patient sera (182). A vicilin from macadamia nut seed with anti-bacterial properties has

been isolated, but its allergenicity was not determined (183).

Cross-Reactivity

Evaluating the cross-reactivity of Abs used for immunoassays is of importance as many

foods containing almond and macadamia nut seeds may also contain other ingredients/foods

which may interfere and possibly cause errors with accurate detection and labeling.

Earlier studies report cross-reactivity of rabbit anti-total soluble almond proteins and

rabbit anti-AMP pAbs with other plant seed proteins from basmati rice, Brazil nut, cashew and

purified Ana o 2, Great Northern bean, hazelnut, macadamia nut, pistachio, black walnut,

sesame, and tepary bean (110, 134, 140, 141, 184). Lee et al. (185) found rabbit anti-AMP pAbs

and human IgE from almond sensitive patients to cross-react with the 50 kDa maize -zein

protein. Human IgE from almond sensitive patients has also been found to cross-react with rye

grass pollen profilin (174), which is not surprising as pollen profilins are believed to sensitize

individuals to food profilins (111, 186, 187). Cross-reactivity of human IgE is reported in foods

of the same botanical family, such as walnut and pecan (Juglandaceae family), cashew,

pistachio, and mango (Anacardiaceae family), and apricot, almond, cherry, plum, strawberry,

apple, peach, and pear (Rosaceae family) (184, 188-191). Fink et al. (189) report one patient

with an earlier reported allergic reaction to cherries later developed anaphylaxis to almond seeds.

Kewalraman et al. (192) also reported human IgE from almond allergenic patients to cross-react

with apricot seed, sunflower, pine nut, walnut, and pecan (192). Tawde et al. (193) report 33% of

tested almond and walnut sensitive patient IgE (26 total) cross-reacted with Fus c 1, a fungal 60S

RP aeroallergen from Fusarium culmorum. Sera IgE from patients with sensitivity to several tree

27

nuts (almond, Brazil nut, cashew, and hazelnut) were found to cross-react with peanut proteins

(194).

One macadamia nut sensitive patient’s sera was found to cross-react with a ~60 kDa

polypeptide from raw and roasted hazelnut and the patient also had positive skin prick tests

(SPT) to rye grass pollen and house dust mites (177). Lerch et al. (176) reported two macadamia

nut sensitive patients with positive SPTs to tree pollens (birch, alder, hazel, ash, beech, oak), rye

grass, plantain pollen, sorrel pollen, latex and hazelnut.

Allergen Stability

Tree nuts are typically subjected to a variety of processing conditions (e.g. roasting and

frying) in order to improve their sensory qualities (e.g. flavor, taste, appearance, and texture).

Since several Salmonella outbreaks in 2001 and 2004 were traced back to raw almond seeds

grown in California, ABC and the USDA have created a mandatory program requiring

pasteurization of all raw almond seeds. The final rule was published in the Federal Register on

March 30, 2007 and mandatory compliance with this rule began September 1, 2007 (195).

A rabbit pAb-based inhibition ELISA found almond seed proteins stable to various

thermal processing treatments (139, 140, 196, 197). Su et al. (198) using pAb rabbit anti-AMP

immunoassays report the stability of AMP to -irradiation alone (1, 5, 10, 25 kGy) or in

combination with various thermal processing treatments (pressure cooking, blanching, frying,

microwaving, and dry roasting). Binding of human IgE from patients with sensitivity to several

tree nuts (almond, Brazil nut, cashew, and hazelnut) to almond seed proteins did not significantly

differ between the raw and roasted (180 °C for 15 min) almond seed samples (194). However,

Venkatachalam et al. (196) report a loss of IgE binding to a low molecular weight polypeptide

(~18.8-20.6 kDa) in commercially purchased dry roasted almonds.

A decrease in the antigenicity of AMP as a result of acidic pH exposure has been reported

(140, 167).

To date, stability of macadamia nut proteins to thermal processing treatments and

extreme pH conditions has yet to be investigated.

28

Table 5. Immunoassays for tree nut protein detection

Limit of Detection

(LOD) in food matrix

Limit of Detection

(LOD) of pure allergen

Competitive Inhibition ELISA (rabbit IgG) 5-37 ppm 0.087 ± 0.016 ppm 139

Competitive Inhibition ELISA (rabbit IgG) NA 0.3 ppm 140

Western blotting (rabbit IgG) 5 ppm NA 134

Sandwich ELISA (rabbit and sheep IgG) NA 1 ppm 141

Brazil nut 2S protein Competitive Inhibition ELISA (rabbit IgG) NA 1 ppm 142

Cashew major protein (CMP, Ana o 2)

Sandwich ELISA (goat and rabbit IgG) 1-31 ppm 0.02 ppm 143

Time-resolved floroimmunoassay (rabbit IgG, sandwich) 0.1-0.33 ppm 0.005-0.16 ppm 144

Sandwich ELISA (rabbit and sheep IgG) 10 ppm NA 145

Competitive Inhibition ELISA (chicken pAb IgY) 0.01-0.03 ppm NA 146

Competitive Inhibition ELISA (rabbit IgG) 0.25-0.45 ppm 0.002-0.008 ppm 147

Biosensor-based Assay (rabbit IgG) 10 ppm NA 148

Competitive Inhibition ELISA (human IgE) 6 ppm 0.03-1 ppm 149

Sandwich ELISA (mAb IgG1 and rabbit IgG) 0.2-1.2 ppm 0.006 ppm 150

Sandwich ELISA (rabbit IgG) 0.5-1 ppm 0.0007 ppm 151

Sandwich EIA (chicken pAb IgY) 0.12-1 ppm 0.03 ppm 152

Sandwich ELISA (pAb IgG) 1 ppm 0.005-1 ppm 149

Sandwich ELISA (rabbit IgG) 120-200 ppb 60-110 ppb 153

Western blotting (rabbit IgG) 5 ppm NA 134

Rocket Immunoelectrophoresis Assay 20 ppm NA 154

Pecan proteins Competitive Inhibition ELISA (rabbit IgG) NA 0.03-0.8 ppm 155

Competitive Inhibition ELISA (rabbit IgG) 0.8-2 ppm 0.14 ppm 156

Competitive Inhibition ELISA 0.4 ppm NA 157

Sandwich ELISA (mAb IgG1 and rabbit IgG) 0.2-0.8 ppm 0.007 ppm 150

Sandwich ELISA 0.15 ppm 0.07 ppm 136

Prolisa Peanut PAK (Pro-Lab Diagnostics, Inc.,Neston, UK)

1.6 ppm NA

Ridascreen Peanut (R-Biopharm, Darmstadt, Germany) 2.5 ppm NAVeratox Peanut

Protein Test Kit (Neogen, Lansing, MI, USA)0.875 ppm NA

mAb Ara h 1 ELISA (INDOOR Biotechnologies, Charlottesville, VA.)

30-2000 ng/ml NA 159

Surface Plasmon Resonance (SPR) Immunoassays 7 ppm NA 160

Sandwich ELISA (mAb IgM and rabbit IgG) 40-2000 ppm NA 161

Walnut proteins Sandwich ELISA (sheep and rabbit IgG) 1 ppm NA 162

0.003 ppm (almond)0.006 ppm (Brazil nut)

0.011 ppm (cashew)0.008 ppm (hazelnut)

0.04 ppm (peanut)0.1-0.5 ppm (Brazil nut) 0.03 ppm (Brazil nut)

0.1-1 ppm (hazelnut) 0.03 ppm (hazelnut)0.1 ppm (peanut) 0.01 ppm (peanut)

Hazelnut proteins

Almond proteins

Allergen Method Reference

Almond major protein (AMP, amandin)

Sensitivity

158Peanut proteins

Reverse dot blot EIA (chicken IgY)Combined (Brazil nut,

hazelnut, peanut) proteins

164

Combined (almond, Brazil nut, cashew, hazelnut, peanut)

proteins

Competitive Inhibition ELISA (rabbit IgG) at least 1 ppm 163

29

MATERIALS AND METHODS

Materials

Almond seed samples (Table 7) were provided by ABC (Modesto, CA). Macadamia nut

seed samples were provided by Dr. Catherine G. Cavaletto of the University of Hawaii (Manoa,

HI).

Raw, unprocessed Nonpareil Supreme almond seeds (major commercial cultivar) and

commercially available macadamia nut seeds purchased in a retail store (Tallahassee, FL) were

used as the source of proteins for raising antibodies and as a control in all immunoassays. Whole

almond and macadamia nut seeds were flushed with nitrogen and stored in air-tight plastic

screw-top bottles at 4 °C to inhibit microbial spoilage and rancidity.

Sources of chemicals were as follows: Electrophoresis grade acrylamide was from

Polsciences, Inc., Warrington, PA. TEMED (N,N,N’N’-tetramethylenediamhe) and bis (N,N ‘-

methylene bis-acrylamide) were from Bio-Rad Laboratories, Richmond, CA. Cellulose

extraction thimbles (25 mm x 100 mm) and filter paper #4 were from Whatman International

Ltd., Maidstone, UK. Coomassie Brilliant Blue R (CBBR), glycerol, bovine serum albumin

(BSA), alkaline phosphatase (AP)-labeled goat anti-rabbit IgG and goat anti-mouse IgG,

horseradish perioxidase (HRP)-labeled goat anti-rabbit IgG and goat anti-mouse IgG, Ponceau S,

phosphatase substrate [p-nitrophenyl phosphate, disodium (PNPP)], methylene blue, methylene

red, luminol (97.0%), and standard low molecular weight marker kit [phosphorylase b (97.4

kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa),

soybean Kunitz trypsin inhibitor (20.1 kDa), �-lactalbumin (14.2 kDa), myoglobin backbone

polypeptide (16.95 kDa), rnyoglobin fragment I + II (14.4 kDa)] were from Sigma Chemical

Company, St. Louis, MO. Nitrocellulose membrane (NC, 0.2 �m) was purchased from

Schleicher and Schuell, Inc., Keene, NH. RIBI adjuvant system (RIBI ImmunoChem Research,

Inc., product code R-730) for rabbit immunization was purchased from Corixa Corporation,

Hamilton, MT. Microtiter ELISA plates (96-well, round bottom) was purchased from Costar,

Cambridge, MA. Tris [trisfiyroxymethyl aminomethane], glycine sulfate (99.0%), boric acid,

glycine, �-mercaptoethanol (�-ME), sodium hydroxide, sodium chloride, ethanol, methanol,

petroleum ether, Tween 20, lead acetate, formaldehyde, bromophenol blue, vanillin, sodium

thiosulfate, sodium azide, magnesium chloride, hydrogen peroxide (30%), SDS (sodium dodecyl

sulfate), acetic acid, sulfuric acid, hydrochloric acid, ammonium persulfate and all other

30

chemicals and supplies (reagent or better grade) were purchased from Fisher Chemical Co.,

Orlando, FL. Several specific chemical, reagent, and equipment sources are indicated along with

the methods.

Methods


Individual seed weight. All nut seeds (total 100 seeds) were weighed using a balance

(Mettler Toledo PB balance, 5-5,100 g range, 0.01 g sensitivity, Model Number PB5001,

Greifensee, Switzerland) and the mean of triplicate readings was calculated and analyzed for

total seed weight.

Seed dimensions. The length, width, and thickness of almond seed varieties and the

diameter of macadamia nut seed varieties (total of 10 from each sample set) were measured (in

mm) using a digital vernier caliper (Guogen®, 0-150 mm range, Shanghai, China) with 0.01 mm

sensitivity. The mean of the readings was calculated and analyzed for seed dimensions.

Preparation of Nut Seed Flours

All nut seeds were ground in an Osterizer blender (Galaxie Model Number 869-18R,

Jaden Consumer Solutions, Boca Raton, FL) with setting ‘grind’ until homogenous flour (~40

mesh) was obtained. The full fat flours were stored in airtight containers (under nitrogen) at -20

ºC until further use.


Moisture. (AOAC Official Method 925.40): A known weight of full fat nut seed flour

(~1 g) was placed in an aluminum pan and dried in a previously heated vacuum oven (95-100 ºC,

25 inch Hg) to a constant weight (199).

Lipid. (AOAC Official Method 948.22): Full fat nut seed flours (~10 g/ thimble) were

defatted in a Soxhlet apparatus using petroleum ether (boiling point range 39.0-53.8 ºC) as the

solvent (flour to solvent ratio of 1:10 w/v) for 6-8 h. Defatted flours were dried overnight (~10-

12 h) at room temperature (RT, ~25 °C) in a fume hood to remove petroleum ether and then

weighed to calculate lipid content (199).

Lipid content (%) = [Initial wt. of full fat flour (g) - final wt. of defatted flour (g)] x100

Initial wt. of full fat flour (g)

31

**Note: Defatted flours were powdered using either a mortar and pestle or Osterizer blender to

obtain homogenous flour sample (~40 mesh) and stored in plastic screw-capped bottles at 4 ºC

until further analysis.

Protein. (AOAC Official Method 950.48): A known weight of full fat nut seed flour

(~0.2-0.25 g) was placed in a micro-Kjeldahl flask. The catalyst [mixture of 0.42 g CuSO4 + 9.0

g K2 SO4], with a few glass beads (to prevent sample bumping), and 15 ml concentrated H2SO4

(36 N) was added to each sample. Sample digestion was done at 410 ºC for 45 min [or until clear

green solution was obtained which ensures complete oxidation of all organic matter]. The digest

was diluted with 50 ml of distilled, deionized water (DI water) and the micro-Kjeldahl flask was

attached to the distillation unit. After adding 45 ml of 15 N NaOH, sample distillation was done

to collect released ammonia into a boric acid solution (2% w/v) containing the indicators

methylene blue (0.0006% w/v) and methyl red (0.0013% w/v). Borate anion (proportional to the

amount of nitrogen) was titrated with standardized with 0.1 N H2SO4 (standardized using 0.1 N

Na2CO3 as primary standard). A reagent blank was run simultaneously with all sample sets.

Sample nitrogen content was calculated using the following formula:

% N = (ml H2SO4 for sample - ml of H2SO4 for blank) x Normality of H2SO4 x 1.4007 weight of sample (g)

Protein (%) = N (%) x 5.18 (almond seed) and 5.32 (macadamia nut seed) (199).

Amino Acid Composition.

Amino acid composition of defatted almond and macadamia nut seed flours was

determined using a Pico-Tag Column Amino Acid Analyzer (Waters Chromatograph Division,

Milford, MA). PITC (phenyl isothiocyanate, 99.9%) derivatization protocol and HPLC

conditions were the same for both hydrolyzed and free amino acid analysis (details stated

below).

Total amino acids. A known weight of defatted seed flour (~10 mg) was hydrolyzed in a

CEM microwave digestion system (CEM Corporation, Matthews, NC. Model # 920150) using

30 �l of 6 N HCl (constant boil, sequanal grade, Pierce, Rockford, IL) and sample was then

dried. To the dried sample, 125 µl of 10-2 norleucine (internal standard to calculate % recovery

for each amino acid.) was added and all amino acids were derivatized as described below under

‘PITC derivatization’ protocol. The derivatized sample was re-suspended in 1 ml of suspension

32

buffer [0.1379 g NaH2PO4 (monobasic), 188 ml DI water, 12 ml acetonitrile, pH 5.05],

centrifuged (12,000 x g, 10 min, RT) and the supernatant filtered through a YM-10 Microcon

centrifugal filter device (Millipore Corporation, Bedford, MA). The filtered supernatant (20 µl)

from each sample was injected onto the HPLC column (see ‘HPLC conditions’ below) for

analysis.

Free amino acids. A known weight of defatted almond seed flour (~10 mg) was

extracted with 1 ml of 70% aqueous ethanol (v/v) for 30 min at RT. Sample was centrifuged

(12,000 x g, 10 min, RT, Eppendorf Centrifuge 5415D, Brinkman Instruments, Westbury, NY)

and supernatant collected. Residue was extracted two more times and the combined supernatant

from 3 extractions was used to determine final free amino acids.

An aliquot (150 µl) of the pooled sample extract was filtered through a YM-10 Microcon

centrifugal filter device. A known amount of L-asparagine and norleucine (internal standards)

were added to 60 µl of the filtrate and sample was dried and derivatized (see ‘PITC

derivatization’ protocol below). The derivatized sample was re-suspended in 200 µl buffer A

(recipe described below) and 20 µl of each sample was injected on to HPLC (see ‘HPLC

conditions’ below) for analysis.

PITC derivatization:

1) 100 µl of ethanol:triethylamine (TEA):DI water (2:2:1 v/v) was added to the dried

sample (details stated under Total and Free Amino Acids section) and then sample was dried on a

speed vacuum centrifuge (Heto Vacuum Centrifuge, Model VR1, ATR Inc., Laurel, MD) for 1-2

h.

2) After drying, 30 µl of ethanol:TEA:DI water:PITC (7:1:1:1 v/v) was added and

samples was held for 20 min at RT in a nitrogen atmosphere and then dried on a speed vacuum

centrifuge.

HPLC conditions:

− Reversed-phase HPLC column: Pico-Tag column (3.9 mm x 300 mm, Part #

WATO10950)

− Mobile phase: Gradient conditions set [as per Waters Operating Manual- The Pico Tag®

Method (200)]

− Buffer A = sodium acetate buffer [19.5 mg sodium acetate, 0.5 ml TEA, final volume 1 L

(pH 6.4)]

33

− Buffer B = 60% acetonitrile and 40% water (v/v)

− Flow rate: 1 ml/min

− Temperature: 38 °C

Tryptophan content was determined by the colorimetric method (No. 3) of Spies and

Chambers (201). Total amino acid composition is reported as g of amino acid per 100 g of

protein and free amino acid composition as mg of amino acid per 100 g edible portion.

Ash. (AOAC Official Method 923.03): Full fat nut seed flour (~0.1 g) was accurately

weighed in a ceramic crucible (previously heated and cooled till constant weight was obtained)

and heated in a muffle furnace maintained at 550 ºC till a constant weight was obtained (199).

Total soluble sugars. Soluble sugars were analyzed by the method of Dubois et al. (202).

A known weight of the defatted nut seed flour (~0.1 g) was extracted with 1 ml DI water

containing 1 mM NaN3 for 1 hr at RT, centrifuged (16,100 x g, 10 min, RT) and the supernatant

collected. To one hundred �l of the supernatant, 100 �l of DI water followed by 200 �l of lead

acetate (20% w/v) was added and vortexed to thoroughly mix the contents. To this mixture 200

�l Na2SO4 (20% w/v) was added, vortexed and then centrifuged (16,100 x g, 10 min, RT). The

supernatant was diluted (10-fold for almond seeds and 100-fold for macadamia nut seeds) prior

to sugar analysis. Forty �l of the diluted supernatant was made to 400 �l with DI water and 10 �l

of 80% w/v phenol and 1 ml concentrated H2SO4 were added. Sample was vortexed to

thoroughly mix the contents and cooled to RT prior to reading the absorbance. Absorbance was

read at 490 nm. A glucose standard curve (0-70 �g glucose) was prepared simultaneously. Total

soluble sugars are expressed as glucose equivalents.

Tannins. Tannin analysis of all nut seeds were done using vanillin assay as described by

Deshpande and Cheryan (203) with some modifications. Full fat nut seed flour (0.1 g) was

extracted with 1.5 ml of 1% v/v HCl in absolute methanol and vortexed continuously at RT for 1

h and then centrifuged (16,100 x g, 10 min, RT). An aliquot of the supernatant was immediately

analyzed for tannins using the 2% vanillin assay. To 100 µl of supernatant, 100 µl of acidified

methanol was added and then color developed by addition of 1.0 ml vanillin reagent (2% vanillin

in acetified methanol). Color was read after desired incubation time at 500 nm. Sample readings

were corrected for background color due to the sample or solvent by taking appropriate blanks

(solvent blank: vanillin reagent, sample blank: acidified methanol). A catechin standard curve (0

to 1 mg/ml) was prepared simultaneously. Tannin content is expressed as catechin equivalents.

34

Soluble Protein Preparation and Estimation

Defatted flours were extracted (1:10 v/v) with borate saline buffer (BSB, 0.1 M H3BO3,

0.025 M Na2B4O7, 0.075 M NaCl, pH 8.45) for 1 h at RT and then samples were centrifuged (15

min at 16,000 x g, RT) and the supernatants collected. Estimation of soluble protein content of

the supernatants was determined by the protein determination assays of Lowry et al. (204) and

Bradford (205). Standard curves were simultaneously prepared in respective buffers using BSA

as the standard protein (0-200 µg range for Lowry and 0-6 �g range for Bradford). The Bradford

assay was modified to a microtiter plate assay as described by Bio-Rad Protein Assay (Bio-Rad

Laboratories, Hercules, CA).

SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis)

SDS-PAGE was done by the method of Fling and Gregerson (206) and Hoefer (207) as

described by Sathe (69). Typically, samples were electrophoresed on an 8-25% linear monomer

acrylamide gradient separating gel (14.5 cm x 16.5 cm x 1.5 mm) and 4% stacking gel (1.0 cm x

16.5 cm x 1.5 mm). The linear acrylamide gradient of the separating gel was prepared by mixing

8% and 25% acrylamide solutions (15 ml each) using a gradient maker (Hoefer SG100, Hoefer

Scientific Instruments, San Francisco, CA) and a peristaltic pump (Model 7014.20, 6-600 RPM,

Cole-Parmer Instruments Co., Chicago, IL).

Proteins were mixed with suitable volumes of SDS-PAGE sample buffer (0.05 M Tris-

HCl, pH 6.8; 1% (w/v) SDS; 0.01% (w/v) bromophenol blue as the tracking dye; and 30% (v/v)

glycerol) containing 2% (v/v) β-ME (for reducing gels when required) and heated for 10 min in a

boiling water bath (95-100 °C). Suitable aliquots of protein samples were loaded on the gels.

Standard low molecular weight (MW) marker kit (vial content reconstituted in 300 µl and 10 µl

were used, name and kDa of specific proteins are listed under ‘Materials’) were included in each

gel run. All gels were run at a constant current for each application (15-20 mA/gel) until the

tracking dye reached the gel edge. Gels were cooled by running cold tap water (~15 ºC) during

the gel run. All gels were stained with Coomassie Brilliant Blue R (CBBR) unless otherwise

indicated. Typically, gels were stained in 50% (v/v) methanol containing 10% (v/v) acetic acid

and 0.25% (w/v) CBBR overnight. Gels were then destained with 50% (v/v) methanol containing

10% (v/v) acetic acid for 4 h followed by 25% (v/v) methanol containing 5% (v/v) acetic acid

until blue background was removed. Glycoprotein staining was done on SDS-PAGE gels using

35

the Gelcode Glycoprotein staining (Pierce Chemical Co., Rockford, IL) procedure as per the

manufacturer’s instructions with suitable modifications.

Polyclonal Antibody Production and Characterization

Rabbit anti-almond seed and anti-AMP was produced and characterized previously as

described by Acosta et al. (140) and Sathe et al. (49). Rabbit anti-total soluble macadamia nut

seed proteins pAb production was done as per the recommendations of Animal Care and Use

Committee, Florida State University (FSU, Approved Protocol #0207) and is described by

Acosta et al. (140). Five hundred �l of a 1 mg/ml stock solution of total soluble macadamia nut

seed proteins extracted with BSB (pH 8.45) was mixed with 500 �l of RIBI adjuvant

(reconstituted in 0.9% w/v saline per the recommendation of the manufacturer). The final

mixture (1 ml dose, 500 �g total macadamia nut seed proteins) was administered in a white New

Zealand rabbit (as per the recommendation of the manufacturer), 100 �l intradermal at 6 sites,

300 �l intramuscular into the hind leg, and 100 �l subcutaneous into the loose skin around the

neck and shoulder region. Following the primary immunization, 5 booster doses (in RIBI

adjuvant as described for primary immunization) were administered at 4-week intervals. Pre- and

post-immunization blood samples were drawn from the marginal ear vein of the immunized

rabbit and collected in glass tubes. Blood was allowed to clot overnight at 4 °C and clear serum

collected. ELISA (direct binding format) and Western blots were used to determine the pAb

serum titer and to assess the specific reactivity of the pAb with macadamia nut seed proteins.

Serum aliquots for immediate use were stored at 4 °C and at -20 °C for long-term storage.

Monoclonal Antibody 4C10 Production and Characterization

Mouse mAb 4C10 was raised previously using standard techniques (208) in the core

Hybridoma Facility at FSU as described by Sathe et al. (49). Briefly, pairs of mice were each

immunized with 40 �g of AMP (column purified) in RIBI adjuvant (RIBI ImmunoChem

Research, Inc., Hamilton, MT) equally split between intravenous and subcutaneous routes. After

the primary immunization, mice were boosted with 20 �g of AMP (in RIBI adjuvant as described

for primary immunization) at 3 week intervals and given a final injection of 25 �g of AMP in

saline (0.9% w/v). Following fusion, the resultant hybridomas were screened and assayed for

relative strength (titer) and specificity to AMP by noncompetitive ELISA (207) and Western

blotting. Serum aliquots for immediate use were stored at 4 °C and at -20 °C for long-term

storage.

36

Protein G Purification of Rabbit anti-Macadamia Nut Protein IgG

A modified protocol previously standardized for purification of rabbit and goat anti-

cashew sera was followed (143). Briefly, IgG (2.5 ml of original, undiluted rabbit serum) was

bound to and eluted from a protein G affinity column (Protein G Sepharose® 4B fast flow resin,

Sigma, St. Louis, MO, Product # P-3296) using 0.2 M glycine sulfate (pH 2.3) as the elution

buffer. The eluate was immediately neutralized with 1.0 M TRIS (pH 9.0). The purified IgG was

dialyzed against coupling buffer (0.1 M NaHCO3 and 0.5 M NaCl, pH 8.5) to remove any

amines present in the buffers. The purified IgG fraction was concentrated (~5-fold) using a YM-

10 Microcon ® centrifugal filter unit (Millipore Corp., Billerica, MA) to a total volume of 478

�l. Bradford protein determination assay estimated 2.23 mg protein/ml in the purified IgG

fraction.

Competitive Inhibition pAb-based Enzyme Linked Immuno-Sorbent Assay (ELISA)

Development

Almond Protein Detection. Competitive inhibition pAb-based ELISAs (rabbit anti-AMP

and anti-soluble almond seed proteins) were previously standardized and described by Acosta et

al. (140), Sathe et al. (49), Roux et al. (139) and Venkatachalam et al. (196) with a few

modifications. The summarized conditions of the two standardized assays are as follows:

Coating 500 ng/well of either AMP or almond seed proteins in ELISA coating buffer (citrate/phosphate, pH 5.0)

Blocking 200 �l/well of blocking solution; 5% w/v of non-fat dry milk (NFDM) in Tris-buffered saline (TBS-T; 10 mM Tris, 0.9% w/v NaCl, 0.05% v/v Tween 20, pH 7.2]

Dilution of Primary (1°) Ab

Rabbit anti-AMP pAbs (50.5 mg protein/ml) and anti-almond seed protein pAbs (41.9 mg protein/ml) diluted 1:100,000 v/v in 1% w/v NFDM in TBS-T

Inhibitory Antigen Concentrations

10-fold antigen titration beginning with 100,000 ng/ml

Dilution of Secondary (2°) Ab

AP-labeled goat anti-rabbit IgG diluted 1:5,000 v/v in 1% w/v NFDM in TBS-T

Macadamia Nut Protein Detection. Competitive inhibition pAb-based ELISA was

performed to standardize a macadamia nut seed protein inhibition curve as described by Acosta

et al. (140). Direct binding ELISA using checkerboard titration format was used for optimization

37

(210) by evaluating the signal strength. Macadamia nut seed proteins extracted in BSB were

coated at different concentrations using several coating buffers, citrate/phosphate (pH 5.0), PBS

(pH 7.2), BSB (pH 8.45), and carbonate buffer (pH 9.8) to assess the optimum coating

concentration and buffer. Optimization of Protein G purified rabbit anti-macadamia nut protein

IgG was done by evaluating the IC50 values of several purified IgG dilutions (1:102 to 1:106, 2.23

mg protein/ml in purified IgG fraction). The optimized competitive inhibition ELISA protocol is

as follows:

Microtiter plates (96-well, round bottom, Costar® 2797, Corning Incorporated, Corning,

NY) were coated with 125 ng/well of soluble macadamia nut proteins diluted in PBS. Plates

were incubated (1 h, 37 °C), washed (3X with TBS-T), then patted dry on paper towels. Two

hundred �l of blocking solution per well (5% w/v NFDM in TBS-T) was added and plates were

again incubated (1 h, 37 °C). Blocking solution was removed, plates were patted dry with paper

towels, covered with Parafilm (American National Can™, Neenah, WI) and stored frozen (-20

°C) until further use. Eighty �l/well of diluted Protein G purified rabbit anti-macadamia nut

protein IgG (1:25,000 v/v in 1% w/v NFDM, approximately 7.14 ng/well) was added to separate

uncoated microtiter plates. To the top row, 20 �l of a standard protein solution (0.5 mg/ml of

macadamia total protein) or an inhibitory test protein solution was added and serially diluted 10-

fold in successive wells and discarded before last row (positive control, only pAb). Plates were

incubated, washed, and dried as described above. Fifty �l of solution from the inhibitor plate was

then transferred to the appropriate wells of a previously washed and dried coated plate. Plates

were incubated, washed, and dried as stated above. Fifty �l/well of diluted AP-labeled goat anti-

rabbit IgG (2° Ab, 1:5,000 v/v in 1% NFDM w/v in TBS-T) was added and the plates were

incubated, washed, and dried as stated above. Color was developed by the addition of 50 �l/well

of phosphatase substrate [one (p-nitrophenyl phosphate PNPP) tablet dissolved in 5 ml of

alkaline phosphatase substrate buffer (0.0049% w/v MgCl2, 0.096% v/v diethanolamine in DI

water with pH adjusted to 9.8 with 3 M HCl)]. Plates were incubated at RT (in the dark) for 17-

20 min then color development was stopped by adding 50 �l of 3.0 M NaOH per well. Plates

were read at 405 nm using a Bio-Tek ELISA reader (Model Power Wave 200, Biotek

Instruments, Inc., Winooski, VT).

38

Sandwich mAb-based ELISA Development for AMP Detection

A sandwich ELISA was developed to standardize an AMP standard curve. The

optimization of pAb coating dilution was done by coating several dilutions of rabbit anti-total

soluble almond seed protein pAb [1:50 to 1:100,000 v/v of original, unpurified sera, (41.9 mg

protein/ml)] on a microtiter plate with ELISA coating buffer, (citrate/phosphate, pH 5.0) and

using the direct binding ELISA checkerboard titration format (210) to determine optimum pAb

coating dilution by evaluating the signal strength. Optimization of almond seed protein

concentration and titration fold were done by evaluating the standard curve of different standard

almond seed protein solutions (10 �g/ml to 100 �g/ml) titrated at different folds (2- to 10-fold)

using the direct binding ELISA format. Finally, optimization of mAb 4C10 (unpurified

supernatant, 10.3 mg protein/ml) was done by evaluating the signal strength of several Ab

dilutions with fixed coating and almond seed protein titration concentrations. The standardized

procedure for the AMP sandwich ELISA is as follows:

Coating

50 �l/well of 1:5,000 v/v diluted unpurified rabbit anti-total almond seed protein pAbs (41.9 mg protein/ml) in ELISA coating buffer (citrate/phosphate, pH 5.0). Plates were incubated, washed, and dried as stated above.

Blocking 200 �l/well of blocking solution (5% w/v NFDM in TBS-T). Plates were incubated, washed, and dried as stated above.

Incubation of Ag

80 �l/well (except top row which contains 98 �l/well) of 1% w/v NFDM in TBS-T. To the top row, 2.5 �l of a 1.0 mg/ml AMP (control) or inhibitory test protein solution was added and serially diluted 5-fold in successive wells and discarded before the last row (positive control, no antigen). Protein titration concentration were 25,000, 5,000, 1,000, 200, 40, 8, and 1.6 ng/ml. Plates were incubated, washed, and dried as stated above.

Incubation of 1°Ab

50 �l/well of mAb 4C10 supernatant (10.3 mg protein/ml) diluted 1:500 v/v in 1% w/v NFDM in TBS-T. Plates were incubated, washed, and dried as stated above.

Incubation of 2°Ab

50 �l/well of AP-labeled goat anti-mouse IgG diluted 1:5,000 v/v in 1% w/v NFDM in TBS-T. Plates were incubated, washed, and dried as stated above.

Color development and ELISA analysis are described under ‘Macadamia Nut Protein

Detection’.

Western Blot Analysis

Western blotting was done as described by Acosta et al. (140). Proteins from SDS-PAGE

gels were transferred onto NC membrane as described by Towbin et al. (211). Transferred

proteins were stained with 0.1% w/v Ponceau S in DI water to ensure proper transfer. Stained

39

Solution 1 Solution 2

0.044% luminol 0.06% H2O2 (30% v/v)

0.007% p-coumaric acid 10.00% TRIS-HCL (1 M, pH 8.5)1.44% dimethyl sulfoxide (DMSO) 88.51% DI water10.00% TRIS-HCL (1 M, pH 8.5)

88.51% DI water

NC membranes containing transferred proteins were scanned and labeled for future reference.

Unbound sites on the NC paper were blocked using 5% (w/v) NFDM in TBS-T for 1 h at RT.

The NC membrane was washed twice, 5 min each, in TBS-T and then incubated with suitably

diluted 1° Ab [rabbit anti-AMP IgG (1:10,000 v/v), Protein G purified rabbit anti-macadamia nut

protein IgG (1:16,000 v/v) or mAb 4C10 IgG (1:5,000 v/v)] in TBS-T overnight (12-16 h) at 4

°C with constant rocking. NC membrane was rinsed twice with TBS-T, washed once for 15 min,

and then 3X for 5 min each in TBS-T. NC membrane was then incubated with suitably diluted 2°

Ab [horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:40,000 v/v) or HRP-labeled

goat anti-mouse IgG (1:10,000 v/v)] in TBS-T for 1 h at RT. The NC membrane was washed as

stated above, developed using a luminol/p-coumaric acid substrate system* and exposed to X-ray

film (Kodak BioMax XAR-5, Eastman Kodak Co., Rochester, NY).

** Luminol/p-coumaric acid substrate system: Blots were immersed in freshly prepared substrate

solution [Solution 1 + Solution 2 mixed in equal volumes just before use] for 3-5 min at RT.

Dot Blot Analysis

Proteins extracted in BSB from each seed sample were pipetted (500 ng) onto a NC

membrane and allowed to dry (37 ºC for 10 min). The NC membrane was rehydrated with TBS-

T solution (5 min) and then blocked with blocking solution (5% w/v NFDM in TBS-T) for 1 h at

RT with constant rocking. The NC membrane was washed 3X (5 min each) with TBS-T. The

addition of 1° and 2° Ab, washing and development protocols are the same as described under

‘Western blot analysis’. Unprocessed Nonpareil Supreme almond seed, AMP (column purified),

and macadamia nut seed extracted proteins (used as controls, as these proteins were used to

generate the Abs) were used to generate standard curves (range from 1-1,000 ng) and to

determine Ab detection sensitivity limit.

40

CATEGORY FOOD MATRIX

Tree Nutsalmond, Brazil nut, cashew, hazelnut, macadamia nut,

pine nut, pistachio, walnut

Cerealsamaranth, barley, Basmati rice, corn, millets, oats,

sorghum, wheat

Legumes/Dry Beans

Inca peanut, Spanish peanut, Virginia peanut, black bean,

black-eyed pea, chickpea, cow pea, fava bean, green

lentil, green pea, Great Northern bean, horse bean, lima

bean, lupine seed, moth been, mung bean, navy bean,

pigeon pea, pinto bean, red kidney bean, tepary bean, urd

bean, val bean, winged bean

Oilseeds flaxseed, sesame, soybean, sunflower

Miscellaneous pepitas, poppy seed

Spices/Cooking Aids

egg white, egg yolk, baking powder, sugar, salt, black

pepper, cardamom, cinnamon, nutmeg, cocoa, vanilla

extract

Dairy NFDM, vanilla ice cream

Fruits/Vegetables cherries, raisins, cauliflower, spinach

Densitometric Quantification

A densitometer [Molecular Imager ChemiDoc XRS System equipped with ChemiDoc

software (Version 4.2), Bio-Rad Laboratories, Hercules, CA] was used to quantify dot intensity

of select seed and standard (control) proteins. Control proteins ranging in concentration from 1-

1,000 ng were used to generate standard curves, which were used to quantify the %

immunoreactivity of test seed proteins when compared to unprocessed control proteins (100%).

Cross-Reactivity Studies

Using immunoassays, cross-reactivity of rabbit anti-macadamia nut protein pAbs and

mouse anti-AMP mAb 4C10 with select food proteins from tree nuts, oilseeds, legumes, cereals,

spices, fruits, vegetables, and other common baking aids was evaluated.

Table 6: Cross-reactivity testing matrices

41

All food samples were defatted as described under the ‘Lipid’ section (Chemical

Composition), with the exception of spices and common baking aids which contain a trivial

amount of fat and vanilla ice cream which was first lyophilized and then defatted. Proteins were

extracted from defatted flours using BSB (1:10 w/v), analyzed for soluble proteins using

Bradford protein determination assay (205), and evaluated for cross-reactivity using the

immunoassays (ELISA, Western and Dot blotting).

Thermal Processing of Almond and Macadamia Nut Seeds

Whole unprocessed nut seeds (~5 g) were subjected to thermal processing methods as

described by Venkatachalam et al. (196) with some modifications. The treatments used and the

preparation details are:

1. Autoclaving at 121 °C, 15 psi for 5 and 30 min each for almond seeds and 5, 10, 20, and

30 min each for macadamia nut seeds. Samples were placed in an aluminum dish and

autoclaved (Consolidated Stills and Sterilizers, Boston, MA). Following completion of

the autoclave treatment, samples were removed, cooled to RT and air-dried at RT in a

fume hood until constant weight was achieved.

2. Blanching in boiling water (95-100 °C) for 3 and 10 min each for almond seeds and 1, 4,

7, and 10 min each for macadamia nut seeds. The ratio of nut seed to water was 1:10 w/v.

After moist heat treatment, samples were patted dry with paper towels and air-dried at RT

in a fume hood until constant weight was achieved.

3. Dry Roasting at 140 °C for 30 min, 160 °C for 30 min, 168 °C for 12 min, and 177 °C

for 12 min for almond seeds and at 140 °C for 20 and 30 min each, 170 °C for 15 and 20

min each, and 200 °C for 10 and 15 min each for macadamia nut seeds. Sample were

placed in an aluminum dish and subjected to roasting in an oven (Thermolyne

Corporation, Dubuque, IA).

4. Frying in vegetable oil (191 °C) for 1 and 2 min each for almond seeds only. The ratio of

nut seed to oil was 1:10 w/v. After frying, samples were patted dry with paper towels and

air-dried at RT in a fume hood until constant weight was achieved.

5. Microwave Heating at 50% power (500 Watts) for 1 and 3 min each for almond seeds

and at 50% power (500 Watts) for 1 and 2 min each, and at 100% power (1000 Watts) for

42

1 and 2 min each for macadamia nut seed in a Panasonic microwave oven (Panasonic

Company, Secaucus, NJ)

Unprocessed almond and macadamia nut seed samples were used as controls. All samples,

processed and unprocessed, were stored at 4 °C in air-tight plastic bottles until further use.

pH Treatments

Defatted almond and macadamia nut seed flours were dispersed in DI water (flour-to-

water ratio of 1:10 w/v), pH was adjusted to the desired value (pH 1, 3, 5, 7, 9, 11, and 13) with

1.0 M HCl and/or NaOH and magnetically stirred for 3 h at RT. Flour slurries (either analyzed

directly or neutralized to pH 7.0) were centrifuged (16,100 x g, RT, 20 min), and the

supernatants were collected and stored in air tight containers until further use.

Spiking Analysis

Almond and macadamia nut-spiked food samples were made by mixing appropriate

ratios of either defatted nut seed flour or nut seed proteins extracted (in BSB) with selected food.

The spiking levels were 100, 10, and 1 ppm (parts per million). The selected food matrices for

the sandwich mAb 4C10-based ELISA were Hershey’s dark chocolate, Nestle white chocolate,

Hershey’s milk chocolate, sweetened coconut, Green Giant frozen green beans with or without

slivered almonds, Honey Bunches of Oats with or without slivered almonds, Almond Joy candy

bar, Mounds candy bar, Planter’s trail mix (containing peanuts, raisins, banana chips, cashews,

pineapple, cranberries, and papaya), vanilla extract, almond extract, vanilla ice cream, almond

praline ice cream, all-purpose wheat flour, salt and sugar. The selected food matrices for the

inhibition pAb anti-macadamia nut based ELISA were Hershey’s dark chocolate, Nestle white

chocolate, Hershey’s milk chocolate, Godiva Milk Chocolate bar with coconut and macadamia

nuts, sweetened coconut, Pepperidge Farms chocolate chip cookie with or without macadamia

nuts, Planter’s trail mix, all-purpose wheat flour, vanilla ice cream, vanilla extract, salt, and

sugar.

Data Analysis and Statistical Procedures

Statistical analyses were done using one-way ANOVA procedures provided by SPSS

statistical software (SPSS for Windows 2006, Microsoft Corporation, Version 15.0, Chicago, IL)

and Fisher’s Least Significant Difference (LSD, p � 0.05) as described by Ott (212). All

experiments were performed at least in duplicate and data expressed as mean ± SEM. When

necessary, data plots and regression analysis were done using Microsoft Office Excel (Windows

43

Vista, 2007). For correlation analyses between sample sets, Pearson’s correlation coefficient (r

value) and p-value (two-tailed) were calculated. Almond cultivars, Carmel and Nonpareil, from

the 12 California counties were analyzed to assess the effect of growth location on seed physical

characteristics, chemical composition and protein antigenicity. Similarly, to assess the effect of

harvest year only certain cultivars from the same California counties were included.

44

1 California County Cultivar (# of accessions per cultivar in parenthesis) Total # of samples

Butte Butte (1), Carmel (1), Mission (1), Nonpareil (1), Padre (1), Price (1) 6

Colusa Carmel (1), Mission (1), Nonpareil (1) 3

Fresno Butte (3), Carmel (3), Mission (1), Nonpareil (2), Padre (1), Price (1) 11

Glenn Carmel (1), Mission (1), Nonpareil (1) 3

Kern Butte (2), Carmel (1), Mission (1), Nonpareil (1), Padre (1), Price (1) 7

Madera Butte (1), Carmel (1), Nonpareil (1) 3

Merced Butte (1), Carmel (1), Mission (1), Nonpareil (1), Padre (1), Price (1) 6

Sacramento Butte (1), Carmel (3), Nonpareil (3) 7

San Joaquin Butte (1), Carmel (2), Mission (1), Nonpareil (1), Padre (1), Price (1) 7

Stanislaus Butte (1), Carmel (1), Mission (2), Monterey (1), Nonpareil (3), Padre (1), Price (1), Sonora (1) 11

Tulare Butte (1), Carmel (1), Nonpareil (1), Padre (1), Price (1) 5

Yolo Butte (1), Carmel (1), Mission (1), Nonpareil (2) 5

Total 74

Table 7. Almond cultivar and history of origin

45

2

Figure 1. Location of almond seed samples included in the current investigation.

46

3

Table 8. Average climatic conditions in select California counties during the 2003/2004 and 2005/2006 almond seasona

Annual

PRCP (in)

Tmax

(°F)Tmin

(°F)PRCP

(in)Tmax

(°F)Tmin

(°F)PRCP

(in)Tmax

(°F)Tmin

(°F)PRCP

(in)Tmax

(°F)Tmin

(°F)PRCP

(in)

NORTH VALLEY

Butte 14.7 57.0 39.1 6.6 72.9 47.9 0.0 89.4 59.7 3.9 74.4 46.9 25.3Colusa 13.7 56.8 39.5 5.0 72.8 47.3 0.0 92.9 58.9 3.0 75.6 48.3 21.7Glenn 11.8 56.8 38.8 9.8 73.9 49.8 0.1 90.2 59.0 2.9 74.4 48.1 24.5

Sacramento 14.1 57.9 41.3 6.0 72.8 48.2 0.0 91.2 59.4 3.9 74.8 49.7 24.1Yolo 14.2 57.0 40.5 5.1 73.3 48.6 0.1 92.0 58.3 3.6 75.1 49.6 23.0

Average 13.7 57.1 39.8 6.5 73.1 48.4 0.0 91.1 59.0 3.4 74.9 48.5 23.7

SOUTH VALLEY

Fresno 5.4 58.1 37.9 3.7 74.7 47.5 0.0 93.5 61.4 1.8 75.8 48.1 10.9Kern 2.8 61.6 41.5 2.4 77.2 52.4 0.0 95.7 68.9 1.0 76.7 53.2 6.2

Madera 6.0 57.4 39.3 3.9 74.4 47.9 0.0 94.4 62.6 3.0 74.2 48.7 12.9Merced 7.6 58.9 40.0 4.6 76.7 47.9 0.0 95.9 61.4 3.2 77.2 47.7 15.4

San Joaquin 8.2 58.3 39.9 4.6 72.8 46.8 0.1 88.3 57.2 1.7 73.9 47.0 14.7Stanislaus 7.1 58.5 39.7 3.3 77.0 48.6 0.0 95.2 61.1 2.1 76.9 49.4 12.5

Tulare 5.6 59.5 38.6 5.6 76.8 49.6 0.0 95.5 63.6 1.5 76.9 49.4 12.7Average 6.1 58.9 39.6 4.0 75.6 48.7 0.0 94.1 62.3 2.0 75.9 49.1 12.2

Winter Spring Summer Fall

California Counties

aPRCP = precipitation, Tmax = maximum temperature, and Tmin = minimum temperature. Seasons: Winter [December (2003 and 2005), January (2004 and 2006), and February (2004 and 2006)], Spring [March, April, and May (2004 and 2006)], Summer [June, July, and August (2004 and 2006)], and Fall [September, October, and November (2004 and 2006)]. Daily weather measurements recorded by the California Irrigation Management Information System (CIMIS) and the National Climatic Data Center (NCDC) of the National Oceanic and Atmospheric Administration (NOAA) were used to calculate county averages. California county weather stations: CIMIS #12 for Butte, CIMIS #32 for Colusa, CIMIS #61 for Glenn, CIMIS #131 for Sacramento, NCDC #9781 and CIMIS #6 for Yolo, CIMIS #2 and #80 for Fresno, NCDC #0442 for Kern, NCDC #5233 for Madera, NCDC #5532 for Merced, CIMIS #70 for San Joaquin, NCDC #6168 and #5738 for Stanislaus, and NCDC #4957 and #9367 for Tulare.

47

4

Figure 2. Estimated timeline for California almond seed production. ** Season varies according to region, variety and weather conditions.

CROP STAGE JAN APR MAY JUN JUL SEPT OCT NOV DECFEB MAR AUG

Irrigation

Dormant

Bloom-Petal Fall

Post-Bloom

Shell Hardening

Hull-Split

Harvest

48

Figure 3. Climatic conditions in Fresno (A), Kern (B), and Stanislaus (C) counties during the 2003/2004 and 2005/2006 almond season. Daily weather measurements recorded by the California Irrigation Management Information System (CIMIS) and the National Climatic Data Center (NCDC) of the National Oceanic and Atmospheric Administration (NOAA) were used to calculate county averages. Weather stations: CIMIS #2 and #80 for Fresno County, NCDC #0442 for Kern County, and NCDC #6168 and #5738 for Stanislaus County. Tmax = maximum temperature and Tmin = minimum temperature.

A

B

C

49

RESULTS AND DISCUSSION

Environmental Conditions

California’s Central Valley extends roughly 400 miles (Figure 1, area stretching from

Shasta County to Kern County) and is one of the most productive agricultural regions in the

world. The Central Valley’s northern region (also known as the Sacramento Valley or North

Valley) encompasses 10 counties and the southern region (also known as the San Joaquin Valley

or South Valley) encompasses 8 counties. The almond seed samples analyzed in the current

investigation were produced in both the North Valley (Butte, Glenn, Colusa, Yolo, and

Sacramento counties) and South Valley (San Joaquin, Stanislaus, Merced, Madera, Fresno,

Tulare, and Kern counties) (Figure 1 and Table 7). Climatic conditions in California vary greatly

from region to region and from year to year. In general, northern California has lower

temperatures and greater precipitation than southern California. Annual precipitation during the

2003/2004 and 2005/2006 almond season varied greatly, with precipitation in the North Valley

almost double the amount received in the South Valley (Table 8). Drought is rarely a problem as

majority of California almond seeds are produced in orchards with irrigated soil. Majority of

precipitation in California typically occurs from late September through April. Almond trees are

dormant from November through January, but begin to bloom in February (Figure 2). Flowers

begin to lose their petals in March and the post-bloom period (April through May) is when

almond seeds grow and develop. By June almond shells hardens and in July the hull (a grayish-

green coat surrounding the shell) splits open and depending on the region almond harvest occurs

in mid-August through October (Figure 2).


Individual seed weight. The tested almond and macadamia nut seeds had individual seed

weights of 0.78-1.44 g and 2.41-3.36 g, respectively (Tables 9-11). Results found in the current

investigation compare favorably with several published studies reporting ranges of 0.45-2.64 g

for almond (59-69) and 2.3-3.3 g for macadamia nut seeds (54, 70). Seed weight varied

significantly by cultivar with almond cultivars Monterey (1.41 g) and Sonora (1.43 g) and the

two commercial macadamia nut varieties [Blue Diamond (3.16 g) and Trader Joe’s (3.36 g)]

weighing more than all other tested cultivars (Tables 9 and 11). Seed weight variation by cultivar

is reported for almonds (59-63, 65, 67-69), hazelnuts (213, 214), macadamia nuts (54), sunflower

seeds (215) and walnuts (216). Almond seeds from the North and South Valley did not differ in

50

weight; however seed weight did vary by county and by cultivar x county interaction (Tables 9

and 10). Generally classified as medium-small seeds (25-30 nuts/oz), cultivars Butte, Mission,

and Padre grown in Stanislaus County were above average size (medium size, 20-25 nuts/oz).

However, cultivar Carmel (medium size) and Price (medium-small size) grown in Stanislaus

County were below average size. Similarly, Merced County grown cultivars Carmel, Butte, and

Padre were above average size, while cultivar Mission was below average. Influence of

geographic location on seed weight is reported in macadamia nut (70), pistachio (217), and

soybean (218). Harvest year did not significantly influence individual seed weight of tested

almond samples.

Seed dimensions. Almond seed samples ranged from 18.3-27.8 mm in length, 9.8-13.8

mm in width, and 6.9-10.2 mm in thickness and macadamia nut seeds ranged from 18.7-20.1 mm

in diameter and 14.4-15.9 mm in thickness (Tables 9-11). Results of the current investigation are

consistent with reported ranges of 20.1-35.6 mm in length, 11.6-18.7 mm in width, and 6.6-18.1

mm in thickness for almond seeds (62, 64, 67, 68) and 22.2-25.4 mm in diameter for Hawaiian

macadamia nut cultivars (29). The seed dimensions of almond and macadamia nut seed samples

varied by cultivar. Of tested almond cultivars, Monterey and Sonora were the longest while

Butte, Mission, and Padre were the shortest (Table 9). The widest almond cultivars were

Mission, Monterey, and Sonora, while cultivar Price was the most narrow (Table 9). The thickest

almond cultivars were Mission, Monterey, and Padre and the thinnest cultivars were Sonora,

Price, and Nonpareil (Table 9). Although similar in weight, length, and width, the thickness of

almond cultivars Monterey and Sonora differed considerably. Cultivar Monterey was one of the

thickest (9.41 mm) almond seeds, while Sonora was one of the thinnest (7.77 mm) seeds.

Overall, the largest almond cultivars were Monterey and Sonora and the smallest was cultivar

Price. Almond seed weight was positively correlated with seed length (r = 0.61, p � 0.05, Figure

4A) and seed width (r = 0.79, p � 0.05, Figure 4B) (Table 17). Commercial macadamia nut

samples were larger than tested Hawaiian cultivars and a positive correlation (r = 0.93, p � 0.05,

Figure 4C) was observed between seed weight and seed diameter (Table 18). Influence of

cultivar variation on seed dimension is reported for almond (62), hazelnut (214), pistachio (219),

and walnut (220). Almond seeds grown in either North or South Valley did not differ in length,

width, or thickness, however seed dimensions varied by county and cultivar x county interaction.

Most noticeably, majority of tested cultivars (Butte, Carmel, Mission, Padre, and Price) grew

51

largest in Fresno County while Nonpareil grew largest in Colusa County. In contrast, majority of

tested cultivars grew smallest in Kern (Butte, Mission, and Price) and Merced (Butte, Carmel,

and Padre) counties while Nonpareil grew smallest in Glenn and Tulare counties (Table 10).

Since Kern and Fresno counties have similar weather conditions (Table 8 and Figure 3), the

observed difference in seed size may be influenced by other factors (i.e. soil composition and

elevation). Annual variations in Kern, Fresno, and Stanislaus counties during the 2003/2004 and

2005/2006 almond season had an influence on seed length and thickness. Almond seeds from the

2005/2006 season were longer (8.4%) and thinner (4.2%) than seeds from the 2003/2004 season

(Table 9). Although almond orchards are irrigated during periods of limited precipitation, the

2003/2004 almond season was unusually dry during the spring months (March, April, and May)

and may partly explain the change in seed size as almond seed growth and development occur

during these months (Figure 2). Influence of geographic location and annual variation on seed

dimension is also reported for pistachio nut (217).


Moisture. Tested almond and macadamia nut seeds ranged from 2.9-5.6% and 1.7-2.6%

moisture, respectively (Tables 9-11). Similar ranges of 3.1-6.9% moisture for almond seeds (60,

65, 71-75) and 1.4-6.0% moisture for macadamia nut seeds (27, 71, 75, 76) are reported. The

present investigation found the moisture content of the tested seeds varied by cultivar. Almond

cultivar Mission had the highest moisture content (4.6%), while cultivar Monterey had the lowest

(3.8%) of all the tested cultivars. Interestingly, commercial macadamia nut variety, Trader Joe’s,

had the highest moisture content (2.6%), while the other commercial variety, Blue Diamond, had

the lowest (1.7%) amount of moisture (Table 11). Cultivar influence on moisture content is not

surprising as seed moisture is known to vary in several tree nut cultivars including almonds (65,

68, 72-77), chestnuts (95, 100, 103), hazelnuts (214, 221, 222) macadamia nuts (27, 76, 83),

pecans (96), pistachios (102, 219), and walnuts (73, 220, 224). Seeds with low moisture content

are at reduced risk of microbial growth, enzyme activity, and spoilage and are therefore desired

by the food industry (83). Geographic location and cultivar x county interaction had a significant

influence on the moisture content of almond cultivars (Tables 9 and 10). Not surprisingly, seeds

from the North Valley (average precipitation of 23.7 inches/year) were higher in moisture

(~9.0% more) than seeds from the South Valley (average precipitation of 12.2 inches/year, Table

8). Similarly, another study found macadamia nut cultivars grown in the Kau district (annual

52

rainfall ~125 cm) of Hawaii to have lower moisture content than cultivars grown in the Hamakua

and Puna districts (annual rainfall ~300-330 cm) (83). One study reported almond seeds from

irrigated soil are higher in moisture than seeds from non-irrigated soil (65). Majority of tested

almond cultivars had higher moisture when grown in Sacramento (Butte, Carmel, and Nonpareil)

and Stanislaus (Butte, Mission, and Price) counties, whereas Merced County grown cultivars

Butte, Carmel, Nonpareil, and Padre were lowest in moisture (Table 10). Cultivars Mission and

Price had the lowest moisture content when grown in Butte and Fresno counties (Table 10).

Geographic location is also reported to significantly influence the moisture content of chestnuts

(95), macadamia nuts (83), pecans (96), and pistachios (217). Annual fluctuations in Fresno,

Kern, and Stanislaus counties during the 2003/2004 and 2005/2006 almond seasons did not

significantly impact seed moisture (Table 9). Seed maturity at harvest (early or late maturing

seeds) can also influenced seed moisture. Late maturing macadamia nut cultivars (Kakea and

Keauhou) grown in Hawaii and harvested between October/January were exposed to the high-

rainfall months and contained more moisture than early maturing macadamia nut cultivars (Kau

and Keaau) harvested between July/September (83). However, another study found the late

harvest almond cultivar, Texas, was lower in moisture than the early harvest cultivar Ferragnes

(65).

Lipid. The tested almond and macadamia nut seeds ranged from 50.9-66.7% and 60.0-

66.2% lipid, respectively (Tables 9-11). Several studies report ranges of 21.8-61.7% and 66.2-

78.4% lipid for almond (61, 65, 66, 72-73, 75, 78-80, 85) and macadamia nut seeds (27, 71, 75,

76, 85), respectively. In the present investigation, lipid content was highest in almond cultivars

Price (61.9%) and Sonora (62.2%) and macadamia nut cultivar Mauka (66.2%). Lipid content

was lowest in almond cultivar Padre (54.5%) and Blue Diamond commercial macadamia nut

variety (60.0%) (Tables 9 and 11). Cultivar variation in lipid content of several tree nuts, almond

(61, 63, 65, 68, 72-75, 77), chestnut (95, 99, 100, 103), hazelnut (214, 221, 222) macadamia nut

(27, 76), pecan (96), pistachio (226), and walnut (73, 220, 223, 224) is reported. The lipid

content of almond seeds from North and South Valley did not vary significantly, however lipid

content of cultivars was influenced by individual counties. Particularly, several cultivars from

Fresno (Mission and Padre), Stanislaus (Nonpareil and Price), and Yolo (Carmel and Nonpareil)

counties were noticeably higher in total lipid, while the lipid content was lower in several

cultivars from Butte (Butte and Carmel) and San Joaquin (Butte, Mission, and Nonpareil)

53

counties (Table 10). Geographic location is also reported to influence the lipid content of lupine

seed (227), pecan (94), pistachio (217), cowpea (228), and soybean (218). Lipid content was also

affected by harvest year with a 9.8% higher lipid content in almond seeds from the 2005/2006

season compared to seeds from the 2003/2004 season (Table 9). The lipid content of chestnuts

(99) and oat seeds (225) is also reported to differ significantly between harvest years.

Protein. The tested almond and macadamia nut seeds ranged from 15.7-26.7% and 5.6-

7.1% protein, respectively (Tables 9-11). Results of the current investigation compare favorably

with the reported range of 11.5-31.5% for almond seeds (63, 66, 68, 71-74, 79, 85) and 7.9-

13.0% protein for macadamia nut seeds (71, 79, 85). A positive correlation between protein and

lipid content (r = 0.646, p � 0.05, Figure 5A) of almond seeds was observed (Table 17). Of

tested almond cultivars, protein content was higher in Monterey (25.6%) and Price (24.8%) and

the lower in Padre (20.4%) (Table 9). Similarly, protein content was higher in Trader Joe’s

commercial macadamia nut variety and cultivar Keauhou (both 7.1%) and lower in cultivar

Keaau (5.6%) (Table 11). Cultivar influence on protein content of several tree nuts, almond (63,

68, 72-74), chestnut (95, 100, 103), hazelnut (214, 221, 222), pecan (96), pistachio (100, 226),

and walnut (73, 220, 223, 224) is reported. Almond seed protein also varied by geographic

location, most notably seeds grown in San Joaquin County were significantly lower in protein

(~10.4-23.5%) compared to seeds from all other tested locations (Table 9). However, no

difference in seed protein was observed in seeds from North and South Valley (Table 9).

Interaction of almond cultivar x county influenced almond seed protein, most noticeably protein

content was higher in cultivars Carmel, Mission, Nonpareil, and Padre from Fresno County and

lower in cultivars Butte, Carmel, and Nonpareil from San Joaquin County (Table 10).

Geographic location is also reported to significantly impact the protein content of buckwheat

flour (87), cowpea (228), durum wheat (229), pistachio (217), and soybean (218). Several reports

suggest the nitrogen composition of soil influences protein content with high quantities of

nitrogen increasing protein content in buckwheat (87), rapeseed (88) lupine seed (89), pearl

millet (90), and rice (91). Additionally, higher protein was reported in chestnuts produced in

schist-based soil compared to chestnuts from granite-based soil (92). Harvest year also impacted

the protein content of almond seeds with 7.1% more protein in seeds from the 2005/2006 season

compared to seeds from the 2003/2004 season (Table 9). Influence of harvest year on protein

content is reported for buckwheat (87), oat seed (225), and rye (230).

54

Ash. The tested almond and macadamia nut seeds ranged from 2.6-3.5% and 1.0-1.4%

ash, respectively (Tables 9-11). Results in the current investigation are consistent with the

reported ranges of 2.3-6.8% for almond seeds (68, 71-74, 79, 85) and 1.1-4.0% ash for

macadamia nut seeds (27, 71, 79, 85). Cultivar variation had a significant influence on the ash

content of tested almond and macadamia nut seeds. Ash content was highest in almond cultivar

Mission (3.3%) and macadamia nut cultivar Keaau (1.4%) and lowest in almond cultivar Sonora

(3.1%) and macadamia nut cultivar Purvis (1.0%). Ash content is reported to vary by cultivar in

almonds (68, 72-74), chestnuts (95, 100, 103), hazelnuts (214, 221, 222), pecans (96), pistachios

(102), and walnuts (73, 210, 223, 224). The ash content of tested almond seeds was found to vary

by geographic location, in particular ash content was higher in almond seeds from the North

Valley (3.23%) compared to South Valley seeds (3.09%). The interaction of almond cultivar x

county was also significant for ash content. Interestingly, ash content was higher in cultivars

Butte and Padre from San Joaquin County but lower in cultivars Carmel and Price from the same

county (Table 10). Similarly, Fresno County produced higher ash in cultivars Mission and Price,

but lower ash in cultivars Nonpareil and Padre (Table 10). Interaction of cultivar and county

factors such as soil composition, irrigation practices, and fertilization regimes may partly explain

the observed variation. Influence of geographic location on ash content of chestnut (95, 100),

hazelnut (98), lupine seed (227), oat seed (225), pecan (96), pistachio (217), and rye (230) is

reported. Harvest year was not found to significantly influence ash content of tested almond seed

samples (Table 9).

Total soluble sugars. Almond and macadamia nut seeds ranged from 3.0-6.5% and 5.3-

8.7% soluble sugars, respectively (Tables 9-11). Ranges of 2.1-7.5% for almond seeds (65, 66,

68, 70, 71, 73, 81, 82, 85) and 1.4-6.5% soluble sugars for macadamia nut seeds (27, 71, 81, 83,

85) are reported. Soluble sugars were negatively correlated with protein content of almond (r = -

0.61, p � 0.05, Figure 5B) and macadamia nut seed samples (r = -0.78, p � 0.05, Figure 5D) and

lipid content of almond seed samples (r = -0.61, p � 0.05, Figure 5B and Table 17). Soluble

sugars varied by cultivar with sugars significantly lower in almond cultivars Monterey (3.0%)

and Sonora (3.4%) and macadamia nut cultivar Mauka (5.3%). In contrast, almond cultivar Padre

(5.1%) and macadamia nut cultivar Keaau (8.7%) had the highest content of soluble sugars

(Tables 9 and 11). Cultivar influence on sugar content is reported in almonds (65, 68, 72-74, 81,

82), chestnuts (100, 103), macadamia nuts (83), pecans (96), and walnuts (73). Soluble sugars

55

did not vary in seeds from the North and South Valley, however interaction of almond cultivar x

county was significant. For instance, soluble sugars were high in almond cultivars Nonpareil,

Padre, and Price from Butte County and cultivars Butte and Mission from San Joaquin County.

On the other hand, Fresno County grown cultivars Butte, Carmel, and Mission were lower in

soluble sugars. Soluble sugars were highest in cultivar Carmel from Stanislaus County (Table

10). Sugar content of chestnut (95) is also reported to vary by geographic location. Harvest year

also influenced soluble sugar content of tested almond seeds, with seeds from the 2003/2004

season containing 25.4% more soluble sugars than seeds from the 2005/2006 season (Table 9).

Harvest year significantly affected the sugar content of pecans with pecans harvested in 1997

containing 44-56% less sugar than pecans harvested in 1995 and 1996 (95). Severe weather

conditions (e.g. flooding) during 1997 were believed to be a contributing factor to the reported

variation in sugar content of pecans (95). Precipitation was greater in Fresno, Kern, and

Stanislaus counties during spring 2005/2006 almond season and may partly explain the decrease

in total soluble sugars (Figure 2).

Tannins. Almond and macadamia nut seed samples ranged from 0.07-0.36% and 0.03-

0.04% tannins, respectively (Tables 9-11). Tannins are reported to range from 0.12-0.46% for

almond seeds (71, 72, 75, 84) and 0.01-0.05% tannin for macadamia nut seeds (71, 75). Cultivar

variation influenced the tannin content of almond and macadamia nut seed samples. Almond

cultivar Sonora had a significantly larger amount of tannins (0.36%) compared to all other tested

cultivars which ranged from 0.13-0.19% (Table 9). Macadamia nut cultivar Mauka had the

lowest amount of tannins (0.03%) compared to all other tested seeds (Table 11). Cultivar

influence on tannin content is reported for almond (72, 84), common bean (231), field pea (232),

pecan (96, 233), and walnut (73). Geographic location and cultivar x county interaction

significantly influenced the tannin content of almond seeds. Although tannin content did not

differ in seeds from North and South Valley, notably higher tannins were observed in Yolo

County grown cultivars Butte, Carmel, and Nonpareil (Tables 9 and 10). Tannin content was also

lower in Merced County grown cultivars Butte, Carmel, Nonpareil, and Padre and Kern County

grown cultivars Mission and Price (Table 10). Geographic location is reported to impact tannin

content of common bean (203), field pea (232), and pecan (96). Harvest year also significantly

influenced tannin content of tested almond seeds with 40.0% more tannins in seeds from the

2005/2006 season (Table 9).

56

1

Table 9. Cultivar, geographic location, and harvest year variation on the physical characteristics and chemical composition of select almond seedsa

aData are expressed as mean ± SEM (n = 3). For each column, means with different letters are significantly different (p � 0.05) according to Fisher’s LSD test. Lipid, protein, ash, total soluble sugars, and tannins are expressed as gram per 100 gram dry weight.

Cultivar

Butte 1.03 ± 0.02 d 20.05 ± 0.15 e 11.98 ± 0.06 c 9.00 ± 0.06 b 4.14 ± 0.11 bc 57.21 ± 0.61 b 21.61 ± 0.27 cd 3.10 ± 0.03 bc 4.50 ± 0.12 bc 0.15 ± 0.01 cd

Carmel 1.14 ± 0.02 bc 23.52 ± 0.16 c 11.75 ± 0.07 c 8.18 ± 0.06 c 4.04 ± 0.09 bcd 57.28 ± 0.58 b 21.34 ± 0.29 d 3.20 ± 0.04 ab 4.23 ± 0.11 bcd 0.13 ± 0.01 d

Mission 1.14 ± 0.03 bc 20.01 ± 0.17 e 12.80 ± 0.08 a 9.54 ± 0.09 a 4.58 ± 0.16 a 58.28 ± 0.48 b 23.24 ± 0.23 b 3.26 ± 0.02 a 4.03 ± 0.14 d 0.15 ± 0.01 cd

Nonpareil 1.17 ± 0.02 b 23.17 ± 0.16 c 12.42 ± 0.07 b 7.95 ± 0.05 d 3.96 ± 0.08 cd 57.63 ± 0.72 b 20.91 ± 0.27 de 3.12 ± 0.03 bc 4.64 ± 0.16 b 0.19 ± 0.01 b

Padre 1.10 ± 0.03 cd 19.63 ± 0.18 e 12.38 ± 0.11 b 9.53 ± 0.07 a 3.88 ± 0.09 cd 54.53 ± 0.47 c 20.37 ± 0.13 e 3.18 ± 0.02 abc 5.14 ± 0.16 a 0.17 ± 0.01 bc

Price 0.99 ± 0.02 d 21.47 ± 0.18 d 11.46 ± 0.09 d 7.88 ± 0.09 d 4.12 ± 0.13 bcd 61.92 ± 0.72 a 24.84 ± 0.34 a 3.17 ± 0.07 abc 4.09 ± 0.15 cd 0.18 ± 0.01 b

Monterey 1.41 ± 0.02 a 26.51 ± 0.57 b 12.76 ± 0.13 a 9.41 ± 0.24 a 3.79 ± 0.07 d 57.49 ± 0.83 b 25.55 ± 0.16 a 3.19 ± 0.02 abc 3.00 ± 0.02 e 0.14 ± 0.01 d

Sonora 1.43 ± 0.00 a 27.75 ± 0.39 a 12.83 ± 0.21 a 7.77 ± 0.19 d 4.30 ± 0.09 ab 62.23 ± 0.32 a 22.50 ± 1.08 bc 3.08 ± 0.03 c 3.40 ± 0.02 e 0.36 ± 0.02 a

LSD

County

Butte 1.22 ± 0.02 ab 22.51 ± 0.46 bc 12.37 ± 0.20 ab 8.38 ± 0.16 a 3.73 ± 0.10 cd 55.07 ± 1.00 c 21.19 ± 0.43 bcd 3.21 ± 0.04 abc 5.20 ± 0.61 ab 0.18 ± 0.01 b

Colusa 1.27 ± 0.08 a 23.46 ± 0.30 ab 12.71 ± 0.24 a 8.35 ± 0.14 ab 3.70 ± 0.06 cd 56.19 ± 0.70 bc 21.20 ± 0.23 bcd 3.19 ± 0.07 abc 4.81 ± 0.29 bc 0.12 ± 0.01 ef

Fresno 1.16 ± 0.03 abcd 23.95 ± 0.32 a 12.06 ± 0.11 bc 8.22 ± 0.09 ab 4.13 ± 0.14 bc 59.66 ± 1.25 ab 23.14 ± 0.64 a 3.01 ± 0.09 cd 3.89 ± 0.29 de 0.15 ± 0.01 bcde

Glenn 1.11 ± 0.04 bcd 21.10 ± 0.46 d 11.71 ± 0.19 cd 8.21 ± 0.11 ab 4.20 ± 0.13 b 55.21 ± 0.56 c 19.75 ± 0.15 d 3.19 ± 0.07 abc 4.60 ± 0.09 bcd 0.17 ± 0.02 bc

Kern 1.17 ± 0.02 abcd 23.15 ± 0.38 b 12.51 ± 0.23 ab 7.77 ± 0.17 d 3.50 ± 0.16 d 55.17 ± 0.31 c 21.46 ± 0.24 bc 3.33 ± 0.05 a 4.82 ± 0.13 abc 0.13 ± 0.02 def

Madera 1.20 ± 0.07 abc 23.28 ± 0.42 b 12.49 ± 0.26 ab 8.12 ± 0.10 abc 4.03 ± 0.25 bc 56.26 ± 1.15 bc 19.93 ± 0.58 d 3.16 ± 0.04 abc 5.69 ± 0.30 a 0.15 ± 0.02 bcde

Merced 1.07 ± 0.04 d 21.90 ± 0.30 cd 11.50 ± 0.11 d 8.29 ± 0.15 ab 2.97 ± 0.04 e 54.08 ± 0.54 c 21.02 ± 0.25 bcd 3.31 ± 0.12 ab 4.70 ± 0.44 bcd 0.10 ± 0.01 f

Sacramento 1.10 ± 0.03 cd 23.73 ± 0.23 a 11.56 ± 0.09 d 8.02 ± 0.09 bcd 4.73 ± 0.11 a 56.74 ± 0.39 bc 21.74 ± 0.45 abc 3.30 ± 0.02 ab 4.49 ± 0.06 bcd 0.15 ± 0.01 bcde

San Joaquin 1.16 ± 0.02 abcd 23.67 ± 0.40 a 12.44 ± 0.18 ab 8.05 ± 0.15 abcd 3.86 ± 0.24 bcd 54.19 ± 1.77 c 17.70 ± 0.58 e 2.90 ± 0.11 d 4.67 ± 0.35 bcd 0.16 ± 0.02 bcd

Stanislaus 1.17 ± 0.07 abcd 23.73 ± 0.40 a 12.07 ± 0.24 bc 7.85 ± 0.11 cd 3.97 ± 0.17 bc 60.63 ± 1.82 a 20.40 ± 0.32 cd 3.10 ± 0.08 bcd 4.19 ± 0.35 cde 0.22 ± 0.02 a

Tulare 1.06 ± 0.03 d 22.57 ± 0.35 bc 12.26 ± 0.20 ab 7.53 ± 0.20 cd 3.82 ± 0.06 bcd 56.63 ± 0.67 bc 21.57 ± 0.41 bc 3.19 ± 0.09 abc 4.24 ± 0.19 cde 0.14 ± 0.02 cde

Yolo 1.20 ± 0.02 abc 24.30 ± 0.25 a 12.31 ± 0.10 ab 8.05 ± 0.12 abcd 3.87 ± 0.09 bcd 58.96 ± 1.96 ab 22.06 ± 0.22 ab 3.14 ± 0.07 abc 3.41 ± 0.39 e 0.24 ± 0.01 a

LSD

Region

North Valley 1.16 ± 0.02 a 23.29 ± 0.16 a 11.99 ± 0.07 a 8.14 ± 0.05 a 4.22 ± 0.08 a 56.68 ± 0.48 a 21.39 ± 0.22 a 3.23 ± 0.02 a 4.43 ± 0.14 a 0.17 ± 0.01 a

South Valley 1.15 ± 0.02 a 23.37 ± 0.15 a 12.17 ± 0.08 a 8.00 ± 0.05 a 3.84 ± 0.08 b 57.38 ± 0.64 a 20.92 ± 0.30 a 3.09 ± 0.04 b 4.46 ± 0.14 a 0.16 ± 0.01 a

LSD

Year

2003-2004 1.11 ± 0.05 a 21.28 ± 0.28 b 12.23 ± 0.13 a 8.89 ± 0.13 a 4.21 ± 0.25 a 55.71 ± 0.63 b 21.26 ± 0.44 b 3.18 ± 0.04 a 4.61 ± 0.13 a 0.12 ± 0.01 b

2005-2006 1.19 ± 0.02 a 23.22 ± 0.29 a 12.47 ± 0.08 a 8.52 ± 0.10 b 4.43 ± 0.12 a 61.73 ± 0.89 a 22.89 ± 0.37 a 3.08 ± 0.05 a 3.44 ± 0.13 b 0.20 ± 0.01 a

LSD 0.10

0.07

0.12

0.05

0.030.460.120.912.11

0.44 0.21 0.15

0.340.210.250.54

1.00 0.48 0.35 0.44 3.74 1.45 0.23 0.88 0.04

0.24 1.65 0.77 0.10 0.39 0.02

0.030.370.151.152.390.490.310.290.81

Weight

(g)

Length

(mm)

Width

(mm)

Thickness

(mm)

Moisture

(g/100 g)

Lipid

(g/100 g)

Tannins

(g/100 g)

Protein

(g/100 g)

Ash

(g/100 g)

Sugars

(g/100 g)

57

2

Table 10. Interaction of cultivar and geographic location on the physical characteristics and chemical composition of select almond seedsa


Cultivar County

Butte 1.11 ± 0.01 a 19.25 ± 0.75 cd 12.04 ± 0.18 bc 9.04 ± 0.16 abc 3.67 ± 0.39 bc 53.99 ± 0.92 d 21.40 ± 0.27 bc 3.06 ± 0.02 bcde 4.82 ± 0.10 b 0.08 ± 0.00 e

Fresno 1.11 ± 0.02 a 20.72 ± 0.20 b 12.50 ± 0.07 a 9.17 ± 0.09 ab 4.37 ± 0.26 ab 58.58 ± 1.63 bc 21.18 ± 0.63 cd 3.02 ± 0.07 de 4.05 ± 0.25 c 0.14 ± 0.03 cd

Kern 0.88 ± 0.05 c 19.26 ± 0.37 cd 11.38 ± 0.18 d 8.59 ± 0.15 c 3.98 ± 0.23 bc 55.37 ± 0.27 bcd 22.15 ± 0.27 abc 3.16 ± 0.05 abcd 4.25 ± 0.16 bc 0.15 ± 0.01 bcd

Madera 1.00 ± 0.02 b 22.90 ± 0.76 a 12.03 ± 0.17 bc 8.08 ± 0.18 d 4.23 ± 0.05 b 65.23 ± 0.14 a 23.49 ± 0.24 a 2.89 ± 0.03 e 4.14 ± 0.21 bc 0.19 ± 0.01 abc

Merced 0.91 ± 0.01 c 18.98 ± 0.29 cd 11.10 ± 0.10 d 9.19 ± 0.10 ab 3.37 ± 0.08 c 54.60 ± 0.96 cd 19.15 ± 0.20 e 3.23 ± 0.01 abc 5.86 ± 0.32 a 0.07 ± 0.01 e

Sacramento 1.17 ± 0.01 a 20.41 ± 0.38 b 12.40 ± 0.11 ab 9.10 ± 0.20 ab 5.14 ± 0.12 a 57.77 ± 0.40 bcd 23.66 ± 0.39 a 3.18 ± 0.01 abcd 4.47 ± 0.13 bc 0.20 ± 0.01 ab

San Joaquin 1.02 ± 0.03 b 19.15 ± 0.65 cd 11.80 ± 0.16 c 9.37 ± 0.27 a 3.69 ± 0.05 bc 53.84 ± 1.21 d 19.35 ± 0.24 e 3.25 ± 0.03 ab 5.87 ± 0.17 a 0.10 ± 0.02 de

Stanislaus 1.14 ± 0.01 a 20.04 ± 0.28 bc 12.38 ± 0.13 ab 9.38 ± 0.26 a 5.12 ± 0.06 a 54.43 ± 0.56 d 19.65 ± 0.29 de 3.06 ± 0.00 bcde 5.02 ± 0.24 b 0.12 ± 0.02 de

Tulare 0.84 ± 0.02 c 18.27 ± 0.43 d 11.32 ± 0.29 d 9.47 ± 0.19 a 3.95 ± 0.03 bc 56.47 ± 0.51 bcd 22.85 ± 0.50 ab 3.29 ± 0.02 a 3.90 ± 0.04 c 0.11 ± 0.01 de

Yolo 1.07 ± 0.01 ab 20.50 ± 0.24 b 12.17 ± 0.08 abc 8.83 ± 0.15 bc 3.85 ± 0.31 bc 59.10 ± 1.39 b 22.58 ± 0.57 abc 3.03 ± 0.05 de 4.13 ± 0.28 bc 0.22 ± 0.01 a

Butte 1.21 ± 0.03 a 22.12 ± 0.65 d 12.11 ± 0.25 ab 8.82 ± 0.14 a 3.55 ± 0.12 cde 53.00 ± 0.13 d 22.00 ± 0.46 bc 3.28 ± 0.00 ab 3.87 ± 0.16 de 0.16 ± 0.01 b

Colusa 1.10 ± 0.02 cd 22.82 ± 0.37 d 11.95 ± 0.19 abc 8.13 ± 0.18 b 3.73 ± 0.02 bcde 57.59 ± 0.14 bc 21.15 ± 0.38 cd 3.31 ± 0.02 ab 4.20 ± 0.18 cde 0.12 ± 0.01 cd

Fresno 1.20 ± 0.02 ab 24.74 ± 0.32 a 12.18 ± 0.14 ab 8.31 ± 0.12 ab 4.26 ± 0.23 ab 60.36 ± 1.26 b 23.63 ± 0.73 a 3.17 ± 0.09 b 3.65 ± 0.31 ef 0.12 ± 0.01 cd

Glenn 1.18 ± 0.02 ab 22.28 ± 0.65 d 12.07 ± 0.16 abc 8.37 ± 0.13 ab 3.94 ± 0.09 bcd 56.04 ± 0.83 cd 19.82 ± 0.30 def 3.31 ± 0.02 ab 4.62 ± 0.04 bc 0.12 ± 0.00 cd

Kern 1.21 ± 0.02 a 22.95 ± 0.26 d 12.16 ± 0.32 ab 7.92 ± 0.31 bc 3.82 ± 0.15 bcd 55.38 ± 0.66 cd 21.07 ± 0.28 cd 3.25 ± 0.01 ab 4.72 ± 0.21 bc 0.10 ± 0.03 de

Madera 1.06 ± 0.02 d 23.18 ± 0.71 cd 11.54 ± 0.16 cde 7.87 ± 0.12 bc 3.46 ± 0.04 de 53.75 ± 0.51 cd 18.67 ± 0.15 f 3.22 ± 0.00 b 5.06 ± 0.17 b 0.13 ± 0.02 c

Merced 0.98 ± 0.02 e 21.99 ± 0.35 d 11.13 ± 0.09 e 8.04 ± 0.17 bc 3.06 ± 0.02 e 53.98 ± 0.04 cd 20.74 ± 0.19 cde 3.52 ± 0.02 a 3.76 ± 0.07 de 0.07 ± 0.01 e

Sacramento 1.15 ± 0.02 bc 24.45 ± 0.28 ab 11.28 ± 0.12 de 8.26 ± 0.13 b 4.89 ± 0.17 a 57.35 ± 0.47 bc 23.08 ± 0.27 ab 3.37 ± 0.02 ab 4.43 ± 0.09 bcd 0.13 ± 0.01 c

San Joaquin 1.19 ± 0.02 ab 24.22 ± 0.51 ac 12.37 ± 0.23 a 8.13 ± 0.22 b 4.03 ± 0.34 bcd 55.83 ± 2.42 cd 18.72 ± 0.40 f 2.82 ± 0.15 c 4.09 ± 0.28 cde 0.13 ± 0.02 c

Stanislaus 0.78 ± 0.02 f 20.50 ± 0.44 e 9.84 ± 0.12 f 7.56 ± 0.13 c 3.55 ± 0.12 cde 56.71 ± 1.25 bcd 19.28 ± 0.26 ef 2.62 ± 0.01 c 5.92 ± 0.17 a 0.21 ± 0.02 a

Tulare 1.12 ± 0.02 c 23.20 ± 0.53 bcd 12.04 ± 0.21 abc 8.12 ± 0.26 b 3.87 ± 0.07 bcd 55.86 ± 1.24 cd 20.76 ± 0.35 cde 3.35 ± 0.02 ab 4.43 ± 0.34 bcd 0.10 ± 0.01 de

Yolo 1.22 ± 0.01 a 24.76 ± 0.32 a 11.78 ± 0.08 bcd 8.35 ± 0.22 ab 4.16 ± 0.02 bc 66.69 ± 0.79 a 21.83 ± 0.31 bc 3.25 ± 0.14 ab 2.96 ± 0.05 f 0.22 ± 0.01 a

Butte 1.03 ± 0.03 cd 19.86 ± 0.51 bc 12.83 ± 0.34 ab 8.82 ± 0.25 e 3.67 ± 0.05 e 57.52 ± 0.10 c 22.43 ± 0.09 c 3.33 ± 0.01 a 4.30 ± 0.14 bc 0.15 ± 0.00 abc

Colusa 1.12 ± 0.01 bc 20.45 ± 0.29 b 12.64 ± 0.20 b 9.39 ± 0.23 bcde 3.87 ± 0.08 de 56.84 ± 0.81 c 24.37 ± 0.27 a 3.06 ± 0.01 d 3.34 ± 0.12 d 0.16 ± 0.01 ab

Fresno 1.33 ± 0.01 a 21.55 ± 0.29 a 13.31 ± 0.15 a 9.54 ± 0.19 bcd 4.61 ± 0.08 bcd 61.28 ± 0.77 a 24.07 ± 0.32 ab 3.37 ± 0.09 a 3.42 ± 0.02 d 0.13 ± 0.01 bc

Glenn 0.91 ± 0.01 e 19.85 ± 0.37 bc 12.41 ± 0.17 bc 8.93 ± 0.19 de 5.27 ± 0.08 ab 57.94 ± 0.16 bc 20.80 ± 0.39 d 3.28 ± 0.01 abc 4.83 ± 0.07 ab 0.21 ± 0.02 a

Kern 1.00 ± 0.03 de 18.43 ± 0.40 d 12.02 ± 0.23 c 9.25 ± 0.25 cde 4.03 ± 0.12 cde 60.36 ± 0.46 a 23.81 ± 0.18 ab 3.17 ± 0.02 cd 3.46 ± 0.15 d 0.09 ± 0.00 c

Merced 1.14 ± 0.02 b 19.04 ± 0.48 cd 12.89 ± 0.06 ab 9.98 ± 0.13 ab 4.64 ± 0.07 bc 57.83 ± 0.47 bc 22.69 ± 0.24 c 3.32 ± 0.00 ab 3.92 ± 0.13 cd 0.14 ± 0.01 bc

San Joaquin 1.14 ± 0.05 b 19.13 ± 0.58 cd 12.43 ± 0.24 bc 9.59 ± 0.39 abc 4.02 ± 0.07 cde 53.44 ± 0.85 d 23.27 ± 0.33 bc 3.19 ± 0.03 bcd 5.28 ± 0.19 a 0.12 ± 0.01 bc

Stanislaus 1.30 ± 0.04 a 20.89 ± 0.24 ab 13.32 ± 0.12 a 10.20 ± 0.15 a 5.56 ± 0.37 a 59.65 ± 0.76 ab 23.85 ± 0.29 ab 3.27 ± 0.04 abc 3.86 ± 0.27 cd 0.16 ± 0.03 ab

0.87 0.14 0.61 0.07

0.20 0.75 0.06

0.030.740.30

(g/100 g)(g/100 g)(mm)(mm)(mm)

Protein Ash Sugars

(g/100 g)(g/100 g)(g/100 g)

Length Width Thickness Moisture Lipid Tannins

(g/100 g)

LSD

LSD 0.08 1.11 0.40 0.46 0.80 4.08 1.65

1.614.030.690.510.531.260.06

0.10 1.08 0.54 0.62 0.76 2.06

(g)

Mission

LSD

Carmel

Butte

Weight

58

3


Table 10 continued. Interaction of cultivar and geographic location on the physical characteristics and chemical composition of select almond seedsa

Cultivar County

Butte 1.23 ± 0.03 c 22.91 ± 0.67 abc 12.62 ± 0.31 b 7.95 ± 0.20 cd 3.92 ± 0.02 cd 57.15 ± 0.84 abc 20.37 ± 0.25 bc 3.13 ± 0.01 bc 6.52 ± 0.27 a 0.20 ± 0.01 bc

Colusa 1.44 ± 0.04 a 24.10 ± 0.38 a 13.47 ± 0.26 a 8.57 ± 0.20 a 3.66 ± 0.13 d 54.79 ± 0.69 bc 21.26 ± 0.36 abc 3.08 ± 0.00 bc 5.42 ± 0.11 bcd 0.11 ± 0.01 f

Fresno 1.10 ± 0.06 defg 22.77 ± 0.55 abc 11.87 ± 0.17 c 8.07 ± 0.13 bcd 3.94 ± 0.07 cd 58.60 ± 2.60 ab 22.41 ± 1.19 a 2.76 ± 0.11 d 4.26 ± 0.57 ef 0.20 ± 0.01 bc

Glenn 1.03 ± 0.01 fg 19.93 ± 0.41 d 11.35 ± 0.31 c 8.06 ± 0.17 bcd 4.46 ± 0.09 ab 54.38 ± 0.46 bc 19.67 ± 0.15 c 3.08 ± 0.03 bc 4.58 ± 0.20 def 0.23 ± 0.01 ab

Kern 1.13 ± 0.02 def 23.35 ± 0.72 ab 12.86 ± 0.31 ab 7.63 ± 0.15 d 3.17 ± 0.08 ef 54.97 ± 0.03 bc 21.84 ± 0.23 ab 3.40 ± 0.00 a 4.91 ± 0.19 cde 0.16 ± 0.01 de

Madera 1.35 ± 0.02 ab 23.38 ± 0.50 ab 13.45 ± 0.25 a 8.37 ± 0.12 abc 4.59 ± 0.04 a 58.76 ± 0.26 ab 21.20 ± 0.22 abc 3.09 ± 0.00 bc 6.32 ± 0.13 ab 0.17 ± 0.02 de

Merced 1.15 ± 0.00 de 21.81 ± 0.50 c 11.86 ± 0.10 c 8.53 ± 0.23 ab 2.89 ± 0.04 f 54.18 ± 1.20 bc 21.30 ± 0.46 abc 3.10 ± 0.04 bc 5.65 ± 0.24 abc 0.13 ± 0.01 ef

Sacramento 1.05 ± 0.04 efg 23.00 ± 0.32 abc 11.84 ± 0.12 c 7.78 ± 0.12 d 4.57 ± 0.11 a 56.13 ± 0.57 abc 20.41 ± 0.59 bc 3.23 ± 0.02 ab 4.56 ± 0.08 def 0.16 ± 0.01 de

San Joaquin 1.12 ± 0.00 def 22.56 ± 0.50 bc 12.59 ± 0.27 b 7.88 ± 0.16 d 3.54 ± 0.03 de 50.90 ± 0.61 c 15.65 ± 0.33 d 3.08 ± 0.01 bc 5.84 ± 0.22 abc 0.24 ± 0.02 ab

Stanislaus 1.30 ± 0.02 bc 24.07 ± 0.33 a 12.82 ± 0.15 b 7.95 ± 0.13 cd 4.11 ± 0.20 bc 61.94 ± 2.26 a 20.78 ± 0.33 abc 3.22 ± 0.04 ab 3.61 ± 0.24 f 0.23 ± 0.01 ab

Tulare 1.00 ± 0.02 g 21.93 ± 0.38 c 12.49 ± 0.34 b 6.93 ± 0.16 e 3.77 ± 0.09 cd 57.40 ± 0.29 ab 22.38 ± 0.24 a 3.04 ± 0.02 c 4.06 ± 0.16 ef 0.18 ± 0.00 cd

Yolo 1.19 ± 0.02 cd 24.07 ± 0.33 a 12.58 ± 0.10 b 7.90 ± 0.12 cd 3.72 ± 0.08 cd 61.67 ± 3.25 a 22.18 ± 0.31 ab 3.09 ± 0.08 bc 3.63 ± 0.57 f 0.25 ± 0.02 a

Butte 1.13 ± 0.01 c 19.63 ± 0.63 bc 11.97 ± 0.23 c 9.09 ± 0.20 b 3.76 ± 0.05 c 54.64 ± 0.68 cd 20.84 ± 0.04 a 3.13 ± 0.01 cd 6.15 ± 0.24 a 0.16 ± 0.02 b

Fresno 1.32 ± 0.00 a 21.40 ± 0.30 a 13.80 ± 0.13 a 9.23 ± 0.20 b 3.66 ± 0.06 c 57.27 ± 0.44 a 20.98 ± 0.21 a 3.09 ± 0.00 d 5.56 ± 0.29 ab 0.24 ± 0.02 a

Kern 1.03 ± 0.03 d 19.54 ± 0.41 bcd 12.04 ± 0.15 c 9.71 ± 0.13 a 3.68 ± 0.06 c 52.29 ± 0.38 e 19.82 ± 0.12 b 3.23 ± 0.05 b 5.00 ± 0.23 c 0.14 ± 0.01 b

Merced 0.91 ± 0.02 e 18.45 ± 0.34 d 11.40 ± 0.12 d 9.21 ± 0.09 b 3.58 ± 0.10 c 56.79 ± 0.43 ab 20.36 ± 0.23 ab 3.12 ± 0.01 cd 4.34 ± 0.05 d 0.21 ± 0.00 a

San Joaquin 1.05 ± 0.01 d 19.19 ± 0.44 bcd 11.99 ± 0.17 c 9.72 ± 0.11 a 4.72 ± 0.10 a 55.56 ± 0.68 bc 20.82 ± 0.33 a 3.34 ± 0.02 a 4.09 ± 0.08 d 0.17 ± 0.00 b

Stanislaus 1.22 ± 0.01 b 20.21 ± 0.25 b 13.23 ± 0.15 b 10.06 ± 0.22 a 4.22 ± 0.15 b 53.23 ± 0.60 de 19.72 ± 0.03 b 3.17 ± 0.01 c 5.77 ± 0.08 a 0.15 ± 0.01 b

Tulare 1.07 ± 0.01 d 19.01 ± 0.28 cd 12.23 ± 0.24 c 9.73 ± 0.13 a 3.57 ± 0.07 c 51.91 ± 0.12 e 20.05 ± 0.38 b 3.17 ± 0.00 c 5.09 ± 0.11 bc 0.14 ± 0.01 b

Butte 0.92 ± 0.02 c 21.69 ± 0.59 ab 11.34 ± 0.28 bcd 7.83 ± 0.23 bc 3.83 ± 0.10 bc 59.23 ± 0.98 bc 22.70 ± 0.51 e 2.85 ± 0.05 d 4.92 ± 0.14 a 0.25 ± 0.01 a

Fresno 1.07 ± 0.01 a 21.30 ± 0.56 b 11.74 ± 0.25 abc 8.74 ± 0.10 a 3.40 ± 0.13 d 56.77 ± 0.51 c 24.24 ± 0.04 cd 3.45 ± 0.02 a 4.86 ± 0.20 a 0.13 ± 0.00 de

Kern 0.83 ± 0.01 d 21.16 ± 0.31 b 11.14 ± 0.22 cd 7.50 ± 0.25 c 4.05 ± 0.16 b 60.97 ± 1.31 b 25.65 ± 0.32 b 3.54 ± 0.04 a 3.99 ± 0.04 b 0.11 ± 0.02 e

Merced 1.10 ± 0.02 a 22.67 ± 0.67 a 12.20 ± 0.19 a 8.03 ± 0.13 bc 5.04 ± 0.05 a 64.80 ± 0.83 a 26.74 ± 0.16 a 3.27 ± 0.00 b 3.50 ± 0.22 cd 0.20 ± 0.01 c

San Joaquin 1.12 ± 0.01 a 21.69 ± 0.36 ab 11.76 ± 0.22 ab 8.33 ± 0.18 ab 3.61 ± 0.06 cd 61.79 ± 0.29 b 24.68 ± 0.08 c 2.91 ± 0.00 cd 4.04 ± 0.09 bc 0.23 ± 0.01 ab

Stanislaus 0.90 ± 0.01 c 20.89 ± 0.33 b 10.78 ± 0.14 d 7.29 ± 0.16 c 4.88 ± 0.08 a 65.38 ± 1.23 a 23.35 ± 0.31 de 3.20 ± 0.04 b 4.25 ± 0.30 b 0.22 ± 0.01 bc

Tulare 0.99 ± 0.03 b 20.86 ± 0.31 b 11.27 ± 0.22 bcd 7.45 ± 0.25 c 4.06 ± 0.12 b 64.53 ± 0.50 a 26.54 ± 0.45 ab 2.97 ± 0.01 c 3.07 ± 0.18 d 0.14 ± 0.01 d

0.55 0.030.06 1.30 0.61 0.54 0.31

0.270.450.491.100.05

Lipid Protein Ash Sugars TanninsWeight Length Width Thickness Moisture

(g) (mm) (mm) (mm) (g/100 g) (g/100 g) (g/100 g) (g/100 g) (g/100 g) (g/100 g)

LSD

LSD

Price

Padre

0.12 1.41

0.040.530.060.671.50

2.60 0.93 0.10

Nonpareil

LSD

0.61 0.48 0.43 6.28 1.87 0.19 1.05 0.05

59

4 Table 11. Physical characteristics and chemical composition of select macadamia nut seedsa


3.16 ± 0.05 a 20.04 ± 0.35 a 15.19 ± 0.31 ab 1.67 ± 0.01 f 60.01 ± 0.39 b 6.41 ± 0.23 ab 1.12 ± 0.01 cd 7.28 ± 0.12 c 0.04 ± 0.00 a

3.36 ± 0.22 a 20.10 ± 0.41 a 15.49 ± 0.41 a 2.64 ± 0.01 a 61.65 ± 0.63 b 7.07 ± 0.24 a 1.12 ± 0.03 cd 6.28 ± 0.01 f 0.04 ± 0.00 a

2.71 ± 0.05 bc 19.39 ± 0.38 ab 15.46 ± 0.32 a 2.33 ± 0.06 b 60.99 ± 0.45 b 7.08 ± 0.52 a 1.16 ± 0.02 c 6.61 ± 0.13 e 0.04 ± 0.00 a

2.77 ± 0.07 b 19.51 ± 0.49 ab 15.59 ± 0.38 a 1.79 ± 0.02 e 63.04 ± 0.39 ab 6.29 ± 0.23 ab 1.01 ± 0.01 e 6.84 ± 0.03 d 0.04 ± 0.00 a

2.41 ± 0.06 c 18.68 ± 0.41 b 14.46 ± 0.49 b 1.91 ± 0.02 d 61.81 ± 1.47 b 6.27 ± 0.48 ab 1.28 ± 0.00 b 8.34 ± 0.05 b 0.04 ± 0.01 a

2.41 ± 0.10 c 18.73 ± 0.17 b 14.44 ± 0.16 b 1.83 ± 0.02 e 60.32 ± 1.26 b 5.56 ± 0.65 b 1.40 ± 0.02 a 8.68 ± 0.02 a 0.04 ± 0.00 a

2.64 ± 0.12 bc 19.60 ± 0.45 ab 15.87 ± 0.32 a 2.04 ± 0.02 c 66.16 ± 2.91 a 6.76 ± 0.25 ab 1.11 ± 0.01 d 5.25 ± 0.01 g 0.03 ± 0.00 b

Moisture

(g/100 g)

Lipid

(g/100 g)

Protein

(g/100 g)

4.14

Ash

(g/100 g)

# 246 Keauhou# 294 Purvis # 508 Kakea# 660 Keaau# 741 Mauka

LSD

Macadamia nut

Blue DiamondTrader Joe's

Weight

(g)

Diameter

(mm)

Thickness

(mm)

0.081.001.110.34

Sugars

(g/100 g)

Tannins

(g/100 g)

0.010.210.051.22

60

Amino Acid Composition

Total amino acids. The total amino acid composition of tested almond and macadamia

nut seeds is summarized in Tables 12-16. Almond and macadamia nut seed proteins are

composed mainly of non-essential amino acids, with aspartic acid, glutamic acid, and arginine

accounting for 36.0-50.3% and 44.6-51.1% of total amino acids, respectively. Comparable

ranges of 44.5-57.7% and 44.9-49.5% are reported for almond (60, 71-74, 85) and macadamia

nut seed proteins (71, 85). Among almond seed samples, glutamic acid and aspartic acid were

positively correlated (r = 0.64, p � 0.05, Figure 6A), while glutamic acid and arginine were

negatively correlated (r = -0.88, p � 0.05, Figure 6B) (Table 17). Protein quality, assessed by

essential-to-total amino acid ratio (E/T), ranged from 25.4-33.8% in almond seeds and 22.5-

26.1% in macadamia nut seeds (Tables 12-16). Similar ranges of 22.7-32.8% and 27.2-29.5% are

reported for almond (60, 71-74, 85) and macadamia nut seed proteins (71, 85). Essential amino

acids varied by cultivar, with the highest E/T ratio in almond cultivar Price (30.1%) and

macadamia nut cultivar Purvis (26.1%) and lowest E/T ratio in almond cultivar Monterey

(24.9%) and Trader Joe’s macadamia nut variety (22.5%) (Tables 12 and 16). Based on the

FAO/WHO recommended essential amino acid amount for pre-school aged children (2-5 years),

lysine was the first limiting essential amino acid (LEAA) in almond and macadamia nut seeds

(Tables 12, 14, and 16). Compared to the FAO/WHO recommended essential amino acid amount

for adults (18+ years), lysine or the sulfur amino acids (methionine and cysteine) were the first

limiting essential amino acid in almond and macadamia nut seeds (Tables 12, 14, and 16).

Similarly, lysine was the first limiting essential amino acid for pre-school aged children (2-5

years) in Spanish (60), Italian (73), and American (85) grown almond and macadamia nut seeds

(85). Other studies report sulfur amino acids (methionine and cysteine) as the first limiting

essential amino acid (for pre-school children and adults) in California grown almond seeds (71,

72) and tryptophan as the first limiting essential amino acid (for pre-school children) in U.S.

purchased macadamia nut seeds (71). In the current investigation, lysine varied by cultivar with

the lowest amount in almond cultivars Monterey and Sonora (0.68-0.70 g/100 g protein) and

macadamia nut cultivar Purvis (0.47 g/100 g protein) and the highest amount in almond cultivar

Padre (1.32 g/100 g protein) and macadamia nut cultivar Mauka (1.12 g/100 g protein) (Tables

12 and 16). Sulfur amino acid also varied by cultivar in tested almond and macadamia nut seed

samples. The essential amino acid content of almond seed samples varied by geographic

61

location, with a significantly higher E/T ratio in seeds from the South Valley compared to seeds

from the North Valley (Table 12). Low E/T ratios were observed in almond cultivars Butte,

Carmel, and Mission from Fresno County, cultivars Butte, Carmel, and Nonpareil from Yolo

County, and cultivars Padre and Price from Merced County.

Almond and macadamia nut seed proteins are also rich in arginine containing 7.3-11.9%

and 9.4-11.5%, respectively (Tables 12-16). An arginine-rich diet may reduce the risk of

cardiovascular disease as arginine can be converted to nitric oxide, a potent vasodilator and

antioxidant (234). Additionally almond and macadamia nut seed samples were low in lysine,

which foods high in lysine are believed to decrease the bioavailability of arginine, and had

lysine-to-arginine ratios of 0.08-0.18 and 0.05-0.10, respectively. Lysine and arginine were

positively correlated in both almond (r = 0.67, p � 0.05, Figure 6C) and macadamia nut (r = 0.84,

p � 0.05, Figure 6D) seed proteins (Tables 17 and 18). Similarly, a positive correlation was

reported between lysine and arginine in cowpea proteins (228). Arginine significantly varied by

cultivar, geographic location, and harvest year. In particular, arginine was significantly higher in

almond cultivars Price and Padre and macadamia nut cultivar Mauka and lowest in almond

cultivars Monterey and Sonora. Cultivar variation is also reported to significantly impact

individual amino acids from peanut (235). Almond seeds from the 2003/2004 season contained

~21.0% more arginine than seeds from the 2005/2006 season.

Free amino acids. Free amino acid composition of select almond seeds are summarized

in Tables 19-22. Total free amino acids in tested almond seeds ranged from 172.3 to 723.2 mg

per 100 g dry weight, comparable with the large variation (98.1-1,507.0 mg per 100 g)

previously reported (39, 109). Soler et al. (60) found the highest free amino acid content in

almond seeds occur 85 days after fruit set and as seeds mature and develop the amount

decreases. Thus, the large variation in total free amino acids observed in the tested almond seeds

may be dependent on time of harvest and seed maturity. Asparagine, aspartic acid, glutamic acid,

and arginine dominate (49.0-79.1%) the total free amino acid composition of tested almond

seeds, similar to the reported range of 57.0-81.9% (86, 109). The current investigation, like

previous studies (39, 86, 109), found a large variation in free arginine which accounted for ~6-

24% of total free amino acids. Alanine, proline, tyrosine, and isoleucine account for less than

10% each and the remaining free amino acids (Ser, Gln, Gly, His, Thr, Val, Met, Cys, Leu, Phe,

and Lys) amount for less than 5% each. Total free amino acids in almond cultivars ranged from

62

237.5-604.9 mg for Butte, 296.8-723.2 mg for Carmel, 353.9-483.0 mg for Mission, 476.5 mg

for Monterey, 219.5-491.6 mg for Nonpareil, 278.8-479.3 mg for Padre, 172.3-573.2 mg for

Price, and 260.6 mg/100 g dry weight for Sonora. Martín Carratalá et al. (39) developed a

decision tree to identify almond cultivars based on their free amino acid composition, however

cultivars Mission and Nonpareil in the current study were incorrectly identified.

The free asparagine content of raw almond seeds is of importance as free asparagine is

reported to be a precursor for acrylamide formation (37, 42). Free asparagine in tested almond

seeds ranged from 38.7-325.0 mg/100 g dry weight and accounted for 14.9-44.9% of total amino

acids. A positive correlation was observed between free asparagine and majority of free amino

acids (Asp, Glu, Gln, Gly, His, Arg, Thr, Ala, Pro, Val, and Lys) (Table 22). The free asparagine

content of tested almond cultivars differ significantly, with the highest amount in cultivar Carmel

(163.9 mg/100 g) and lowest amount in cultivar Sonora (38.7 mg/100g) (Table 20). Free

asparagine content of tested almond seeds varied by geographic location, with 18.3% more free

asparagine in almond seeds from the North Valley compared to seeds from the South Valley

(Table 20). Interaction of cultivar x county also influenced the free asparagine content of tested

almond seeds, with the exception of cultivar Mission. Most notably cultivars Butte from Tulare

County, Carmel from Madera County and cultivar Padre from Kern County were significantly

higher in free asparagine, whereas San Joaquin County produced less free asparagine in cultivars

Butte, Carmel, and Nonpareil (Table 22). Influence of harvest year on free asparagine content

was also significant, with 45.6% more free asparagine in almond seeds from the 2003/2004

season (Table 20).

63

1

Table 12. Cultivar, geographic location, and harvest year variation on the essential amino acid composition of select almond seedsa

aAmino acid values are expressed as gram per 100 g protein and data are expressed as mean ± SEM (n = 3). For each column, means with different letters are significantly different (p � 0.05) according to Fisher’s LSD test. E/T (%) represents essential-to-total amino acid ratio and LEAA represents limiting essential amino acids based on the recommended pattern report of a joint FAO/WHO expert consultation (93) for bpre-school child (2-5 years) and cadult (18+ years).

LEAAb

LEAAc

Cultivar

Butte 4.02 ± 0.16 bc 2.39 ± 0.02 b 4.31 ± 0.06 bc 0.97 ± 0.04 b 2.92 ± 0.06 c 5.59 ± 0.07 c 6.23 ± 0.18 a 0.99 ± 0.04 c 1.19 ± 0.07 bc 28.62 bc Lys LysCarmel 3.35 ± 0.14 d 2.46 ± 0.04 ab 4.39 ± 0.05 b 1.13 ± 0.06 ab 2.99 ± 0.04 bc 5.93 ± 0.08 b 5.97 ± 0.14 ab 1.00 ± 0.03 c 1.05 ± 0.05 c 28.29 c Lys LysMission 3.36 ± 0.22 d 2.60 ± 0.06 a 4.80 ± 0.06 a 1.69 ± 0.13 a 3.20 ± 0.06 a 6.34 ± 0.08 a 5.18 ± 0.06 c 1.12 ± 0.04 bc 1.07 ± 0.05 c 29.35 ab Lys Lys

Monterey 2.44 ± 0.06 e 2.38 ± 0.03 b 3.95 ± 0.08 d 1.12 ± 0.05 ab 2.49 ± 0.03 e 5.17 ± 0.05 d 5.46 ± 0.09 c 0.70 ± 0.05 d 1.18 ± 0.02 bc 24.89 e Lys LysNonpareil 3.55 ± 0.14 cd 2.51 ± 0.05 ab 4.39 ± 0.06 b 1.13 ± 0.04 ab 2.94 ± 0.04 c 5.73 ± 0.07 bc 5.98 ± 0.14 ab 1.03 ± 0.04 bc 1.12 ± 0.04 bc 28.37 bc Lys Lys

Padre 4.49 ± 0.11 ab 2.37 ± 0.06 b 4.43 ± 0.03 b 1.12 ± 0.03 ab 3.14 ± 0.08 ab 5.85 ± 0.14 bc 5.35 ± 0.09 c 1.32 ± 0.09 a 1.30 ± 0.14 b 29.36 ab Lys LysPrice 4.72 ± 0.06 a 2.45 ± 0.04 b 4.44 ± 0.06 b 0.96 ± 0.02 b 3.21 ± 0.05 a 5.91 ± 0.09 b 5.60 ± 0.09 bc 1.17 ± 0.03 b 1.65 ± 0.07 a 30.10 a Lys Lys

Sonora 2.42 ± 0.02 e 2.45 ± 0.02 b 4.16 ± 0.07 c 1.15 ± 0.14 ab 2.72 ± 0.08 d 5.68 ± 0.18 bc 5.42 ± 0.15 c 0.68 ± 0.03 d 1.29 ± 0.07 b 25.96 d Lys LysLSD

County

Butte 3.66 ± 0.55 bc 2.26 ± 0.11 d 4.36 ± 0.02 de 0.99 ± 0.01 de 3.06 ± 0.02 bcde 5.89 ± 0.18 c 5.44 ± 0.09 def 1.00 ± 0.05 cd 0.78 ± 0.14 e 27.43 ef Lys LysColusa 3.81 ± 0.65 b 3.09 ± 0.13 a 4.90 ± 0.17 ab 1.86 ± 0.33 a 3.14 ± 0.10 bcd 6.09 ± 0.15 bc 5.02 ± 0.21 f 1.16 ± 0.06 bc 1.21 ± 0.20 abc 30.27 ab Lys LysFresno 2.96 ± 0.13 c 2.38 ± 0.05 c 4.28 ± 0.09 de 1.16 ± 0.08 cd 2.86 ± 0.08 ef 5.79 ± 0.17 c 5.95 ± 0.23 bc 0.96 ± 0.05 d 1.11 ± 0.06 bcd 27.46 def Lys LysGlenn 3.77 ± 0.06 bc 2.46 ± 0.08 cd 4.39 ± 0.01 cde 1.36 ± 0.02 bc 2.96 ± 0.03 de 5.82 ± 0.09 c 5.61 ± 0.10 cde 1.22 ± 0.08 b 0.78 ± 0.05 e 28.37 cde Lys LysKern 3.70 ± 0.51 bc 2.62 ± 0.17 bc 4.55 ± 0.11 cd 1.12 ± 0.04 cd 3.20 ± 0.07 abc 6.11 ± 0.05 bc 5.57 ± 0.13 cde 1.05 ± 0.04 cd 1.30 ± 0.24 ab 29.23 abc Lys Lys

Madera 4.07 ± 0.24 ab 2.44 ± 0.17 cd 4.53 ± 0.12 cd 1.32 ± 0.13 bc 3.08 ± 0.04 bcd 5.94 ± 0.01 c 5.06 ± 0.16 f 1.47 ± 0.07 a 0.99 ± 0.03 cde 28.91 bcde Lys LysMerced 4.90 ± 0.07 a 2.27 ± 0.06 d 4.66 ± 0.13 bc 0.99 ± 0.02 de 3.24 ± 0.05 ab 5.95 ± 0.07 c 5.87 ± 0.03 bcd 1.24 ± 0.08 b 1.47 ± 0.09 a 30.59 a Lys Met/Cys

Sacramento 2.97 ± 0.04 c 2.49 ± 0.04 bcd 4.17 ± 0.04 ef 0.72 ± 0.02 e 2.66 ± 0.06 fg 5.37 ± 0.14 d 7.77 ± 0.05 a 0.73 ± 0.04 e 0.91 ± 0.05 de 27.79 cde Lys LysSan Joaquin 3.33 ± 0.39 bc 2.73 ± 0.14 b 4.51 ± 0.17 cd 1.22 ± 0.06 bcd 3.17 ± 0.06 bc 6.39 ± 0.12 ab 5.61 ± 0.07 cde 1.06 ± 0.06 cd 1.07 ± 0.11 bcd 29.08 abcd Lys LysStanislaus 3.68 ± 0.36 bc 2.35 ± 0.05 d 4.20 ± 0.08 ef 1.12 ± 0.05 cd 3.00 ± 0.07 cde 5.82 ± 0.07 c 5.41 ± 0.08 def 1.09 ± 0.06 bcd 1.24 ± 0.04 abc 27.92 cde Lys Lys

Tulare 3.73 ± 0.50 bc 2.58 ± 0.17 bc 5.12 ± 0.11 a 1.44 ± 0.20 b 3.40 ± 0.12 a 6.59 ± 0.08 a 5.21 ± 0.12 ef 1.11 ± 0.05 bcd 1.41 ± 0.22 a 30.58 a Lys LysYolo 2.80 ± 0.08 c 2.37 ± 0.06 c 3.98 ± 0.03 f 1.01 ± 0.06 d 2.58 ± 0.03 g 5.21 ± 0.08 d 6.27 ± 0.37 b 0.75 ± 0.03 e 0.97 ± 0.05 cde 25.93 f Lys LysLSD

Region

North Valley 3.24 ± 0.12 b 2.51 ± 0.05 a 4.28 ± 0.05 b 1.05 ± 0.07 b 2.80 ± 0.04 b 5.56 ± 0.08 b 6.50 ± 0.18 a 0.89 ± 0.04 b 0.93 ± 0.04 b 27.78 b Lys LysSouth Valley 3.62 ± 0.14 a 2.47 ± 0.12 a 4.47 ± 0.05 a 1.19 ± 0.04 a 3.08 ± 0.04 a 6.03 ± 0.06 a 5.58 ± 0.07 b 1.10 ± 0.03 a 1.20 ± 0.04 a 28.74 a Lys Lys

LSD

Year

2003-2004 4.23 ± 0.23 a 2.43 ± 0.04 a 4.62 ± 0.07 a 1.24 ± 0.06 a 3.15 ± 0.07 a 6.08 ± 0.14 a 5.43 ± 0.11 b 1.18 ± 0.05 a 1.03 ± 0.06 a 29.39 a Lys Lys2005-2006 2.75 ± 0.05 b 2.35 ± 0.03 a 4.00 ± 0.03 b 1.01 ± 0.06 b 2.64 ± 0.03 b 5.35 ± 0.07 b 6.24 ± 0.23 a 0.80 ± 0.03 b 1.14 ± 0.05 a 26.29 b Lys Lys

LSD

0.15 0.19 0.61 0.17

0.37

0.29 0.58 0.11 0.160.39 0.10 0.14 0.18 0.14

0.190.110.140.150.12

His Thr Val Met Ile Leu Phe Lys Trp E/T

1.05

0.84 0.26 0.28 0.27 0.21 0.40 0.50 0.16 0.28 1.65

0.27 0.47 0.14 0.200.49

0.74

0.780.120.090.35

64

2

Table 13. Cultivar, geographic location, and harvest year variation on the non-essential amino acid composition of select almond seedsa

aAmino acid values are expressed as gram per 100 g protein and data are expressed as mean ± SEM (n = 3). For each column, means with different letters are significantly different (p � 0.05) according to Fisher’s LSD test.

Cultivar

Butte 8.06 ± 0.23 c 27.32 ± 0.43 bc 3.07 ± 0.04 abc 9.36 ± 0.24 b 7.59 ± 0.06 a 4.96 ± 0.07 b 7.87 ± 0.22 ab 2.86 ± 0.05 b 0.29 ± 0.01 b

Carmel 8.03 ± 0.21 c 28.24 ± 0.40 b 3.00 ± 0.04 c 8.94 ± 0.18 b 7.30 ± 0.08 bc 5.11 ± 0.07 ab 7.91 ± 0.16 ab 2.93 ± 0.09 b 0.25 ± 0.01 b

Mission 7.00 ± 0.33 d 26.94 ± 0.47 c 3.02 ± 0.06 c 9.57 ± 0.30 b 6.91 ± 0.09 d 5.16 ± 0.07 ab 8.06 ± 0.21 a 3.63 ± 0.21 a 0.36 ± 0.02 a

Monterey 11.49 ± 0.26 a 31.94 ± 0.12 a 3.02 ± 0.04 c 7.55 ± 0.07 c 6.30 ± 0.12 f 4.86 ± 0.05 c 7.01 ± 0.09 cd 2.75 ± 0.01 b 0.18 ± 0.01 c

Nonpareil 8.27 ± 0.27 c 28.49 ± 0.46 b 3.11 ± 0.04 abc 8.95 ± 0.19 b 7.20 ± 0.09 bc 5.01 ± 0.06 bc 7.63 ± 0.19 abc 2.69 ± 0.05 b 0.29 ± 0.01 b

Padre 7.96 ± 0.39 c 26.38 ± 0.38 c 3.19 ± 0.13 ab 10.47 ± 0.24 a 7.40 ± 0.11 ab 5.30 ± 0.15 a 6.79 ± 0.19 d 2.78 ± 0.04 b 0.36 ± 0.02 a

Price 7.72 ± 0.38 cd 26.12 ± 0.26 c 3.23 ± 0.06 a 10.50 ± 0.15 a 7.04 ± 0.09 cd 4.81 ± 0.06 c 7.40 ± 0.19 bcd 2.73 ± 0.04 b 0.37 ± 0.01 a

Sonora 9.75 ± 0.45 b 31.96 ± 0.18 a 3.05 ± 0.06 bc 7.31 ± 0.38 c 6.60 ± 0.10 e 5.11 ± 0.11 ab 7.37 ± 0.13 bcd 2.70 ± 0.05 b 0.19 ± 0.03 c

LSD

County

Butte 9.14 ± 0.26 ab 30.28 ± 1.18 ab 2.90 ± 0.13 c 8.86 ± 0.28 def 7.00 ± 0.05 bc 5.21 ± 0.08 bcd 6.31 ± 0.22 d 2.58 ± 0.08 de 0.30 ± 0.03 a

Colusa 5.28 ± 0.78 g 24.68 ± 1.71 e 3.43 ± 0.19 a 9.91 ± 0.74 abc 7.86 ± 0.66 a 4.61 ± 0.10 f 9.56 ± 0.47 a 4.08 ± 0.39 a 0.32 ± 0.01 a

Fresno 8.79 ± 0.24 abc 29.43 ± 0.69 abc 2.93 ± 0.07 c 8.31 ± 0.18 efg 6.99 ± 0.14 bc 5.24 ± 0.10 bcd 7.72 ± 0.19 b 2.89 ± 0.04 bcd 0.24 ± 0.01 bc

Glenn 8.96 ± 0.08 abc 27.23 ± 0.13 cde 3.11 ± 0.07 bc 9.35 ± 0.14 bcd 7.39 ± 0.17 abc 5.27 ± 0.19 bc 7.15 ± 0.25 bcd 2.85 ± 0.08 bcd 0.31 ± 0.01 a

Kern 6.79 ± 0.70 ef 28.36 ± 2.34 bcd 3.16 ± 0.14 abc 9.26 ± 0.71 cde 7.48 ± 0.33 ab 4.90 ± 0.06 def 7.79 ± 0.52 b 2.72 ± 0.18 de 0.31 ± 0.03 a

Madera 8.16 ± 0.70 bcd 26.21 ± 0.35 de 3.03 ± 0.12 bc 10.61 ± 0.26 a 7.30 ± 0.29 bc 5.49 ± 0.13 ab 7.28 ± 0.70 bc 2.70 ± 0.16 de 0.33 ± 0.04 a

Merced 7.39 ± 0.43 de 25.89 ± 0.25 de 2.95 ± 0.08 c 10.31 ± 0.18 ab 7.37 ± 0.06 abc 5.06 ± 0.07 cd 7.32 ± 0.16 bc 2.82 ± 0.02 cd 0.31 ± 0.01 a

Sacramento 8.08 ± 0.17 bcd 29.01 ± 0.28 abc 2.99 ± 0.05 c 8.16 ± 0.14 fg 7.53 ± 0.07 ab 4.69 ± 0.07 ef 9.12 ± 0.14 a 2.43 ± 0.05 e 0.21 ± 0.01 c

San Joaquin 7.75 ± 0.63 cde 27.77 ± 1.17 bcd 3.26 ± 0.10 ab 8.69 ± 0.48 defg 7.12 ± 0.14 bc 5.08 ± 0.04 cd 7.80 ± 0.21 b 3.17 ± 0.27 bc 0.29 ± 0.04 ab

Stanislaus 9.34 ± 0.52 ab 29.28 ± 0.91 abc 3.11 ± 0.07 bc 9.17 ± 0.41 cdef 6.85 ± 0.13 c 5.00 ± 0.08 cde 6.47 ± 0.27 cd 2.55 ± 0.05 de 0.30 ± 0.03 a

Tulare 5.71 ± 0.23 fg 26.00 ± 1.38 de 2.95 ± 0.21 c 9.95 ± 0.84 abc 7.46 ± 0.25 ab 5.76 ± 0.39 a 8.03 ± 0.21 b 3.21 ± 0.26 b 0.34 ± 0.01 a

Yolo 9.82 ± 0.54 a 31.35 ± 0.42 a 3.01 ± 0.05 bc 7.72 ± 0.13 g 7.02 ± 0.10 bc 4.95 ± 0.17 cdef 7.47 ± 0.53 b 2.51 ± 0.04 de 0.22 ± 0.02 c

LSD

Region

North Valley 8.32 ± 0.26 a 28.83 ± 0.42 a 3.06 ± 0.04 a 8.56 ± 0.16 b 7.38 ± 0.10 a 4.87 ± 0.06 b 8.21 ± 0.22 a 2.74 ± 0.10 a 0.25 ± 0.01 b

South Valley 8.03 ± 0.23 a 28.03 ± 0.43 a 3.05 ± 0.04 a 9.23 ± 0.19 a 7.15 ± 0.07 a 5.19 ± 0.06 a 7.43 ± 0.13 b 2.85 ± 0.06 a 0.29 ± 0.01 a

LSD

Year

2003-2004 7.39 ± 0.34 b 26.34 ± 0.59 b 3.06 ± 0.06 a 10.02 ± 0.34 a 7.53 ± 0.11 a 5.22 ± 0.08 a 7.81 ± 0.17 a 2.88 ± 0.03 a 0.35 ± 0.02 a

2005-2006 9.46 ± 0.21 a 30.74 ± 0.34 a 2.97 ± 0.05 a 7.92 ± 0.11 b 6.99 ± 0.10 b 4.96 ± 0.09 b 7.71 ± 0.30 a 2.75 ± 0.06 a 0.21 ± 0.01 b

LSD

0.24

Asx Glx Ser Arg Gly Ala

0.89 1.43 0.17 0.70 0.28

0.17 0.47

1.26 2.61 0.27 0.89 0.39 0.061.04 0.55 0.36

Pro Tyr Cys

0.64 0.29 0.05

0.21 0.03

0.75 1.26 0.15 0.61 0.30 0.25 0.77 0.16 0.04

0.67 1.20 0.12 0.49 0.23

65

3


Table 14. Interaction of cultivar and geographic location on the essential amino acid composition of select almond seedsa

Cultivar County LEAAb

LEAAc

Butte 4.54 ± 0.03 bc 2.21 ± 0.01 d 4.19 ± 0.01 bcd 0.87 ± 0.02 ab 2.98 ± 0.03 c 5.55 ± 0.01 cd 5.36 ± 0.02 c 1.05 ± 0.02 bc 1.50 ± 0.02 b 28.26 d Lys LysFresno 3.27 ± 0.16 de 2.41 ± 0.03 bc 4.03 ± 0.11 cd 1.10 ± 0.15 a 2.54 ± 0.06 e 5.09 ± 0.11 e 6.23 ± 0.52 bc 0.91 ± 0.08 cd 0.95 ± 0.13 d 26.53 e Lys LysKern 4.06 ± 0.56 cd 2.48 ± 0.06 b 4.25 ± 0.09 bc 0.79 ± 0.08 ab 2.83 ± 0.09 cd 5.41 ± 0.14 de 6.84 ± 0.38 ab 0.69 ± 0.09 d 0.92 ± 0.08 d 28.27 d Lys Lys

Madera 5.22 ± 0.03 ab 2.34 ± 0.01 c 4.91 ± 0.00 a 1.00 ± 0.00 a 3.55 ± 0.01 a 6.10 ± 0.00 b 6.26 ± 0.01 bc 1.00 ± 0.00 c 1.09 ± 0.01 cd 31.45 ab Lys LysMerced 5.44 ± 0.03 a 2.42 ± 0.01 bc 4.39 ± 0.03 b 1.07 ± 0.06 a 2.98 ± 0.01 c 5.79 ± 0.06 bc 5.39 ± 0.03 c 1.26 ± 0.01 ab 2.06 ± 0.04 a 30.79 bc Lys Lys

Sacramento 3.04 ± 0.10 e 2.18 ± 0.04 d 3.98 ± 0.02 d 0.64 ± 0.00 b 2.66 ± 0.05 de 5.37 ± 0.05 de 7.73 ± 0.10 a 0.87 ± 0.16 cd 0.92 ± 0.09 d 27.38 de Lys Met/CysSan Joaquin 4.93 ± 0.03 ab 2.74 ± 0.02 a 5.00 ± 0.03 a 1.05 ± 0.04 a 3.23 ± 0.03 b 5.94 ± 0.02 b 5.37 ± 0.01 c 1.05 ± 0.01 bc 1.42 ± 0.04 b 30.73 bc Lys LysStanislaus 4.52 ± 0.03 bc 2.17 ± 0.02 d 4.44 ± 0.01 b 1.06 ± 0.00 a 3.31 ± 0.02 b 6.03 ± 0.03 b 5.58 ± 0.15 bc 1.42 ± 0.01 a 1.33 ± 0.04 bc 29.86 c Lys Lys

Tulare 4.91 ± 0.03 ab 2.45 ± 0.00 bc 4.83 ± 0.00 a 1.08 ± 0.00 a 3.56 ± 0.02 a 6.55 ± 0.02 a 5.64 ± 0.01 bc 1.41 ± 0.01 a 1.87 ± 0.02 a 32.31 a Lys Met/CysYolo 2.86 ± 0.13 e 2.36 ± 0.04 c 4.00 ± 0.05 cd 0.99 ± 0.08 ab 2.66 ± 0.06 de 5.44 ± 0.17 cde 6.76 ± 0.54 abc 0.86 ± 0.05 cd 0.93 ± 0.08 d 26.87 e Lys LysLSD

Butte 2.43 ± 0.04 gh 2.02 ± 0.01 g 4.31 ± 0.01 de 1.00 ± 0.03 d 3.10 ± 0.00 bcde 6.30 ± 0.02 abc 5.23 ± 0.01 cd 1.11 ± 0.00 bcde 0.47 ± 0.01 g 25.97 e Lys LysColusa 2.37 ± 0.05 h 2.81 ± 0.05 b 4.51 ± 0.02 cd 2.60 ± 0.03 a 2.92 ± 0.02 def 6.42 ± 0.03 ab 4.56 ± 0.01 d 1.29 ± 0.03 ab 0.76 ± 0.00 ef 28.24 c Lys LysFresno 2.72 ± 0.07 f 2.27 ± 0.05 ef 4.16 ± 0.06 ef 1.01 ± 0.08 d 2.87 ± 0.11 efg 5.86 ± 0.26 bcde 6.10 ± 0.37 b 0.99 ± 0.09 def 1.06 ± 0.09 bcd 27.04 d Lys LysGlenn 3.89 ± 0.06 c 2.65 ± 0.02 c 4.38 ± 0.00 de 1.40 ± 0.01 c 2.89 ± 0.01 defg 5.62 ± 0.01 de 5.76 ± 0.00 bc 1.03 ± 0.01 cde 0.66 ± 0.01 fg 28.28 c Lys LysKern 4.83 ± 0.04 b 3.00 ± 0.00 a 4.78 ± 0.01 bc 1.06 ± 0.01 d 3.34 ± 0.02 b 6.00 ± 0.04 bcd 5.87 ± 0.02 bc 1.14 ± 0.02 abcd 1.84 ± 0.01 a 31.86 a Lys Lys

Madera 3.54 ± 0.01 d 2.82 ± 0.01 b 4.79 ± 0.00 b 1.62 ± 0.02 b 3.00 ± 0.01 cde 5.96 ± 0.02 bcde 5.41 ± 0.01 bc 1.31 ± 0.01 a 0.93 ± 0.03 de 29.37 b Lys LysMerced 5.03 ± 0.04 a 2.39 ± 0.01 de 4.37 ± 0.00 de 0.98 ± 0.01 d 3.14 ± 0.01 bcd 5.80 ± 0.01 cde 5.82 ± 0.04 bc 1.06 ± 0.00 cde 1.28 ± 0.02 b 29.88 b Lys Lys

Sacramento 3.01 ± 0.03 e 2.49 ± 0.04 d 4.26 ± 0.05 de 0.67 ± 0.04 e 2.72 ± 0.07 fg 5.46 ± 0.16 de 7.71 ± 0.08 a 0.69 ± 0.06 g 0.98 ± 0.06 cde 27.99 cd Lys LysSan Joaquin 2.56 ± 0.04 fg 2.46 ± 0.07 d 4.28 ± 0.19 de 1.27 ± 0.08 c 3.09 ± 0.07 bcde 6.44 ± 0.19 ab 5.51 ± 0.07 bc 0.94 ± 0.03 ef 0.89 ± 0.09 de 27.44 cd Lys LysStanislaus 5.10 ± 0.03 a 2.49 ± 0.02 d 4.47 ± 0.00 d 0.94 ± 0.00 d 3.21 ± 0.01 bc 5.85 ± 0.01 bcde 5.42 ± 0.00 bc 1.06 ± 0.00 cde 1.16 ± 0.03 bc 29.70 b Lys Lys

Tulare 4.85 ± 0.04 b 2.20 ± 0.01 f 5.32 ± 0.05 a 1.03 ± 0.04 d 3.65 ± 0.03 a 6.74 ± 0.03 a 5.39 ± 0.01 bc 1.22 ± 0.01 abc 1.89 ± 0.04 a 32.28 a Lys LysYolo 2.66 ± 0.11 f 2.30 ± 0.04 ef 3.93 ± 0.01 f 1.00 ± 0.02 d 2.65 ± 0.04 g 5.36 ± 0.11 e 5.62 ± 0.05 bc 0.83 ± 0.03 fg 1.04 ± 0.04 cd 25.39 e Lys LysLSD

Butte 2.24 ± 0.02 d 3.05 ± 0.03 a 4.95 ± 0.02 ab 2.39 ± 0.05 b 3.03 ± 0.03 b 6.39 ± 0.01 ab 4.96 ± 0.01 cd 1.00 ± 0.01 b 0.77 ± 0.00 e 28.78 ab Lys LysColusa 2.45 ± 0.07 cd 3.08 ± 0.05 a 5.20 ± 0.03 a 2.69 ± 0.04 a 3.03 ± 0.02 b 6.42 ± 0.04 ab 4.72 ± 0.01 d 0.89 ± 0.01 b 0.96 ± 0.04 d 29.44 ab Lys LysFresno 2.43 ± 0.14 cd 2.44 ± 0.04 c 4.73 ± 0.08 bc 1.95 ± 0.08 c 3.15 ± 0.18 b 6.47 ± 0.12 ab 4.95 ± 0.09 cd 1.13 ± 0.01 ab 0.76 ± 0.01 e 28.02 b Lys LysGlenn 3.57 ± 0.06 bc 2.34 ± 0.01 c 4.51 ± 0.00 c 1.45 ± 0.01 d 3.04 ± 0.02 b 6.26 ± 0.03 ab 5.68 ± 0.13 a 1.34 ± 0.00 a 0.90 ± 0.02 d 29.09 ab Lys LysKern 2.35 ± 0.07 d 2.77 ± 0.05 b 4.93 ± 0.02 ab 2.40 ± 0.04 b 3.29 ± 0.02 ab 6.41 ± 0.04 ab 5.24 ± 0.02 bc 1.16 ± 0.01 ab 0.98 ± 0.01 d 29.52 ab Lys Lys

Merced 4.86 ± 0.02 a 2.72 ± 0.01 b 4.85 ± 0.01 abc 1.01 ± 0.01 f 3.35 ± 0.03 ab 6.06 ± 0.07 b 5.21 ± 0.02 bc 1.04 ± 0.01 b 1.33 ± 0.02 b 30.42 ab Lys LysSan Joaquin 4.58 ± 0.03 ab 2.29 ± 0.00 c 4.64 ± 0.01 bc 1.00 ± 0.01 f 3.63 ± 0.04 a 6.87 ± 0.09 a 5.46 ± 0.20 ab 1.37 ± 0.01 a 1.45 ± 0.04 a 31.29 a Lys Met/CysStanislaus 3.86 ± 0.59 ab 2.37 ± 0.08 c 4.69 ± 0.20 bc 1.17 ± 0.04 e 3.12 ± 0.22 b 6.09 ± 0.31 b 5.21 ± 0.10 bc 1.06 ± 0.14 b 1.23 ± 0.04 c 28.80 ab Lys Lys

LSD 0.130.420.191.21

0.62 0.73

0.310.640.48

0.19

0.26

0.360.270.12

0.15 0.28 0.20

Leu Phe Lys Trp

0.38

His Thr Val Met Ile

Mission

Carmel

Butte

0.86

0.18

E/T

1.260.320.241.43

0.20 0.23 0.97

2.650.100.28

66

4

Table 14 continued. Interaction of cultivar and geographic location on the essential amino acid composition of select almond seedsa


Cultivar County LEAAb

LEAAc

Butte 4.88 ± 0.02 a 2.50 ± 0.01 cd 4.41 ± 0.00 cd 0.98 ± 0.01 d 3.02 ± 0.01 bc 5.48 ± 0.00 efg 5.64 ± 0.01 cde 0.90 ± 0.01 de 1.09 ± 0.01 cde 28.89 b Lys LysColusa 5.25 ± 0.09 a 3.36 ± 0.03 a 5.29 ± 0.01 a 1.13 ± 0.01 cd 3.35 ± 0.03 a 5.75 ± 0.01 cdef 5.48 ± 0.02 cde 1.02 ± 0.02 cd 1.66 ± 0.07 a 32.29 a Lys LysFresno 3.31 ± 0.26 bc 2.56 ± 0.07 c 4.46 ± 0.19 c 1.39 ± 0.11 b 2.86 ± 0.11 cd 5.69 ± 0.17 def 5.72 ± 0.04 cd 0.92 ± 0.03 de 1.20 ± 0.07 cd 28.11 bcd Lys LysGlenn 3.65 ± 0.04 b 2.27 ± 0.01 ef 4.41 ± 0.01 cd 1.32 ± 0.01 bc 3.04 ± 0.03 bc 6.03 ± 0.05 abcd 5.45 ± 0.15 cde 1.40 ± 0.00 b 0.90 ± 0.02 fg 28.46 bc Lys LysKern 2.57 ± 0.04 d 2.25 ± 0.00 efg 4.31 ± 0.00 cde 1.18 ± 0.06 cd 3.05 ± 0.01 bc 6.22 ± 0.02 abc 5.27 ± 0.01 def 0.96 ± 0.02 cd 0.76 ± 0.01 g 26.59 cd Lys Lys

Madera 4.60 ± 0.01 a 2.06 ± 0.01 g 4.26 ± 0.00 cdef 1.03 ± 0.00 d 3.16 ± 0.01 ab 5.92 ± 0.00 bcde 4.71 ± 0.01 f 1.64 ± 0.00 a 1.06 ± 0.00 def 28.44 bc Lys Met/CysMerced 4.76 ± 0.09 a 2.14 ± 0.01 fg 4.96 ± 0.02 b 1.01 ± 0.03 d 3.34 ± 0.03 a 6.11 ± 0.01 abcd 5.92 ± 0.01 c 1.42 ± 0.00 b 1.66 ± 0.07 a 31.30 a Lys Met/Cys

Sacramento 2.93 ± 0.08 bcd 2.49 ± 0.08 cd 4.08 ± 0.05 ef 0.76 ± 0.02 e 2.60 ± 0.10 de 5.28 ± 0.23 fg 7.83 ± 0.07 a 0.78 ± 0.04 ef 0.84 ± 0.07 g 27.59 bcd Lys LysSan Joaquin 4.88 ± 0.05 a 3.28 ± 0.01 a 4.96 ± 0.07 b 1.12 ± 0.04 cd 3.34 ± 0.03 a 6.29 ± 0.02 ab 5.81 ± 0.00 cd 1.30 ± 0.01 b 1.43 ± 0.05 b 32.40 a Lys LysStanislaus 3.21 ± 0.36 bcd 2.30 ± 0.05 def 4.12 ± 0.10 def 1.18 ± 0.06 cd 2.93 ± 0.08 bc 5.80 ± 0.09 bcde 5.41 ± 0.10 cde 1.10 ± 0.09 c 1.27 ± 0.05 bc 27.32 bcd Lys Lys

Tulare 2.60 ± 0.07 cd 2.96 ± 0.01 b 4.93 ± 0.13 b 1.85 ± 0.15 a 3.15 ± 0.12 ab 6.44 ± 0.10 a 5.02 ± 0.21 ef 1.01 ± 0.00 cd 0.92 ± 0.01 efg 28.88 b Lys LysYolo 2.87 ± 0.10 cd 2.41 ± 0.09 cde 4.00 ± 0.05 f 1.02 ± 0.09 d 2.54 ± 0.04 e 5.13 ± 0.10 g 6.60 ± 0.51 b 0.71 ± 0.04 f 0.93 ± 0.06 efg 26.20 d Lys LysLSD

Butte 4.48 ± 0.01 d 2.46 ± 0.00 c 4.22 ± 0.00 d 0.97 ± 0.01 f 2.98 ± 0.00 c 5.54 ± 0.01 d 5.27 ± 0.01 a 1.07 ± 0.00 d 1.07 ± 0.02 c 28.05 d Lys LysFresno 4.75 ± 0.03 c 1.90 ± 0.01 f 4.69 ± 0.04 a 0.99 ± 0.01 ef 3.31 ± 0.02 b 5.90 ± 0.09 c 5.17 ± 0.14 a 1.43 ± 0.00 c 1.42 ± 0.01 b 29.55 c Lys Met/CysKern 3.86 ± 0.09 f 2.23 ± 0.02 d 4.51 ± 0.04 b 1.37 ± 0.01 a 2.83 ± 0.01 e 5.45 ± 0.06 d 5.00 ± 0.61 a 1.03 ± 0.00 e 0.73 ± 0.00 d 27.02 e Lys Lys

Merced 4.12 ± 0.03 e 2.63 ± 0.02 a 4.27 ± 0.00 d 1.22 ± 0.02 b 2.73 ± 0.00 f 5.31 ± 0.01 e 5.53 ± 0.00 a 0.87 ± 0.00 f 0.74 ± 0.01 d 27.43 e Lys LysSan Joaquin 5.01 ± 0.05 b 2.07 ± 0.01 e 4.40 ± 0.00 c 1.02 ± 0.02 e 3.32 ± 0.05 b 6.17 ± 0.02 b 5.34 ± 0.01 a 1.54 ± 0.01 b 1.44 ± 0.03 b 30.30 b Lys Met/CysStanislaus 3.99 ± 0.04 ef 2.66 ± 0.03 a 4.50 ± 0.01 b 1.16 ± 0.02 c 3.90 ± 0.02 a 7.25 ± 0.03 a 5.51 ± 0.04 a 2.19 ± 0.01 a 2.65 ± 0.00 a 33.82 a Lys

Tulare 5.24 ± 0.05 a 2.61 ± 0.01 b 4.42 ± 0.00 c 1.11 ± 0.01 d 2.91 ± 0.01 d 5.31 ± 0.01 e 5.60 ± 0.02 a 1.08 ± 0.00 d 1.08 ± 0.04 c 29.35 c Lys LysLSD

Butte 4.94 ± 0.03 a 2.65 ± 0.02 a 4.63 ± 0.00 b 0.96 ± 0.01 c 3.19 ± 0.01 d 5.56 ± 0.01 e 5.88 ± 0.01 c 1.15 ± 0.01 cd 1.77 ± 0.02 c 30.72 c Lys LysFresno 4.86 ± 0.02 ab 2.65 ± 0.03 a 4.85 ± 0.01 a 0.96 ± 0.01 c 3.56 ± 0.02 a 6.33 ± 0.03 a 6.04 ± 0.02 b 1.18 ± 0.01 c 2.22 ± 0.05 a 32.65 a Lys LysKern 4.76 ± 0.03 b 2.55 ± 0.01 b 4.48 ± 0.00 c 0.97 ± 0.00 c 3.40 ± 0.01 b 6.32 ± 0.01 a 6.17 ± 0.01 a 1.11 ± 0.01 d 1.41 ± 0.03 e 31.19 b Lys Lys

Merced 4.16 ± 0.10 d 2.12 ± 0.03 e 4.43 ± 0.00 d 1.05 ± 0.00 a 3.28 ± 0.01 c 6.17 ± 0.02 b 4.95 ± 0.03 f 1.35 ± 0.05 a 1.49 ± 0.08 e 29.01 f Lys LysSan Joaquin 4.98 ± 0.05 a 2.37 ± 0.01 c 4.01 ± 0.00 g 0.83 ± 0.00 e 2.84 ± 0.01 f 5.21 ± 0.01 f 5.65 ± 0.01 d 0.85 ± 0.00 e 1.12 ± 0.01 f 27.85 g Lys LysStanislaus 4.55 ± 0.03 c 2.58 ± 0.01 b 4.40 ± 0.00 e 1.03 ± 0.01 b 2.95 ± 0.03 e 5.69 ± 0.01 d 5.26 ± 0.00 e 1.27 ± 0.01 b 1.62 ± 0.04 d 29.35 e Lys Lys

Tulare 4.78 ± 0.04 b 2.23 ± 0.02 d 4.26 ± 0.01 f 0.93 ± 0.01 d 3.24 ± 0.00 c 6.05 ± 0.02 c 5.28 ± 0.01 e 1.27 ± 0.01 b 1.90 ± 0.04 b 29.94 d Lys Met/CysLSD 0.05 0.05 0.04

Price

0.15 0.06 0.02 0.02

Nonpareil

Padre

0.040.070.050.15

His Thr Val Met Ile Leu Phe Lys Trp E/T

0.72

0.73 0.21 0.31 0.21 0.27 0.50 0.64

0.130.07

0.20

1.98

0.620.060.02

0.18 0.19

0.06 0.13

67

5

Table 15. Interaction of cultivar and geographic location on the non-essential amino acid composition of select almond seedsa


Cultivar County

Butte 10.23 ± 0.04 a 27.64 ± 0.13 bc 2.93 ± 0.01 ef 7.28 ± 0.00 de 9.70 ± 0.01 bc 4.99 ± 0.00 bcd 6.02 ± 0.02 c 2.62 ± 0.01 a 0.33 ± 0.02 bcd

Fresno 8.93 ± 0.29 b 29.80 ± 0.61 a 3.16 ± 0.05 cd 7.57 ± 0.12 cd 8.25 ± 0.42 de 4.75 ± 0.17 d 7.77 ± 0.56 b 2.96 ± 0.10 a 0.28 ± 0.02 cde

Kern 8.36 ± 0.20 bc 27.16 ± 0.72 bc 3.10 ± 0.09 d 7.71 ± 0.06 bc 8.82 ± 0.40 cd 4.82 ± 0.11 cd 8.66 ± 0.66 ab 2.87 ± 0.16 a 0.25 ± 0.05 de

Madera 7.55 ± 0.02 c 23.29 ± 0.05 e 2.57 ± 0.00 g 7.50 ± 0.02 cd 10.93 ± 0.01 a 6.11 ± 0.01 a 7.35 ± 0.03 bc 2.94 ± 0.01 a 0.31 ± 0.00 bcde

Merced 6.21 ± 0.00 d 25.18 ± 0.02 de 3.50 ± 0.03 a 7.97 ± 0.02 b 10.90 ± 0.04 a 4.57 ± 0.05 d 7.86 ± 0.01 b 2.69 ± 0.01 a 0.33 ± 0.01 bcd

Sacramento 8.04 ± 0.19 bc 28.49 ± 0.42 ab 2.77 ± 0.06 f 7.58 ± 0.04 cd 8.31 ± 0.35 de 4.97 ± 0.11 bcd 9.56 ± 0.13 a 2.75 ± 0.12 a 0.15 ± 0.01 f

San Joaquin 5.15 ± 0.05 e 23.46 ± 0.07 e 3.33 ± 0.00 b 8.44 ± 0.01 a 11.52 ± 0.02 a 5.20 ± 0.03 bc 8.94 ± 0.01 ab 2.88 ± 0.02 a 0.36 ± 0.01 bc

Stanislaus 8.75 ± 0.04 b 26.25 ± 0.08 cd 2.88 ± 0.01 ef 7.05 ± 0.01 e 10.60 ± 0.02 ab 5.39 ± 0.02 b 6.06 ± 0.05 c 2.69 ± 0.01 a 0.46 ± 0.01 a

Tulare 5.79 ± 0.02 de 23.98 ± 0.04 e 3.29 ± 0.00 bc 7.68 ± 0.01 bc 11.47 ± 0.01 a 4.95 ± 0.01 bcd 7.45 ± 0.03 bc 2.72 ± 0.00 a 0.37 ± 0.00 b

Yolo 8.84 ± 0.45 b 30.23 ± 0.72 a 3.00 ± 0.04 de 7.30 ± 0.11 de 7.62 ± 0.18 e 4.65 ± 0.17 d 8.19 ± 0.48 ab 3.05 ± 0.29 a 0.24 ± 0.04 e

LSD

Butte 8.56 ± 0.01 bc 32.92 ± 0.01 a 2.61 ± 0.01 fg 6.90 ± 0.01 d 8.24 ± 0.02 fg 5.38 ± 0.03 b 6.80 ± 0.03 g 2.40 ± 0.05 e 0.24 ± 0.01 ef

Colusa 7.03 ± 0.08 de 28.49 ± 0.07 c 3.01 ± 0.06 de 6.40 ± 0.04 e 8.25 ± 0.08 fg 4.82 ± 0.07 ef 8.52 ± 0.04 bc 4.94 ± 0.22 a 0.30 ± 0.01 cd

Fresno 8.86 ± 0.29 b 30.07 ± 0.61 b 2.80 ± 0.10 ef 6.83 ± 0.11 d 8.14 ± 0.22 fg 5.25 ± 0.16 bc 7.89 ± 0.27 cd 2.90 ± 0.04 bcde 0.22 ± 0.02 ef

Glenn 8.80 ± 0.05 b 26.95 ± 0.05 d 3.26 ± 0.01 abc 7.76 ± 0.02 b 9.05 ± 0.02 e 4.85 ± 0.02 ef 7.71 ± 0.05 de 3.04 ± 0.00 bcd 0.29 ± 0.00 d

Kern 5.24 ± 0.09 f 23.14 ± 0.09 f 3.47 ± 0.04 a 8.22 ± 0.02 a 10.85 ± 0.01 b 4.78 ± 0.02 f 8.95 ± 0.09 ab 3.12 ± 0.02 bc 0.37 ± 0.01 a

Madera 6.60 ± 0.07 e 25.42 ± 0.10 e 3.29 ± 0.01 ab 7.94 ± 0.05 ab 10.03 ± 0.01 cd 5.20 ± 0.01 bcd 8.85 ± 0.07 ab 3.05 ± 0.00 bcd 0.25 ± 0.00 e

Merced 8.34 ± 0.02 bc 26.45 ± 0.03 de 3.13 ± 0.00 bcd 7.24 ± 0.01 c 9.92 ± 0.00 d 4.90 ± 0.02 def 6.97 ± 0.02 efg 2.85 ± 0.03 bcde 0.33 ± 0.00 bc

Sacramento 7.69 ± 0.25 cd 28.45 ± 0.36 c 2.93 ± 0.05 de 7.75 ± 0.07 b 8.45 ± 0.20 f 4.79 ± 0.06 ef 9.32 ± 0.22 a 2.45 ± 0.08 e 0.18 ± 0.01 g

San Joaquin 8.77 ± 0.57 b 30.03 ± 0.47 b 3.07 ± 0.04 cd 6.88 ± 0.12 d 7.74 ± 0.09 g 5.11 ± 0.06 bcde 7.53 ± 0.25 defg 3.25 ± 0.42 b 0.21 ± 0.01 fg

Stanislaus 9.33 ± 0.02 ab 26.08 ± 0.03 de 3.39 ± 0.03 a 7.37 ± 0.01 c 10.60 ± 0.01 bc 4.94 ± 0.03 cdef 5.58 ± 0.04 h 2.62 ± 0.00 cde 0.39 ± 0.00 a

Tulare 5.21 ± 0.02 f 22.95 ± 0.03 f 2.50 ± 0.01 g 8.00 ± 0.02 ab 11.83 ± 0.01 a 6.63 ± 0.03 a 7.57 ± 0.03 def 2.66 ± 0.02 cde 0.36 ± 0.00 ab

Yolo 10.29 ± 0.44 a 32.05 ± 0.36 a 3.05 ± 0.08 cd 6.76 ± 0.10 d 7.91 ± 0.09 fg 4.98 ± 0.02 cdef 6.83 ± 0.32 fg 2.56 ± 0.02 de 0.18 ± 0.01 g

LSD

Butte 6.07 ± 0.10 cd 28.66 ± 0.18 ab 3.17 ± 0.02 b 6.73 ± 0.01 cd 7.98 ± 0.08 b 4.84 ± 0.06 d 8.53 ± 0.09 bc 4.95 ± 0.20 b 0.29 ± 0.01 b

Colusa 5.06 ± 0.04 d 26.94 ± 0.08 ab 3.03 ± 0.02 bc 6.87 ± 0.03 bc 8.37 ± 0.06 b 4.93 ± 0.08 d 9.62 ± 0.08 a 5.45 ± 0.23 a 0.28 ± 0.00 b

Fresno 7.30 ± 0.10 abc 29.65 ± 0.29 a 2.77 ± 0.01 de 6.40 ± 0.03 d 8.46 ± 0.14 b 5.19 ± 0.10 c 7.92 ± 0.07 c 3.98 ± 0.03 d 0.30 ± 0.03 b

Glenn 9.14 ± 0.03 a 27.14 ± 0.04 ab 2.97 ± 0.01 bcd 6.98 ± 0.01 bc 9.24 ± 0.03 ab 5.42 ± 0.01 ab 6.88 ± 0.03 d 2.78 ± 0.01 e 0.34 ± 0.00 b

Kern 5.76 ± 0.07 cd 27.18 ± 0.05 ab 3.45 ± 0.02 a 6.59 ± 0.04 cd 8.75 ± 0.08 b 4.75 ± 0.11 d 8.94 ± 0.09 ab 4.49 ± 0.15 c 0.58 ± 0.02 a

Merced 6.89 ± 0.06 bcd 25.07 ± 0.04 b 3.41 ± 0.01 a 7.27 ± 0.02 ab 10.99 ± 0.02 a 4.82 ± 0.03 d 7.78 ± 0.06 c 2.92 ± 0.01 e 0.44 ± 0.01 ab

San Joaquin 8.86 ± 0.02 ab 24.98 ± 0.06 b 2.66 ± 0.03 ef 6.41 ± 0.01 d 10.92 ± 0.00 a 5.64 ± 0.02 a 6.16 ± 0.03 d 2.71 ± 0.01 e 0.37 ± 0.01 b

Stanislaus 6.92 ± 1.08 bcd 26.44 ± 1.85 a 2.86 ± 0.11 cde 7.45 ± 0.20 a 10.69 ± 0.95 a 5.41 ± 0.08 bc 8.37 ± 0.40 bc 2.69 ± 0.05 e 0.33 ± 0.08 b

LSD

0.04

0.17

Mission

Butte

Carmel

0.550.750.331.461.03

2.19 3.75 0.23

Gly Arg

0.580.290.22

Ala Pro Tyr Cys

0.92 1.92 0.17 0.31 1.08 0.45 1.62 0.50 0.09

Asx Glx Ser

0.41 1.92 0.23 0.83 0.33

68

6

Table 15 continued. Interaction of cultivar and geographic location on the non-essential amino acid composition of select almond seedsa


Cultivar County

Butte 9.73 ± 0.03 a 27.64 ± 0.04 cde 3.19 ± 0.00 c 7.10 ± 0.01 cdef 9.48 ± 0.01 cd 5.04 ± 0.02 b 5.81 ± 0.02 g 2.76 ± 0.00 de 0.37 ± 0.00 bc

Colusa 3.54 ± 0.05 e 20.86 ± 0.10 g 3.84 ± 0.01 a 9.33 ± 0.00 a 11.57 ± 0.04 a 4.39 ± 0.04 d 10.60 ± 0.07 a 3.23 ± 0.01 b 0.34 ± 0.01 cd

Fresno 8.70 ± 0.44 ab 28.46 ± 1.46 bcd 3.13 ± 0.04 cd 7.24 ± 0.29 bcd 8.56 ± 0.28 ef 5.23 ± 0.10 b 7.45 ± 0.22 cdef 2.88 ± 0.09 cd 0.26 ± 0.02 ef

Glenn 9.12 ± 0.04 a 27.51 ± 0.09 cde 2.97 ± 0.01 def 7.01 ± 0.01 defg 9.65 ± 0.04 c 5.68 ± 0.04 a 6.58 ± 0.05 fg 2.67 ± 0.00 ef 0.34 ± 0.01 cd

Kern 8.35 ± 0.05 ab 33.59 ± 0.08 a 2.84 ± 0.01 ef 6.74 ± 0.01 efg 7.67 ± 0.07 fg 5.03 ± 0.01 b 6.64 ± 0.06 efg 2.31 ± 0.02 h 0.25 ± 0.00 f

Madera 9.72 ± 0.03 a 27.00 ± 0.03 de 2.77 ± 0.00 f 6.66 ± 0.01 g 11.19 ± 0.00 ab 5.77 ± 0.02 a 5.70 ± 0.02 g 2.34 ± 0.00 gh 0.41 ± 0.00 ab

Merced 6.43 ± 0.05 cd 25.34 ± 0.08 ef 2.78 ± 0.00 f 7.50 ± 0.03 bc 10.71 ± 0.01 ab 5.22 ± 0.01 b 7.67 ± 0.02 cde 2.78 ± 0.00 de 0.28 ± 0.01 def

Sacramento 8.48 ± 0.15 ab 29.56 ± 0.35 bc 3.04 ± 0.09 cde 7.30 ± 0.06 bcd 7.87 ± 0.13 efg 4.58 ± 0.12 cd 8.92 ± 0.15 b 2.40 ± 0.05 gh 0.25 ± 0.01 f

San Joaquin 5.70 ± 0.02 d 23.26 ± 0.06 fg 3.64 ± 0.00 a 7.60 ± 0.01 b 10.58 ± 0.03 b 5.02 ± 0.01 b 8.34 ± 0.03 bc 3.01 ± 0.01 c 0.44 ± 0.01 a

Stanislaus 7.34 ± 0.70 bc 30.35 ± 0.98 b 3.02 ± 0.07 cde 6.68 ± 0.14 fg 8.70 ± 0.44 de 5.02 ± 0.11 b 6.76 ± 0.31 defg 2.53 ± 0.06 fg 0.27 ± 0.03 ef

Tulare 6.21 ± 0.11 cd 29.06 ± 0.37 bcd 3.41 ± 0.09 b 6.91 ± 0.10 defg 8.06 ± 0.12 efg 4.89 ± 0.08 bc 8.49 ± 0.08 bc 3.77 ± 0.15 a 0.32 ± 0.00 cde

Yolo 9.58 ± 0.80 a 31.00 ± 0.58 b 3.00 ± 0.07 cde 7.15 ± 0.10 cde 7.63 ± 0.18 g 4.93 ± 0.27 bc 7.78 ± 0.77 cd 2.49 ± 0.05 fgh 0.23 ± 0.02 f

LSD

Butte 10.61 ± 0.04 a 28.07 ± 0.03 a 3.47 ± 0.00 c 6.55 ± 0.01 f 9.91 ± 0.01 e 4.74 ± 0.00 e 5.50 ± 0.03 f 2.67 ± 0.00 d 0.43 ± 0.01 b

Fresno 8.67 ± 0.01 c 25.34 ± 0.02 c 2.04 ± 0.00 f 6.91 ± 0.02 e 11.70 ± 0.01 b 6.79 ± 0.03 a 6.16 ± 0.00 e 2.57 ± 0.00 f 0.27 ± 0.00 e

Kern 8.35 ± 0.14 d 27.83 ± 0.29 a 2.97 ± 0.02 d 8.12 ± 0.07 a 9.34 ± 0.05 f 5.45 ± 0.02 b 7.70 ± 0.04 b 2.99 ± 0.02 a 0.23 ± 0.00 f

Merced 9.08 ± 0.04 b 28.17 ± 0.03 a 3.67 ± 0.01 b 7.79 ± 0.02 b 8.97 ± 0.04 g 4.81 ± 0.04 e 7.01 ± 0.04 d 2.78 ± 0.00 c 0.29 ± 0.00 e

San Joaquin 6.73 ± 0.04 f 25.37 ± 0.10 c 2.93 ± 0.02 e 7.43 ± 0.01 c 11.21 ± 0.01 c 5.17 ± 0.03 d 7.87 ± 0.05 a 2.61 ± 0.01 e 0.40 ± 0.01 c

Stanislaus 4.71 ± 0.03 g 23.22 ± 0.02 d 3.83 ± 0.00 a 7.70 ± 0.01 b 11.94 ± 0.02 a 5.37 ± 0.04 c 6.02 ± 0.03 e 2.84 ± 0.02 b 0.55 ± 0.02 a

Tulare 7.55 ± 0.13 e 26.67 ± 0.06 b 3.46 ± 0.01 c 7.32 ± 0.03 d 10.23 ± 0.03 d 4.78 ± 0.03 e 7.26 ± 0.12 c 3.02 ± 0.01 a 0.37 ± 0.01 d

LSD

Butte 6.90 ± 0.02 d 25.83 ± 0.02 c 3.42 ± 0.01 b 7.31 ± 0.02 b 10.23 ± 0.00 c 4.57 ± 0.03 e 7.89 ± 0.03 b 2.79 ± 0.00 c 0.34 ± 0.01 d

Fresno 5.34 ± 0.04 g 24.34 ± 0.08 e 3.01 ± 0.01 ef 7.61 ± 0.02 a 10.90 ± 0.02 b 4.97 ± 0.02 b 7.88 ± 0.05 b 2.96 ± 0.00 b 0.33 ± 0.01 de

Kern 6.41 ± 0.02 f 25.25 ± 0.01 d 3.30 ± 0.01 c 7.36 ± 0.00 b 9.92 ± 0.01 d 4.88 ± 0.02 c 8.29 ± 0.01 a 3.03 ± 0.00 a 0.36 ± 0.01 c

Merced 9.25 ± 0.06 b 27.29 ± 0.10 b 2.97 ± 0.03 f 6.47 ± 0.03 f 10.92 ± 0.04 b 5.20 ± 0.04 a 6.02 ± 0.02 d 2.46 ± 0.00 g 0.41 ± 0.00 b

San Joaquin 10.35 ± 0.02 a 27.69 ± 0.02 a 3.10 ± 0.01 d 6.82 ± 0.02 d 9.85 ± 0.01 e 4.65 ± 0.02 d 6.74 ± 0.03 c 2.63 ± 0.00 e 0.33 ± 0.00 de

Stanislaus 9.07 ± 0.02 c 27.16 ± 0.04 b 3.72 ± 0.02 a 6.54 ± 0.01 e 9.95 ± 0.01 d 4.43 ± 0.01 f 6.67 ± 0.02 c 2.64 ± 0.00 d 0.48 ± 0.01 a

Tulare 6.71 ± 0.00 e 25.26 ± 0.05 d 3.05 ± 0.02 e 7.19 ± 0.01 c 11.71 ± 0.01 a 4.94 ± 0.04 b 8.28 ± 0.02 a 2.59 ± 0.00 f 0.32 ± 0.01 e

LSD 0.10 0.17

Nonpareil

Padre

Price

1.64 2.56

0.360.23

0.05 0.02

0.20 0.07

0.030.040.170.08

0.22 0.44 0.90

0.090.100.04

Ala Pro Tyr Cys

0.43 1.09

Asx Glx Ser Gly Arg

0.06 0.06 0.08 0.08 0.01

69

7

Table 16. Amino acid composition of select macadamia nut seedsa

aAmino acid values are expressed as gram per 100 g protein and data are reported as mean ± SEM (n = 3). For each row, means with different letters are significantly different (p � 0.05) according to Fisher’s LSD test. LEAA represents limiting essential amino acids based on the recommended pattern report of a joint FAO/WHO expert consultation (93) for bpre-school child (2-5 years) and cadult (18+ years). E/T (%) represents essential-to-total amino acid ratio. dAmino acid distribution.

Amino Acid LSD

Asx 8.93 ± 0.05 a 8.19 ± 0.15 cd 8.79 ± 0.06 ab 7.80 ± 0.10 d 8.51 ± 0.25 abc 8.28 ± 0.35 bc 8.01 ± 0.24 cd 0.58

Glx 31.56 ± 0.21 a 32.00 ± 0.34 a 31.29 ± 0.04 a 27.48 ± 0.19 c 28.18 ± 0.42 bc 28.80 ± 0.63 b 31.05 ± 0.46 a 1.09

Ser 3.77 ± 0.13 c 4.11 ± 0.09 bc 4.19 ± 0.14 ab 4.55 ± 0.03 a 4.09 ± 0.13 bc 3.79 ± 0.17 bc 3.34 ± 0.22 d 0.42

Gly 5.10 ± 0.04 c 5.51 ± 0.01 b 5.50 ± 0.09 b 6.32 ± 0.07 a 5.67 ± 0.17 b 5.75 ± 0.18 b 5.00 ± 0.10 c 0.32

His 2.94 ± 0.14 b 2.93 ± 0.05 b 2.98 ± 0.15 b 3.34 ± 0.06 a 3.12 ± 0.14 ab 3.05 ± 0.09 abc 2.80 ± 0.06 c 0.32

Arg 10.52 ± 0.11 b 10.51 ± 0.32 b 10.23 ± 0.14 bc 9.44 ± 0.08 c 10.59 ± 0.25 b 10.48 ± 0.55 b 11.53 ± 0.46 a 0.93

Thr 2.77 ± 0.11 b 2.97 ± 0.07 b 3.12 ± 0.15 b 4.07 ± 0.07 a 3.73 ± 0.20 a 3.64 ± 0.16 a 2.71 ± 0.26 b 0.46

Ala 4.69 ± 0.13 ab 4.29 ± 0.02 b 4.71 ± 0.14 ab 4.83 ± 0.08 ab 4.97 ± 0.22 a 4.59 ± 0.38 ab 4.97 ± 0.21 a 0.58

Pro 7.30 ± 0.04 b 7.23 ± 0.22 b 7.03 ± 0.37 b 8.31 ± 0.12 a 7.19 ± 0.21 b 8.12 ± 0.41 a 6.93 ± 0.33 b 0.80

Tyr 3.81 ± 0.03 b 4.75 ± 0.11 a 4.28 ± 0.08 ab 4.37 ± 0.14 ab 4.21 ± 0.28 ab 4.03 ± 0.31 ab 4.29 ± 0.54 ab 0.78

Val 4.07 ± 0.04 d 4.01 ± 0.03 d 4.08 ± 0.07 d 4.69 ± 0.02 a 4.48 ± 0.08 b 4.59 ± 0.03 ab 4.23 ± 0.06 c 0.15

Met 1.78 ± 0.05 b 1.78 ± 0.02 b 1.79 ± 0.01 b 2.11 ± 0.03 a 2.10 ± 0.04 a 2.13 ± 0.05 a 1.92 ± 0.10 b 0.15

Cys 0.72 ± 0.07 abc 0.88 ± 0.02 a 0.80 ± 0.08 ab 0.76 ± 0.01 ab 0.73 ± 0.10 ab 0.53 ± 0.07 c 0.66 ± 0.07 bc 0.20

Ile 2.40 ± 0.09 a 2.15 ± 0.02 b 2.20 ± 0.07 b 2.42 ± 0.03 a 2.47 ± 0.05 a 2.56 ± 0.07 a 2.56 ± 0.08 a 0.18

Leu 5.08 ± 0.18 ab 4.48 ± 0.08 d 4.63 ± 0.10 cd 4.73 ± 0.06 bcd 4.96 ± 0.12 abc 5.06 ± 0.16 ab 5.12 ± 0.13 a 0.36

Phe 2.99 ± 0.03 d 3.08 ± 0.01 cd 3.08 ± 0.07 cd 3.58 ± 0.03 a 3.40 ± 0.16 ab 3.28 ± 0.18 bc 3.16 ± 0.05 bcd 0.29

Lys 1.08 ± 0.06 ab 0.69 ± 0.10 c 0.70 ± 0.06 c 0.47 ± 0.02 c 0.75 ± 0.13 bc 0.76 ± 0.19 bc 1.12 ± 0.17 a 0.35

Trp 0.47 ± 0.03 c 0.45 ± 0.02 c 0.60 ± 0.05 b 0.74 ± 0.00 a 0.83 ± 0.06 a 0.54 ± 0.00 bc 0.60 ± 0.04 b 0.10

LEAAb (2-5 yr)

First

Second

Third

LEAAc (18+ yr)

First

Second

Third

AADd (%)

Hydrophobic

Hydrophilic

Acidic

Basic

E/T c d c a a a b 0.61

LysTrpLeu

#508

'Kakea'

#660

'Keaau'

#741

'Mauka'

Blue

Diamond

Trader

Joe's

#246

'Keauhou'

#294

'Purvis'

LysTrpLeu

LysTrpLeu

LysTrpLeu

LysLeuTrp

LysTrpLeu

LysTrpLeu

41.02

LysTrp

LysTrp

Lys Lys

40.1914.13

Lys Lys Lys

38.426.5540.49

38.697.31

42.858.63

35.2813.2526.14

14.5423.58 22.53

38.607.08 7.43

37.0814.3025.62

13.9223.18

40.09

39.446.0539.0615.4524.23

7.8236.7014.4625.84

41.19

70

8

aValues in bold are significant (r � 0.273 and r � -0.273) based on critical values table for Pearson’s correlation coefficients (n = 58, p � 0.05). Physical characteristics: Wt = individual seed weight, SL = seed length, SW = seed width, ST = seed thickness. Chemical composition: M = moisture, L = lipid, P = protein, A = ash, S = total soluble sugars, T = tannins.

Wt SL SW ST M L P A S T Asx Glx Ser Gly His Arg Thr Ala Pro Tyr Val Met Cys Ile Leu Phe Lys Trp

Free

Asn

Wt 1.000SL 0.608 1.000SW 0.785 0.251 1.000ST 0.172 -0.530 0.361 1.000M 0.128 -0.020 0.196 0.187 1.000L 0.041 0.331 -0.038 -0.336 0.396 1.000P 0.037 0.089 0.019 -0.022 0.264 0.645 1.000A 0.033 -0.055 0.100 0.202 0.079 -0.113 0.134 1.000S -0.049 -0.251 0.023 0.070 -0.242 -0.586 -0.614 -0.153 1.000T 0.155 0.319 0.129 -0.290 0.205 0.414 0.032 -0.400 -0.100 1.000

Asx 0.127 0.218 -0.075 -0.108 0.158 0.248 0.080 -0.248 -0.105 0.398 1.000Glx 0.239 0.385 0.108 -0.157 0.054 0.266 0.173 -0.008 -0.445 0.344 0.643 1.000Ser -0.285 -0.204 -0.265 -0.071 -0.156 -0.137 -0.245 -0.086 0.182 -0.074 -0.299 -0.269 1.000Gly -0.143 -0.174 -0.148 0.106 -0.191 -0.407 -0.368 0.030 0.354 -0.318 -0.550 -0.607 0.329 1.000His -0.292 -0.368 -0.276 0.066 -0.148 -0.203 -0.186 -0.070 0.530 -0.174 -0.208 -0.781 0.204 0.473 1.000Arg -0.145 -0.378 -0.067 0.213 -0.004 -0.193 -0.115 0.021 0.470 -0.264 -0.435 -0.878 0.101 0.427 0.862 1.000Thr -0.079 -0.011 0.026 -0.120 -0.239 -0.184 -0.183 -0.018 0.084 -0.115 -0.561 -0.325 0.712 0.329 -0.032 -0.033 1.000Ala 0.213 0.059 0.316 0.136 0.116 -0.059 -0.106 -0.008 0.152 0.069 0.031 -0.186 -0.703 -0.069 0.114 0.343 -0.500 1.000Pro -0.019 0.048 0.021 -0.020 -0.001 -0.041 0.116 0.175 -0.258 -0.240 -0.625 -0.185 0.189 0.483 -0.222 -0.163 0.567 -0.347 1.000Tyr -0.057 -0.155 0.156 0.119 -0.140 -0.018 0.109 0.053 -0.221 -0.225 -0.420 -0.033 0.152 -0.150 -0.400 -0.263 0.595 -0.190 0.467 1.000Val -0.093 -0.222 0.139 0.105 -0.185 -0.263 -0.097 0.114 0.221 -0.315 -0.778 -0.678 0.114 0.365 0.281 0.497 0.509 0.258 0.357 0.466 1.000Met -0.012 -0.099 0.208 0.085 -0.099 0.002 0.029 0.071 -0.228 -0.133 -0.310 0.101 0.098 -0.281 -0.505 -0.297 0.468 -0.061 0.287 0.907 0.422 1.000Cys -0.244 -0.492 -0.036 0.171 0.114 -0.116 -0.121 0.022 0.345 -0.208 -0.311 -0.575 0.435 0.000 0.497 0.621 0.214 0.007 -0.270 0.079 0.397 0.104 1.000Ile -0.123 -0.285 0.072 0.128 -0.060 -0.197 -0.026 0.123 0.300 -0.314 -0.599 -0.732 0.025 0.195 0.523 0.768 0.092 0.411 -0.123 0.042 0.690 0.016 0.644 1.000

Leu -0.064 -0.177 0.177 0.103 0.025 -0.163 0.014 0.236 0.048 -0.296 -0.567 -0.372 -0.072 -0.102 0.001 0.361 0.136 0.385 -0.016 0.343 0.619 0.402 0.443 0.810 1.000Phe -0.127 0.119 -0.236 -0.152 0.169 0.021 0.035 0.008 -0.033 0.073 0.095 0.079 -0.061 0.295 -0.046 -0.248 -0.079 -0.218 0.329 -0.334 -0.367 -0.532 -0.377 -0.333 -0.440 1.000Lys -0.065 -0.334 0.110 0.221 0.093 -0.235 -0.220 0.107 0.393 -0.190 -0.344 -0.520 0.048 0.017 0.379 0.674 -0.128 0.381 -0.331 -0.060 0.318 0.066 0.645 0.714 0.647 -0.411 1.000Trp -0.055 -0.109 -0.081 0.075 -0.076 -0.032 0.059 0.032 0.233 -0.179 -0.465 -0.652 0.254 0.317 0.601 0.716 0.063 0.059 -0.076 -0.230 0.301 -0.314 0.462 0.628 0.343 -0.071 0.539 1.000

Free Asn -0.208 0.019 -0.277 -0.140 -0.088 -0.016 0.160 0.122 -0.100 -0.343 -0.069 -0.110 -0.099 0.113 -0.004 0.068 0.056 -0.026 0.170 0.120 0.106 0.157 -0.115 0.054 0.082 -0.035 0.027 -0.148 1.000

Table 17. Correlation coefficients among various physical and chemical parameters of select almond seeds a

71

9

Table 18. Correlation coefficients among various physical and chemical parameters of select macadamia nut seeds a

aValues in bold are significant (r � 0.754 and r � -0.754) based on critical values table for Pearson’s correlation coefficients (n = 7, p � 0.05). Physical characteristics: Wt = individual seed weight, SD = seed diameter, ST = seed thickness. Chemical composition: M = moisture, L = lipid, P = protein, A = ash, S = total soluble sugars, T = tannins.

Wt SD ST M L P A S T Asx Glx Ser Gly His Arg Thr Ala Pro Tyr Val Met Cys Ile Leu Phe Lys Trp

Wt 1.000SD 0.932 1.000ST 0.500 0.741 1.000M 0.427 0.336 0.363 1.000L -0.173 0.099 0.607 0.080 1.000P 0.569 0.642 0.724 0.753 0.277 1.000A -0.587 -0.755 -0.863 -0.127 -0.419 -0.600 1.000S -0.454 -0.695 -0.959 -0.465 -0.699 -0.779 0.741 1.000T -0.497 -0.700 -0.585 -0.029 -0.412 -0.352 0.397 0.673 1.000

Asx 0.134 0.041 -0.323 -0.053 -0.669 0.132 0.265 0.301 -0.024 1.000Glx 0.672 0.710 0.463 0.564 -0.044 0.696 -0.219 -0.566 -0.694 0.438 1.000Ser 0.110 -0.049 -0.035 0.123 -0.336 0.068 -0.288 0.231 0.725 -0.114 -0.441 1.000Gly -0.255 -0.379 -0.223 -0.140 -0.166 -0.378 -0.028 0.408 0.797 -0.466 -0.789 0.833 1.000His -0.284 -0.383 -0.279 -0.391 -0.200 -0.452 -0.085 0.483 0.754 -0.320 -0.855 0.813 0.942 1.000Arg -0.094 0.033 0.073 0.141 0.442 0.167 0.259 -0.328 -0.645 0.094 0.492 -0.924 -0.850 -0.862 1.000Thr -0.522 -0.637 -0.454 -0.353 -0.144 -0.583 0.191 0.594 0.849 -0.397 -0.942 0.680 0.936 0.941 -0.701 1.000Ala -0.663 -0.437 -0.007 -0.584 0.511 -0.197 -0.059 -0.014 0.031 -0.081 -0.472 -0.176 -0.030 0.178 0.185 0.214 1.000Pro -0.209 -0.282 -0.278 -0.471 -0.226 -0.712 0.086 0.475 0.461 -0.467 -0.711 0.458 0.801 0.773 -0.686 0.767 -0.103 1.000Tyr 0.330 0.266 0.437 0.818 0.369 0.578 -0.342 -0.483 0.102 -0.548 0.133 0.351 0.250 0.013 -0.082 0.038 -0.392 -0.121 1.000Val -0.689 -0.689 -0.414 -0.596 0.114 -0.774 0.252 0.513 0.578 -0.541 -0.969 0.298 0.741 0.773 -0.409 0.886 0.432 0.785 -0.164 1.000Met -0.766 -0.790 -0.527 -0.543 0.131 -0.794 0.409 0.566 0.581 -0.513 -0.946 0.180 0.649 0.667 -0.248 0.841 0.442 0.678 -0.148 0.974 1.000Cys 0.674 0.592 0.489 0.652 -0.014 0.817 -0.636 -0.452 0.011 0.077 0.377 0.538 0.064 0.025 -0.294 -0.171 -0.341 -0.365 0.654 -0.532 -0.588 1.000Ile -0.690 -0.549 -0.350 -0.764 0.333 -0.749 0.380 0.303 -0.079 -0.280 -0.538 -0.501 -0.028 0.091 0.330 0.248 0.646 0.304 -0.557 0.648 0.702 -0.875 1.000

Leu -0.499 -0.352 -0.333 -0.771 0.170 -0.609 0.375 0.258 -0.325 0.103 -0.205 -0.703 -0.388 -0.203 0.509 -0.102 0.593 0.019 -0.777 0.299 0.363 -0.828 0.898 1.000Phe -0.541 -0.545 -0.215 -0.400 0.247 -0.496 -0.017 0.323 0.621 -0.634 -0.953 0.517 0.829 0.858 -0.512 0.914 0.446 0.672 0.121 0.920 0.877 -0.178 0.416 0.029 1.000Lys 0.087 0.253 0.119 -0.205 0.224 0.079 0.075 -0.278 -0.816 0.361 0.558 -0.916 -0.931 -0.808 0.838 -0.793 0.232 -0.590 -0.482 -0.476 -0.397 -0.338 0.358 0.680 -0.638 1.000Trp -0.674 -0.641 -0.247 -0.341 0.284 -0.238 0.031 0.272 0.602 -0.250 -0.792 0.391 0.516 0.635 -0.259 0.699 0.750 0.168 0.002 0.666 0.680 -0.054 0.359 0.117 0.805 -0.398 1.000

72

y = 8.701x + 11.96R² = 0.369

15.00

17.00

19.00

21.00

23.00

25.00

27.00

29.00

0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50

Alm

on

d s

eed

len

gth

(m

m)

Almond seed weight (g)

y = 3.906x + 7.808R² = 0.616

9.00

10.00

11.00

12.00

13.00

14.00

0.70 0.90 1.10 1.30 1.50

Alm

on

d s

eed

wid

th (

mm

)

Almond seed weight (g)

y = 1.466x + 15.36R² = 0.868

18.40

18.80

19.20

19.60

20.00

20.40

2.25 2.75 3.25Ma

cad

am

ia n

ut

seed

dia

met

er

(mm

)

Macadamia nut seed weight (g)

A

B

C

Figure 4. Relationship between (A) seed weight and seed length, and (B) seed weight and seed width in 58 almond seed samples. Relationship between (C) seed weight and seed diameter in 7 macadamia nut seed samples. Data are expressed in grams (seed weight) and millimeters (length, width, and diameter).

73

y = 1.115x + 33.11R² = 0.416

50.00

55.00

60.00

65.00

70.00

15.00 18.00 21.00 24.00 27.00

Lip

id (%

)

Protein (%)A

y = -2.409x + 68.30R² = 0.343

50.00

55.00

60.00

65.00

70.00

2.50 3.50 4.50 5.50 6.50

Lip

id (%

)

Total soluble sugars (%)C

y = -1.461x + 28.39R² = 0.377

15.00

18.00

21.00

24.00

27.00

2.50 3.50 4.50 5.50 6.50

Pro

tein

(%

)

Total soluble sugars (%)B

y = -0.350x + 8.959R² = 0.606

5.50

6.00

6.50

7.00

7.50

5.00 6.00 7.00 8.00 9.00

Pro

tein

(%

)

Total soluble sugars (%)D

Figure 5. Relationship between (A) lipid and protein, (B) protein and total soluble sugars, and (C) lipid and total soluble sugars in 58 almond seed samples. Relationship between (D) protein and total soluble sugars in 7 macadamia nut seed samples. Data are expressed in gram per 100 gram edible portion.

74

y = 1.032x + 19.14R² = 0.438

18.00

22.00

26.00

30.00

34.00

3.00 5.00 7.00 9.00 11.00 13.00

Glu

tam

ic a

cid

Aspartic acidA

y = -1.735x + 43.92R² = 0.770

18.00

22.00

26.00

30.00

34.00

7.00 8.00 9.00 10.00 11.00 12.00

Glu

tam

ic a

cid

Arginine B

y = 3.345x + 5.887R² = 0.400

7.00

8.00

9.00

10.00

11.00

12.00

0.60 1.00 1.40 1.80 2.20

Arg

inin

e

LysineC

y = 2.236x + 8.693R² = 0.702

9.00

10.00

11.00

12.00

0.40 0.60 0.80 1.00 1.20

Arg

inin

e

LysineD

Figure 6. Relationship between (A) glutamic acid and aspartic acid, (B) glutamic acid and arginine, and (C) arginine and lysine in 58 almond seed samples. Relationship between (D) arginine and lysine in 7 macadamia nut seed samples. Data are expressed in gram per 100 gram protein.

75

1

Table 19. Cultivar, geographic location, and harvest year variation on the free (essential) amino acid composition of select almond seedsa

aAmino acid values are expressed as milligram per 100 g dry weight and data are expressed as mean ± SEM (n = 3). For each column, means with different letters are significantly different (p � 0.05) according to Fisher’s LSD test.

Cultivar

Butte 4.41 ± 0.30 c 3.36 ± 0.18 cd 8.41 ± 0.46 ab 2.40 ± 0.27 bc 10.40 ± 0.92 cd 3.14 ± 0.15 cd 6.85 ± 0.39 cd 3.34 ± 0.17 bc

Carmel 6.24 ± 0.34 a 5.28 ± 0.28 a 9.27 ± 0.40 ab 2.48 ± 0.17 bc 12.16 ± 1.35 c 3.26 ± 0.13 bc 7.96 ± 0.31 bc 4.31 ± 0.23 a

Mission 5.97 ± 0.45 ab 4.05 ± 0.19 bc 9.12 ± 0.39 ab 2.02 ± 0.28 bcd 9.51 ± 1.72 cd 2.63 ± 0.14 d 7.17 ± 0.28 cd 4.04 ± 0.17 ab

Monterey 2.34 ± 0.21 de 4.44 ± 0.10 b 9.77 ± 0.75 a 3.67 ± 0.03 a 41.61 ± 5.71 a 1.37 ± 0.31 e 7.90 ± 1.00 bc 3.73 ± 0.31 abc

Nonpareil 4.60 ± 0.29 c 4.06 ± 0.16 bc 9.55 ± 0.44 a 2.00 ± 0.17 cd 13.24 ± 1.30 c 4.39 ± 0.17 a 9.36 ± 0.40 a 4.04 ± 0.19 ab

Padre 4.88 ± 0.23 bc 2.90 ± 0.16 d 8.00 ± 0.28 bc 0.86 ± 0.11 e 6.95 ± 0.20 d 4.37 ± 0.26 a 6.20 ± 0.39 d 3.25 ± 0.17 c

Price 5.00 ± 0.44 bc 5.42 ± 0.50 a 8.69 ± 0.78 ab 1.61 ± 0.25 d 6.63 ± 0.49 d 3.69 ± 0.19 b 6.90 ± 0.56 cd 3.49 ± 0.46 bc

Sonora 1.33 ± 0.29 e 3.43 ± 0.31 cd 6.76 ± 0.20 c 2.70 ± 0.04 b 27.24 ± 4.55 b 2.98 ± 0.18 cd 8.62 ± 1.15 ab 3.93 ± 0.28 abc

LSD

County

Butte 6.43 ± 0.70 abcd 4.87 ± 0.32 bcd 8.76 ± 0.35 bcde 1.96 ± 0.69 cde 7.17 ± 0.31 c 4.44 ± 0.17 ab 6.70 ± 0.58 def 3.57 ± 0.39 bcde

Colusa 6.38 ± 1.32 abcd 5.12 ± 0.66 bc 10.57 ± 0.49 abc 1.82 ± 0.48 cde 7.59 ± 0.27 c 4.38 ± 0.37 ab 7.29 ± 0.24 cdef 3.37 ± 0.12 cde

Fresno 5.01 ± 0.69 cde 4.82 ± 0.20 bcd 9.36 ± 0.56 abcde 3.83 ± 0.36 a 15.85 ± 2.76 ab 3.20 ± 0.17 c 9.25 ± 0.49 bc 5.17 ± 0.36 a

Glenn 5.70 ± 0.46 bcd 4.24 ± 0.28 bcd 7.35 ± 0.38 ef 1.09 ± 0.22 e 6.46 ± 0.28 c 3.56 ± 0.34 bc 5.84 ± 0.63 f 3.06 ± 0.17 def

Kern 7.42 ± 0.61 ab 5.56 ± 1.00 b 11.07 ± 1.25 ab 3.14 ± 0.65 ab 7.94 ± 0.38 c 4.10 ± 0.31 abc 8.72 ± 0.57 bcd 4.44 ± 0.45 abc

Madera 8.07 ± 0.83 a 8.06 ± 1.14 a 11.06 ± 0.74 ab 1.37 ± 0.54 de 7.22 ± 0.59 c 3.21 ± 0.59 c 9.74 ± 1.43 ab 3.87 ± 0.30 bcd

Merced 4.50 ± 0.45 de 4.13 ± 0.51 cd 10.03 ± 0.25 abcd 1.60 ± 0.47 de 8.11 ± 0.24 c 4.01 ± 0.37 abc 6.10 ± 0.37 ef 4.50 ± 0.32 ab

Sacramento 6.58 ± 0.30 abc 4.66 ± 0.25 bcd 11.75 ± 0.45 a 1.73 ± 0.19 cde 10.92 ± 0.30 bc 4.28 ± 0.26 abc 11.45 ± 0.56 a 5.51 ± 0.31 a

San Joaquin 3.56 ± 0.51 e 2.64 ± 0.24 e 6.01 ± 1.13 f 2.82 ± 0.53 abc 16.60 ± 4.41 ab 4.22 ± 0.76 abc 8.30 ± 0.75 bcd 2.16 ± 0.41 f

Stanislaus 3.49 ± 0.83 e 4.45 ± 0.55 bcd 8.72 ± 0.60 bcde 2.27 ± 0.23 bcd 20.11 ± 4.42 a 3.27 ± 0.28 c 8.06 ± 0.87 bcde 4.03 ± 0.32 bcd

Tulare 6.29 ± 0.43 abcd 3.71 ± 0.36 de 7.95 ± 2.77 def 2.33 ± 0.32 bcd 7.55 ± 0.98 c 4.75 ± 0.64 a 7.14 ± 1.30 def 2.61 ± 0.69 ef

Yolo 3.37 ± 0.71 e 4.58 ± 0.45 bcd 8.52 ± 1.01 cdef 3.16 ± 0.58 ab 21.99 ± 3.54 a 3.34 ± 0.34 bc 9.54 ± 0.40 ab 4.56 ± 0.40 ab

LSD

Region

North Valley 5.77 ± 0.32 a 4.67 ± 0.17 a 9.96 ± 0.37 a 1.97 ± 0.20 b 11.60 ± 1.08 b 4.03 ± 0.15 a 9.13 ± 0.42 a 4.45 ± 0.21 a

South Valley 5.11 ± 0.34 a 4.64 ± 0.27 a 9.00 ± 0.43 a 2.68 ± 0.19 a 13.56 ± 1.42 a 3.70 ± 0.17 a 8.34 ± 0.33 a 3.96 ± 0.20 a

LSD

Year

2003-2004 5.33 ± 0.48 a 3.60 ± 0.23 a 8.93 ± 0.42 a 1.68 ± 0.26 b 6.85 ± 0.19 b 3.14 ± 0.20 a 6.25 ± 0.42 b 3.81 ± 0.31 a

2005-2006 3.20 ± 0.41 b 4.06 ± 0.20 a 8.75 ± 0.53 a 3.37 ± 0.29 a 22.94 ± 2.22 a 2.77 ± 0.19 a 8.99 ± 0.53 a 4.51 ± 0.21 a

LSD 0.56 1.441.26 0.62 1.45 0.82 5.42

2.09

0.93 0.66 1.15 0.54 3.66 0.47 1.02

1.34 2.52 1.18 7.99 1.11

0.72

His Thr Val Met Ile Leu Phe

1.11 0.78 1.49 0.70 4.17 0.53 1.26

1.95

Lys

0.73

1.11

0.58

76

2

Table 20. Cultivar, geographic location, and harvest year variation on the free (non-essential) amino acid composition of select almond seedsa


Cultivar

Butte 117.88 ± 8.62 bcd 47.17 ± 4.48 a 55.99 ± 3.04 abc 11.58 ± 0.45 ab 11.56 ± 0.59 bc 5.98 ± 0.33 de 61.51 ± 3.69 c 14.79 ± 0.67 bc 51.10 ± 2.25 ab 10.06 ± 1.01 ab 1.55 ± 0.26 a

Carmel 163.93 ± 10.36 a 48.86 ± 3.50 a 62.44 ± 2.61 a 12.38 ± 0.53 ab 13.10 ± 0.41 ab 7.94 ± 0.32 ab 65.37 ± 3.44 bc 17.62 ± 0.65 a 52.62 ± 1.80 a 6.14 ± 0.50 bc 1.54 ± 0.22 a

Mission 127.59 ± 7.56 bc 45.04 ± 2.81 a 53.23 ± 2.54 abc 11.57 ± 0.64 ab 10.04 ± 0.54 cd 6.58 ± 0.45 cde 60.22 ± 2.57 c 15.76 ± 0.61 ab 45.53 ± 2.01 bcd 5.67 ± 1.05 bc 0.62 ± 0.17 bc

Monterey 126.63 ± 8.53 bc 31.72 ± 3.13 bc 53.98 ± 12.10 abc 11.62 ± 1.07 ab 14.65 ± 1.67 a 5.32 ± 0.33 e 90.04 ± 7.84 a 15.91 ± 0.58 ab 48.45 ± 4.38 abc 3.26 ± 0.26 c 0.05 ± 0.05 c

Nonpareil 102.96 ± 6.66 cd 39.25 ± 2.38 ab 61.80 ± 2.65 ab 13.11 ± 0.49 a 10.74 ± 0.51 c 7.42 ± 0.42 abc 34.81 ± 2.04 d 18.10 ± 0.74 a 42.14 ± 1.78 cd 6.34 ± 0.72 bc 1.13 ± 0.15 ab

Padre 94.19 ± 9.43 d 47.54 ± 5.50 a 52.36 ± 3.09 bc 11.20 ± 0.64 bc 8.93 ± 0.50 d 5.60 ± 0.34 de 26.47 ± 2.64 de 13.11 ± 0.42 cd 41.17 ± 3.05 d 10.73 ± 1.52 a 1.34 ± 0.24 a

Price 136.84 ± 10.68 ab 41.00 ± 7.18 ab 49.15 ± 5.00 c 9.16 ± 0.82 d 10.52 ± 0.74 cd 6.85 ± 0.67 bcd 75.84 ± 7.36 b 14.43 ± 1.41 bc 44.65 ± 3.09 bcd 5.01 ± 0.52 c 1.01 ± 0.17 ab

Sonora 38.70 ± 6.01 e 19.54 ± 0.57 c 48.03 ± 0.56 c 9.66 ± 1.38 cd 11.02 ± 2.25 c 8.39 ± 2.69 a 21.49 ± 1.55 e 10.79 ± 0.38 d 32.38 ± 0.85 e 3.51 ± 0.10 c 0.07 ± 0.04 c

LSD

County

Butte 143.58 ± 24.51 bcd 47.41 ± 4.42 abc 67.57 ± 2.48 abc 14.39 ± 0.75 abc 12.62 ± 1.29 bc 7.34 ± 0.31 cdefg 49.02 ± 9.37 ab 17.73 ± 1.18 bc 55.46 ± 4.04 ab 12.86 ± 3.63 a 3.06 ± 0.48 a

Colusa 192.71 ± 33.72 ab 58.07 ± 9.34 a 74.15 ± 7.21 ab 15.26 ± 1.64 ab 13.30 ± 1.58 ab 7.57 ± 1.14 cdef 55.52 ± 11.94 ab 19.71 ± 1.76 ab 55.02 ± 5.74 ab 5.61 ± 0.36 cd 0.58 ± 0.22 cde

Fresno 133.43 ± 14.49 cde 36.51 ± 4.03 bcd 55.34 ± 3.30 cde 11.15 ± 0.68 de 11.60 ± 0.72 bcd 7.03 ± 0.28 defg 55.84 ± 2.43 ab 17.31 ± 0.61 bc 49.03 ± 3.42 abc 4.85 ± 0.91 cd 1.19 ± 0.42 cd

Glenn 120.61 ± 27.97 cde 40.90 ± 8.30 abcd 62.37 ± 4.21 bcd 12.81 ± 0.68 bcd 11.77 ± 1.30 bcd 7.92 ± 0.45 cd 37.71 ± 9.65 bc 17.42 ± 0.92 bc 45.94 ± 4.53 abcd 10.81 ± 3.86 ab 0.97 ± 0.44 cde

Kern 150.73 ± 19.31 bc 50.56 ± 3.63 abc 72.10 ± 4.27 ab 14.41 ± 1.52 abc 9.75 ± 0.62 de 7.65 ± 0.83 cde 52.32 ± 10.76 ab 18.12 ± 1.43 bc 43.29 ± 4.58 cd 5.87 ± 0.69 cd 1.16 ± 0.42 cd

Madera 231.66 ± 46.15 a 53.31 ± 3.47 ab 79.65 ± 2.83 a 11.91 ± 1.87 cde 13.68 ± 1.39 ab 11.58 ± 1.88 a 53.98 ± 9.58 ab 23.05 ± 1.88 a 56.27 ± 6.93 a 4.28 ± 0.74 cd 0.00 ± 0.00 e

Merced 118.81 ± 12.90 cde 49.98 ± 5.93 abc 59.31 ± 1.32 bcd 10.61 ± 0.42 de 11.21 ± 0.73 bcd 5.39 ± 0.29 g 35.93 ± 7.81 bc 17.06 ± 0.36 bc 51.32 ± 7.72 abc 4.31 ± 0.37 cd 0.36 ± 0.18 de

Sacramento 170.77 ± 10.90 bc 47.01 ± 2.18 abc 74.34 ± 3.32 ab 16.02 ± 0.83 a 15.96 ± 0.68 a 10.20 ± 0.72 ab 60.95 ± 4.30 a 22.16 ± 0.94 a 52.92 ± 1.96 abc 7.47 ± 0.54 bc 2.29 ± 0.17 ab

San Joaquin 52.32 ± 5.32 f 31.52 ± 5.63 cd 40.80 ± 4.67 e 9.72 ± 0.89 e 9.96 ± 0.64 cde 5.81 ± 0.41 efg 22.76 ± 3.92 c 10.80 ± 0.81 d 49.04 ± 4.56 abc 4.24 ± 0.22 cd 1.44 ± 0.48 bc

Stanislaus 86.64 ± 17.02 ef 56.19 ± 12.40 ab 59.36 ± 6.04 bcd 10.90 ± 0.83 de 9.13 ± 0.86 de 5.61 ± 0.39 fg 52.80 ± 13.87 ab 16.91 ± 1.47 bc 30.87 ± 2.09 e 7.00 ± 1.52 bcd 0.74 ± 0.37 cde

Tulare 122.91 ± 7.46 cde 35.94 ± 11.44 bcd 48.75 ± 11.93 de 13.39 ± 1.86 abcd 7.97 ± 0.66 e 9.21 ± 0.32 bc 39.55 ± 2.37 abc 14.83 ± 3.28 c 35.87 ± 1.96 de 3.37 ± 0.89 d 2.73 ± 0.46 a

Yolo 94.26 ± 12.64 def 26.96 ± 5.30 d 54.52 ± 8.59 cde 12.10 ± 1.22 cd 11.77 ± 1.08 bcd 6.36 ± 0.73 defg 55.14 ± 6.03 ab 16.65 ± 1.90 bc 44.49 ± 5.61 bcd 4.82 ± 0.80 cd 0.59 ± 0.28 cde

LSD

Region

North Valley 148.08 ± 9.41 a 43.71 ± 2.55 a 67.85 ± 2.63 a 14.49 ± 0.52 a 13.76 ± 0.53 a 8.40 ± 0.42 a 54.37 ± 3.23 a 19.51 ± 0.69 a 50.92 ± 1.79 a 7.86 ± 0.81 a 1.65 ± 0.18 a

South Valley 120.94 ± 9.27 b 44.07 ± 3.24 a 57.81 ± 2.44 b 11.46 ± 0.43 b 10.48 ± 0.37 b 7.13 ± 0.33 b 46.11 ± 3.54 a 16.63 ± 0.65 b 44.46 ± 1.90 b 5.03 ± 0.42 b 1.09 ± 0.18 b

LSD

Year

2003-2004 148.67 ± 12.68 a 52.48 ± 2.80 a 59.27 ± 2.24 a 10.88 ± 0.55 a 10.45 ± 0.53 a 5.92 ± 0.40 a 58.13 ± 5.86 a 16.20 ± 0.57 a 47.22 ± 3.10 a 8.04 ± 1.66 a 1.45 ± 0.32 a

2005-2006 80.88 ± 6.65 b 28.15 ± 1.80 b 49.47 ± 2.50 b 11.39 ± 0.62 a 11.07 ± 0.71 a 6.16 ± 0.25 a 49.56 ± 2.64 a 15.39 ± 0.70 a 50.14 ± 3.15 a 5.84 ± 0.60 a 1.01 ± 0.30 a

LSD 26.02 0.891.931.747.076.27

1.03 9.60 1.86 5.21 1.65

53.09

26.10 8.42 7.04 1.30

2.032.673.0615.5219.84

1.75 1.34 11.16 2.44 6.91

Asn

29.11 12.57 9.73 1.80

GlyGlnSerGluAsp

0.89

TyrProAlaArg

4.26

3.8511.693.9422.91

3.049.151.9411.40

Cys

0.68

1.05

0.511.21

77

3

Table 21. Interaction of cultivar and geographic location on the free (essential) amino acid composition of select almond seedsa


Cultivar County

Butte 5.63 ± 0.24 a 5.45 ± 0.67 a 8.42 ± 0.43 b 0.96 ± 0.06 bc 6.45 ± 0.29 b 3.80 ± 0.25 ab 6.27 ± 0.34 abc 3.42 ± 0.15 ab

Fresno 4.60 ± 0.83 ab 3.11 ± 0.25 cd 7.71 ± 1.14 b 3.29 ± 0.88 ab 12.42 ± 2.42 ab 2.53 ± 0.21 cd 5.94 ± 1.12 abc 3.61 ± 0.24 ab

Kern 4.30 ± 0.10 ab 3.21 ± 0.22 cd 9.60 ± 0.49 b 2.34 ± 0.35 abc 8.39 ± 0.65 b 3.47 ± 0.15 bc 7.89 ± 0.15 ab 3.61 ± 0.25 ab

Madera 5.85 ± 0.35 a 5.32 ± 0.29 a 8.66 ± 0.45 b 0.73 ± 0.49 c 6.41 ± 0.32 b 3.32 ± 0.18 bc 7.02 ± 0.44 ab 4.45 ± 0.34 a

Merced 2.95 ± 0.71 b 4.18 ± 0.14 b 13.01 ± 1.43 a 1.20 ± 0.18 abc 10.39 ± 0.38 b 4.52 ± 0.44 a 7.70 ± 0.44 ab 4.23 ± 0.24 ab

Sacramento 5.78 ± 0.86 a 2.77 ± 0.50 d 9.44 ± 0.98 b 3.37 ± 1.12 a 10.08 ± 0.63 b 3.68 ± 0.03 ab 8.15 ± 0.03 ab 3.52 ± 0.35 ab

San Joaquin 3.84 ± 0.89 ab 1.71 ± 0.03 e 2.65 ± 0.47 c 2.41 ± 0.34 abc 7.71 ± 0.34 b 3.06 ± 0.15 bc 3.66 ± 0.37 c 0.87 ± 0.07 d

Stanislaus 4.36 ± 0.08 ab 1.99 ± 0.06 e 7.55 ± 0.28 b 1.01 ± 0.13 bc 5.73 ± 0.17 b 3.88 ± 0.03 ab 6.30 ± 0.12 abc 2.25 ± 0.02 c

Tulare 2.22 ± 0.33 b 3.79 ± 0.48 bc 9.38 ± 0.38 b 2.81 ± 0.47 abc 6.83 ± 0.38 b 3.51 ± 0.24 b 5.09 ± 0.24 bc 3.68 ± 0.42 ab

Yolo 4.37 ± 1.34 ab 3.07 ± 0.32 cd 8.11 ± 1.28 b 3.26 ± 0.80 ab 19.02 ± 3.15 a 1.81 ± 0.56 d 9.05 ± 1.60 a 3.17 ± 0.59 bc

LSD

Butte 7.92 ± 0.21 ab 5.40 ± 0.14 c 8.83 ± 0.47 cde 3.47 ± 0.29 a 7.37 ± 0.63 c 4.32 ± 0.32 ab 6.80 ± 1.25 bc 3.92 ± 0.79 bcd

Colusa 9.19 ± 0.84 a 5.93 ± 0.79 bc 11.35 ± 0.59 ab 2.85 ± 0.30 ab 7.81 ± 0.31 c 3.62 ± 0.14 bc 7.21 ± 0.24 bc 3.37 ± 0.27 cd

Fresno 5.13 ± 0.83 c 4.84 ± 0.17 c 9.30 ± 0.88 bcd 3.43 ± 0.24 a 14.71 ± 3.52 bc 2.78 ± 0.15 cde 9.27 ± 0.53 a 5.19 ± 0.29 ab

Glenn 6.47 ± 0.35 bc 4.69 ± 0.25 cd 6.77 ± 0.06 e 1.57 ± 0.01 bcd 6.00 ± 0.10 c 2.86 ± 0.02 cde 4.59 ± 0.09 d 2.92 ± 0.19 cd

Kern 8.32 ± 0.90 ab 7.37 ± 1.24 b 12.48 ± 2.32 a 2.51 ± 0.84 abc 8.04 ± 0.83 c 3.59 ± 0.26 bc 8.38 ± 1.19 ab 5.14 ± 0.55 ab

Madera 9.65 ± 0.73 a 9.95 ± 1.66 a 10.56 ± 1.39 abcd 0.49 ± 0.09 d 6.29 ± 0.86 c 2.06 ± 0.62 e 6.81 ± 0.74 bc 3.48 ± 0.55 cd

Merced 5.50 ± 0.08 c 5.27 ± 0.11 c 9.62 ± 0.13 bcd 1.36 ± 0.66 cd 8.03 ± 0.48 c 3.20 ± 0.19 cd 5.40 ± 0.41 cd 5.17 ± 0.18 ab

Sacramento 6.84 ± 0.26 bc 4.98 ± 0.30 c 11.23 ± 0.46 abc 1.96 ± 0.33 bc 10.38 ± 0.30 c 3.31 ± 0.15 cd 9.81 ± 0.34 a 5.90 ± 0.49 a

San Joaquin 2.82 ± 0.46 d 2.64 ± 0.35 e 8.19 ± 0.45 de 3.51 ± 0.61 a 21.12 ± 5.87 b 3.54 ± 0.21 bc 9.26 ± 0.85 a 2.72 ± 0.46 d

Stanislaus 8.16 ± 0.64 ab 7.23 ± 0.54 b 9.81 ± 0.61 bcd 2.37 ± 0.74 abc 7.00 ± 0.39 c 3.02 ± 0.09 cde 6.64 ± 0.34 bc 4.24 ± 0.35 bc

Tulare 6.82 ± 0.42 bc 3.24 ± 0.31 de 1.95 ± 0.19 f 1.74 ± 0.35 bcd 5.53 ± 0.63 c 4.86 ± 1.42 a 4.43 ± 0.82 d 1.18 ± 0.16 e

Yolo 2.44 ± 0.13 d 5.91 ± 0.76 bc 8.16 ± 0.34 de 2.65 ± 0.22 abc 33.12 ± 4.89 a 2.55 ± 0.09 de 9.25 ± 0.76 a 5.14 ± 0.21 ab

LSD

Butte 8.81 ± 0.13 ab 4.93 ± 0.12 a 10.80 ± 0.58 a 2.71 ± 0.72 abc 7.07 ± 0.31 b 3.72 ± 0.13 a 8.81 ± 0.25 a 4.99 ± 0.19 ab

Colusa 6.29 ± 0.75 bcd 4.30 ± 0.47 ab 9.50 ± 0.70 ab 2.15 ± 0.53 bcd 6.86 ± 0.26 b 3.12 ± 0.18 ab 7.87 ± 0.31 ab 3.65 ± 0.12 cd

Fresno 6.83 ± 0.79 abc 3.84 ± 0.24 bc 9.50 ± 0.66 ab 1.55 ± 0.25 cd 6.29 ± 0.23 b 2.74 ± 0.11 b 7.81 ± 0.44 ab 4.28 ± 0.25 bcd

Glenn 8.83 ± 1.51 a 5.27 ± 0.64 a 11.48 ± 1.86 a 3.56 ± 1.54 ab 6.77 ± 0.22 b 2.69 ± 0.10 b 7.37 ± 0.39 ab 4.44 ± 0.57 abc

Kern 5.50 ± 0.39 cde 2.85 ± 0.01 c 8.10 ± 0.22 bc 4.13 ± 0.47 a 5.72 ± 0.16 b 2.55 ± 0.07 b 7.06 ± 0.27 b 3.50 ± 0.32 de

Merced 3.73 ± 0.12 e 2.99 ± 0.35 c 6.70 ± 0.62 c 0.94 ± 0.25 d 4.93 ± 0.20 b 2.88 ± 0.40 b 4.82 ± 0.45 c 2.71 ± 0.22 e

San Joaquin 6.15 ± 0.41 cde 4.52 ± 0.27 ab 7.83 ± 0.39 bc 0.84 ± 0.09 d 7.07 ± 0.28 b 2.71 ± 0.13 b 7.85 ± 0.31 ab 5.21 ± 0.40 a

Stanislaus 3.78 ± 0.93 de 3.86 ± 0.33 bc 9.07 ± 0.84 abc 1.17 ± 0.17 cd 20.44 ± 6.15 a 1.61 ± 0.25 c 6.49 ± 0.77 bc 3.81 ± 0.18 cd

LSD

Lys

2.67 0.97 3.25 2.34 6.70 0.97 3.23 1.08

Val Met Ile Leu Phe

0.841.06 2.63 1.70 12.20 0.68 1.75

Mission

10.041.312.481.531.94

2.53

Carmel

1.381.980.99

Butte

His Thr

78

4

Table 21 continued. Interaction of cultivar and geographic location on the free (essential) amino acid composition of select almond seedsa


Cultivar County

Butte 4.93 ± 0.44 abc 4.33 ± 0.45 b 8.69 ± 0.62 c 0.46 ± 0.04 g 6.96 ± 0.16 b 4.56 ± 0.15 abcd 6.61 ± 0.37 cd 3.22 ± 0.10 b

Colusa 3.57 ± 0.32 bcd 4.30 ± 0.96 b 9.79 ± 0.47 bc 0.79 ± 0.10 efg 7.37 ± 0.45 b 5.14 ± 0.30 abc 7.36 ± 0.48 bcd 3.38 ± 0.02 b

Fresno 4.84 ± 1.29 abc 4.79 ± 0.45 b 9.44 ± 0.60 bc 3.25 ± 0.72 ab 17.56 ± 4.76 ab 3.84 ± 0.11 bcd 9.22 ± 1.01 bc 5.14 ± 0.84 a

Glenn 4.92 ± 0.58 abc 3.78 ± 0.34 bcd 7.94 ± 0.61 c 0.61 ± 0.08 fg 6.92 ± 0.42 b 4.27 ± 0.28 abcd 7.09 ± 0.63 bcd 3.19 ± 0.30 b

Kern 6.51 ± 0.48 a 3.76 ± 0.45 bcd 9.66 ± 0.68 bc 3.78 ± 1.01 a 7.83 ± 0.16 b 4.61 ± 0.40 abcd 9.06 ± 0.26 bcd 3.75 ± 0.50 ab

Madera 6.48 ± 0.61 a 6.18 ± 0.40 a 11.57 ± 0.74 ab 2.25 ± 0.84 bcd 8.14 ± 0.40 b 4.36 ± 0.19 abcd 12.67 ± 1.06 a 4.25 ± 0.12 ab

Merced 3.50 ± 0.16 cd 2.99 ± 0.13 cd 10.44 ± 0.36 bc 1.84 ± 0.78 cdef 8.19 ± 0.25 b 4.81 ± 0.07 abcd 6.80 ± 0.12 cd 3.83 ± 0.16 ab

Sacramento 6.31 ± 0.54 a 4.33 ± 0.39 b 12.27 ± 0.76 ab 1.49 ± 0.19 defg 11.46 ± 0.48 b 5.26 ± 0.19 ab 13.09 ± 0.74 a 5.11 ± 0.36 a

San Joaquin 5.02 ± 0.70 abc 2.63 ± 0.26 d 1.64 ± 0.40 d 1.46 ± 0.24 defg 7.58 ± 0.39 b 5.59 ± 2.29 a 6.40 ± 0.67 d 1.04 ± 0.05 c

Stanislaus 1.93 ± 0.15 d 3.52 ± 0.32 bcd 8.35 ± 0.75 c 2.23 ± 0.22 bcd 24.48 ± 5.12 a 3.35 ± 0.38 d 8.53 ± 1.12 bcd 3.96 ± 0.42 ab

Tulare 5.76 ± 0.70 ab 4.18 ± 0.58 bc 13.96 ± 1.54 a 2.92 ± 0.16 abc 9.56 ± 0.56 b 4.65 ± 0.20 abcd 9.84 ± 0.65 b 4.04 ± 0.53 ab

Yolo 3.84 ± 1.03 bcd 3.91 ± 0.32 bcd 8.70 ± 1.56 c 2.08 ± 0.33 bcde 16.43 ± 2.60 ab 3.73 ± 0.43 cd 9.69 ± 0.51 b 4.27 ± 0.58 ab

LSD

Butte 4.56 ± 0.08 a 2.83 ± 0.06 b 5.89 ± 0.11 d 0.58 ± 0.06 b 5.66 ± 0.13 e 3.07 ± 0.03 c 5.08 ± 0.19 cd 3.21 ± 0.06 ab

Fresno 4.08 ± 0.12 a 2.34 ± 0.08 b 8.57 ± 0.16 ab 0.51 ± 0.00 b 7.48 ± 0.08 b 4.74 ± 0.06 b 10.10 ± 0.45 a 3.32 ± 0.02 ab

Kern 5.81 ± 1.61 a 3.09 ± 0.81 ab 6.92 ± 0.95 cd 0.68 ± 0.40 b 6.06 ± 0.09 d 3.11 ± 0.05 c 4.37 ± 0.47 d 3.75 ± 1.01 a

Merced 4.78 ± 0.27 a 2.69 ± 0.29 b 7.79 ± 0.14 bc 1.00 ± 0.15 ab 7.91 ± 0.18 a 4.39 ± 0.10 b 5.60 ± 0.00 bc 3.36 ± 0.19 ab

San Joaquin 5.12 ± 0.19 a 4.06 ± 0.18 a 8.78 ± 0.08 ab 1.10 ± 0.31 ab 6.34 ± 0.03 d 4.46 ± 0.04 b 6.02 ± 0.21 b 2.44 ± 0.08 b

Stanislaus 5.05 ± 0.33 a 2.91 ± 0.23 b 8.98 ± 0.15 a 1.56 ± 0.26 a 8.06 ± 0.04 a 4.48 ± 0.05 b 6.16 ± 0.30 b 2.81 ± 0.26 ab

Tulare 4.75 ± 0.26 a 2.40 ± 0.13 b 9.07 ± 0.10 a 0.60 ± 0.09 b 7.13 ± 0.08 c 6.33 ± 0.97 a 6.05 ± 0.27 b 3.86 ± 0.21 a

LSD

Butte 4.76 ± 1.12 b 7.00 ± 0.54 ab 11.21 ± 0.38 a 0.76 ± 0.04 c 8.28 ± 0.22 a 4.90 ± 0.26 a 8.73 ± 0.17 a 4.49 ± 0.33 b

Fresno 3.82 ± 0.16 b 5.18 ± 0.37 c 8.59 ± 0.24 b 1.16 ± 0.24 c 7.28 ± 0.17 a 3.69 ± 0.25 bc 7.50 ± 0.40 a 4.67 ± 0.74 ab

Kern 4.27 ± 0.21 bc 6.77 ± 0.37 ab 11.57 ± 1.30 a 1.16 ± 0.15 c 7.57 ± 0.60 a 3.36 ± 0.24 c 8.13 ± 1.14 a 4.15 ± 0.38 b

Merced 4.57 ± 0.56 b 3.44 ± 0.62 d 2.24 ± 0.18 d 3.93 ± 0.37 a 5.72 ± 1.87 ab 3.51 ± 0.43 bc 5.02 ± 0.63 b 0.68 ± 0.14 c

San Joaquin 6.86 ± 0.29 a 6.09 ± 0.59 bc 9.68 ± 1.27 ab 0.62 ± 0.16 c 7.43 ± 0.72 a 4.32 ± 0.48 ab 7.99 ± 0.92 a 4.38 ± 0.46 b

Stanislaus 2.54 ± 0.72 c 1.42 ± 0.63 e 5.64 ± 0.69 c 2.22 ± 0.53 b 2.99 ± 1.62 b 2.32 ± 0.12 d 2.05 ± 0.57 c 0.22 ± 0.06 c

Tulare 8.19 ± 0.50 a 8.00 ± 0.50 a 11.87 ± 0.44 a 1.39 ± 0.11 c 7.15 ± 0.08 a 3.72 ± 0.07 bc 8.85 ± 0.44 a 5.83 ± 0.47 a

LSD

Phe Lys

2.24 1.29 2.86 1.30 10.87 1.50 2.81 1.55

Thr Val Met Ile Leu

Nonpareil

Padre

His

Price

1.200.901.090.300.661.111.02

2.28 0.82 2.96 0.88 2.00 1.25

1.89

1.76 1.55

79

5

Table 22. Interaction of cultivar and geographic location on the free (non-essential) amino acid composition of select almond seedsa


Cultivar County

Butte 182.67 ± 10.60 b 113.48 ± 5.13 a 74.37 ± 4.15 a 12.99 ± 0.89 ab 14.35 ± 0.13 abc 4.93 ± 0.22 bc 62.71 ± 3.75 bc 18.60 ± 0.91 a 38.54 ± 2.02 bc 14.87 ± 0.88 abc 0.06 ± 0.00 d

Fresno 92.84 ± 12.51 c 30.83 ± 2.43 e 45.50 ± 4.67 c 11.14 ± 1.33 ab 11.83 ± 0.97 abcd 5.43 ± 0.66 bc 66.35 ± 6.72 b 12.97 ± 1.17 c 59.20 ± 4.85 a 10.75 ± 2.57 bcd 1.75 ± 0.52 abcd

Kern 119.88 ± 5.19 c 43.97 ± 2.72 d 61.59 ± 2.92 abc 12.03 ± 0.75 ab 11.26 ± 1.94 abcd 6.33 ± 0.32 abc 52.02 ± 2.73 bc 16.74 ± 0.71 abc 57.03 ± 4.87 a 11.20 ± 1.18 bcd 2.97 ± 0.10 ab

Madera 175.20 ± 2.90 b 33.80 ± 0.87 de 53.16 ± 2.28 bc 10.87 ± 1.21 b 15.63 ± 0.87 a 6.35 ± 0.21 abc 97.56 ± 6.27 a 13.30 ± 0.35 c 50.35 ± 1.44 ab 2.16 ± 0.16 f 0.00 ± 0.00 d

Merced 88.44 ± 5.59 c 70.55 ± 5.49 c 72.60 ± 2.97 a 12.12 ± 0.83 ab 8.00 ± 0.67 d 6.61 ± 0.50 abc 46.83 ± 5.07 bcd 17.93 ± 0.80 ab 28.73 ± 1.59 c 6.18 ± 0.28 def 0.53 ± 0.05 cd

Sacramento 98.43 ± 8.66 c 29.86 ± 2.70 e 64.30 ± 4.12 ab 12.85 ± 0.21 ab 13.77 ± 0.90 ab 8.27 ± 0.80 a 52.11 ± 2.27 bc 17.99 ± 0.44 ab 62.17 ± 0.87 a 18.61 ± 1.61 a 3.16 ± 0.10 a

San Joaquin 77.08 ± 3.68 c 9.38 ± 0.49 f 13.16 ± 1.09 d 6.30 ± 0.52 c 7.94 ± 0.74 d 3.93 ± 0.82 c 27.68 ± 2.11 d 5.70 ± 0.07 d 55.68 ± 1.10 a 3.60 ± 0.19 ef 1.13 ± 0.01 bcd

Stanislaus 91.72 ± 5.14 c 93.90 ± 4.98 b 68.93 ± 5.59 ab 14.57 ± 0.34 a 9.80 ± 0.29 bcd 5.26 ± 0.37 bc 40.92 ± 5.50 cd 14.36 ± 0.44 bc 32.22 ± 1.56 c 17.15 ± 0.87 ab 0.34 ± 0.16 cd

Tulare 242.37 ± 22.63 a 67.05 ± 3.74 c 72.44 ± 4.56 a 11.40 ± 0.62 ab 8.60 ± 1.19 cd 4.42 ± 0.41 c 96.66 ± 16.55 a 19.95 ± 1.51 a 39.63 ± 3.16 bc 4.36 ± 0.23 def 0.71 ± 0.20 cd

Yolo 88.05 ± 26.17 c 30.98 ± 5.67 e 52.58 ± 10.47 bc 11.75 ± 1.29 ab 12.88 ± 2.46 abc 7.50 ± 1.64 ab 66.80 ± 10.70 b 13.45 ± 2.02 c 58.18 ± 6.35 a 9.60 ± 3.11 cde 2.31 ± 1.24 abc

LSD

Butte 198.23 ± 2.44 c 50.95 ± 2.58 cd 66.93 ± 4.87 cde 13.86 ± 0.66 bcd 15.18 ± 0.63 ab 7.98 ± 0.24 bcd 69.88 ± 1.22 bcd 15.28 ± 0.52 e 54.27 ± 4.70 bcd 4.76 ± 0.38 cd 4.08 ± 0.27 a

Colusa 266.22 ± 11.48 b 78.09 ± 4.66 b 87.01 ± 8.80 a 18.14 ± 1.84 a 16.30 ± 0.41 a 9.77 ± 1.06 ab 80.45 ± 8.80 b 22.77 ± 2.28 ab 43.31 ± 3.12 de 5.04 ± 0.44 cd 0.13 ± 0.13 de

Fresno 123.84 ± 12.73 e 38.28 ± 5.14 de 54.19 ± 3.98 efg 11.09 ± 1.05 cdef 12.01 ± 0.90 de 6.76 ± 0.34 cde 58.15 ± 2.68 de 17.30 ± 0.54 cde 48.00 ± 3.62 cde 5.69 ± 1.47 bcd 1.99 ± 0.56 bc

Glenn 182.32 ± 8.20 cd 58.94 ± 3.85 c 70.93 ± 2.04 cd 12.54 ± 1.18 bcde 14.45 ± 0.44 abc 8.78 ± 0.39 b 59.02 ± 2.68 cde 15.62 ± 0.53 e 55.62 ± 0.96 bc 2.31 ± 0.02 d 0.00 ± 0.00 e

Kern 189.64 ± 16.71 c 50.11 ± 6.91 cde 65.93 ± 5.64 cde 14.66 ± 3.38 abc 10.79 ± 0.53 e 8.56 ± 1.60 bc 75.16 ± 7.18 b 20.00 ± 2.12 bc 51.44 ± 6.01 cd 6.63 ± 1.02 bc 0.69 ± 0.06 cde

Madera 325.02 ± 41.61 a 60.70 ± 2.10 c 84.64 ± 3.71 ab 8.40 ± 1.14 f 16.00 ± 1.12 a 11.52 ± 1.00 a 71.46 ± 8.68 bc 19.12 ± 1.09 cd 71.64 ± 1.93 a 5.44 ± 1.17 bcd 0.00 ± 0.00 e

Merced 147.22 ± 1.32 de 37.24 ± 1.13 e 58.38 ± 1.03 def 9.94 ± 0.65 def 12.55 ± 0.88 cde 6.02 ± 0.06 e 53.06 ± 3.13 e 16.76 ± 0.51 de 68.28 ± 3.07 a 4.01 ± 0.75 cd 0.00 ± 0.00 e

Sacramento 201.14 ± 10.33 c 49.04 ± 2.00 cde 71.96 ± 2.74 bc 15.28 ± 1.10 ab 15.60 ± 0.48 ab 9.90 ± 0.73 ab 76.29 ± 2.26 b 22.15 ± 0.91 ab 55.16 ± 1.94 bc 9.16 ± 0.53 b 2.23 ± 0.26 bc

San Joaquin 50.33 ± 7.23 f 40.41 ± 5.15 de 46.47 ± 3.33 fg 9.26 ± 1.29 ef 10.79 ± 0.70 e 5.06 ± 0.16 e 27.95 ± 4.50 f 12.18 ± 0.55 f 55.35 ± 5.00 bc 4.10 ± 0.17 cd 1.63 ± 0.73 bcd

Stanislaus 180.16 ± 11.06 cd 120.95 ± 5.16 a 87.66 ± 6.31 a 14.05 ± 0.50 bc 12.72 ± 0.60 cde 6.24 ± 0.31 de 129.43 ± 8.06 a 23.19 ± 1.26 a 25.14 ± 1.80 f 13.69 ± 3.69 a 2.86 ± 0.05 ab

Tulare 114.90 ± 4.08 e 11.20 ± 1.94 f 23.91 ± 6.57 h 9.40 ± 0.83 ef 6.75 ± 0.12 f 9.71 ± 0.20 ab 36.63 ± 2.73 f 7.73 ± 0.77 g 39.44 ± 2.31 e 4.96 ± 1.19 cd 2.40 ± 0.95 b

Yolo 107.45 ± 7.64 e 19.66 ± 1.46 f 44.72 ± 3.11 g 11.85 ± 0.88 bcdef 13.60 ± 0.69 bcd 6.28 ± 0.34 de 77.03 ± 4.19 b 16.41 ± 0.96 de 65.29 ± 2.59 ab 4.76 ± 0.14 cd 0.06 ± 0.06 e

LSD

Butte 130.70 ± 6.61 a 57.50 ± 3.74 ab 62.68 ± 10.96 ab 15.63 ± 0.41 ab 9.62 ± 0.26 bc 10.22 ± 0.87 a 57.77 ± 4.45 ab 19.17 ± 1.18 a 38.75 ± 0.75 b 4.06 ± 0.32 c 0.00 ± 0.00 d

Colusa 103.43 ± 12.39 a 44.68 ± 3.82 bcd 53.67 ± 8.04 bcd 11.29 ± 0.98 cd 9.57 ± 1.06 bc 5.93 ± 0.48 bc 47.84 ± 4.74 b 18.68 ± 1.67 a 43.60 ± 3.86 b 3.94 ± 0.26 c 1.03 ± 0.76 bc

Fresno 136.82 ± 17.42 a 32.13 ± 4.06 d 38.95 ± 4.82 d 8.30 ± 0.53 d 5.27 ± 0.50 d 7.17 ± 0.90 b 71.14 ± 6.80 a 14.80 ± 1.96 b 44.55 ± 2.82 b 3.21 ± 0.19 c 0.25 ± 0.13 cd

Glenn 147.11 ± 19.05 a 64.02 ± 4.83 a 72.87 ± 3.39 a 16.15 ± 2.24 a 12.09 ± 1.56 ab 10.18 ± 1.32 a 51.44 ± 2.61 ab 14.66 ± 0.54 b 40.92 ± 0.86 b 3.19 ± 0.31 c 0.00 ± 0.00 d

Kern 113.38 ± 5.78 a 37.89 ± 1.52 cd 43.60 ± 3.07 c 8.52 ± 0.37 d 7.33 ± 0.83 cd 5.60 ± 0.30 bc 68.33 ± 11.53 ab 13.47 ± 0.36 bc 38.79 ± 2.08 b 6.62 ± 0.10 b 2.10 ± 0.20 a

Merced 124.87 ± 9.17 a 29.59 ± 1.20 d 41.02 ± 2.40 d 9.20 ± 0.70 d 10.86 ± 1.30 ab 4.30 ± 0.29 c 51.80 ± 6.34 ab 10.58 ± 0.82 c 40.29 ± 3.21 b 1.70 ± 0.22 d 0.00 ± 0.00 d

San Joaquin 123.86 ± 8.78 a 38.12 ± 1.37 cd 51.57 ± 1.84 bcd 10.03 ± 0.50 cd 13.20 ± 0.90 a 5.10 ± 0.37 c 62.93 ± 2.87 ab 18.17 ± 0.90 a 63.41 ± 3.40 a 20.25 ± 0.51 a 1.71 ± 0.32 ab

Stanislaus 134.06 ± 31.42 a 50.72 ± 7.87 abc 57.37 ± 2.79 bc 12.50 ± 1.14 bc 11.22 ± 0.73 ab 5.33 ± 0.23 c 65.38 ± 6.67 ab 16.15 ± 0.85 ab 49.73 ± 5.87 b 4.03 ± 0.65 c 0.24 ± 0.10 d

LSD

Carmel

Mission

2.78

Asn Asp Glu Ser

Butte

Gln Gly

18.6511.6547.59

41.34 13.47 12.96

13.24

3.9923.72

3.2619.81

11.08

Cys

2.00

1.55

0.79

Tyr

7.12

3.73

1.47

Arg Ala Pro

4.853.63 15.10

4.04 2.33 1.94 12.76 2.93

68.67 1.802.773.2914.7617.37

80

6

Table 22 continued. Interaction of cultivar and geographic location on the free (non-essential) amino acid composition of select almond seedsa


Cultivar County

Butte 88.93 ± 3.14 cde 43.87 ± 8.86 abc 68.21 ± 2.57 abc 14.92 ± 1.45 abcd 10.06 ± 1.16 b 6.71 ± 0.19 cd 28.17 ± 1.58 def 20.18 ± 0.81 bcd 56.64 ± 7.62 ab 20.96 ± 0.08 a 2.03 ± 0.07 b

Colusa 119.20 ± 12.20 abcd 38.04 ± 3.67 bcd 61.28 ± 4.08 abc 12.37 ± 1.32 cdef 10.31 ± 1.82 b 5.38 ± 0.72 cd 30.60 ± 3.70 cde 16.64 ± 0.94 cd 66.74 ± 4.23 a 6.18 ± 0.34 b 1.03 ± 0.16 cd

Fresno 147.82 ± 31.82 a 33.85 ± 6.90 bcd 57.05 ± 6.12 abc 11.24 ± 0.77 def 11.00 ± 1.25 b 7.43 ± 0.46 bcd 52.38 ± 4.48 a 17.32 ± 1.40 bcd 50.56 ± 7.03 bc 3.59 ± 0.27 cde 0.00 ± 0.00 e

Glenn 58.89 ± 5.99 e 22.85 ± 1.93 de 53.81 ± 3.36 bc 13.07 ± 0.93 bcdef 9.10 ± 1.02 b 7.06 ± 0.32 bcd 16.40 ± 1.99 fg 19.23 ± 0.84 bcd 36.27 ± 2.81 d 19.31 ± 1.50 a 1.94 ± 0.12 b

Kern 111.83 ± 8.50 abcd 51.02 ± 4.23 ab 78.27 ± 4.63 a 14.17 ± 0.31 abcde 8.70 ± 0.73 b 6.74 ± 0.21 cd 29.48 ± 2.38 cdef 16.23 ± 1.49 d 35.14 ± 1.59 d 5.12 ± 0.86 bcd 1.63 ± 0.82 bc

Madera 138.31 ± 14.31 abc 45.92 ± 1.10 abc 74.67 ± 1.24 ab 15.41 ± 1.98 abc 11.35 ± 1.75 b 11.64 ± 4.08 a 36.50 ± 8.81 bcd 26.98 ± 0.98 a 40.89 ± 0.33 cd 3.12 ± 0.09 de 0.00 ± 0.00 e

Merced 90.40 ± 4.82 bcde 62.72 ± 3.50 a 60.24 ± 2.61 abc 11.28 ± 0.07 def 9.87 ± 0.30 b 4.76 ± 0.13 d 18.79 ± 1.17 efg 17.37 ± 0.53 bcd 34.35 ± 1.06 d 4.61 ± 0.11 bcd 0.73 ± 0.18 de

Sacramento 140.39 ± 12.94 ab 44.97 ± 3.89 abc 76.72 ± 6.16 a 16.76 ± 1.26 ab 16.31 ± 1.30 a 10.50 ± 1.28 ab 45.61 ± 3.83 ab 22.17 ± 1.72 ab 50.68 ± 3.36 bc 5.78 ± 0.47 bc 2.34 ± 0.23 ab

San Joaquin 56.31 ± 8.14 e 13.74 ± 3.17 e 29.44 ± 10.45 d 10.64 ± 0.72 ef 8.29 ± 0.65 b 7.31 ± 0.48 bcd 12.38 ± 1.66 g 8.02 ± 0.73 e 36.41 ± 2.33 d 4.50 ± 0.64 bcd 1.08 ± 0.10 cd

Stanislaus 55.46 ± 5.94 e 34.61 ± 6.83 bcd 49.93 ± 4.38 cd 9.84 ± 0.84 f 7.93 ± 0.79 b 5.39 ± 0.49 cd 27.25 ± 4.60 def 14.82 ± 1.28 d 32.78 ± 2.43 d 4.77 ± 0.80 bcd 0.03 ± 0.02 e

Tulare 130.92 ± 14.05 abcd 60.67 ± 6.24 a 73.60 ± 7.15 ab 17.37 ± 0.87 a 9.18 ± 0.82 b 8.71 ± 0.48 abc 42.47 ± 3.47 abc 21.92 ± 1.70 abc 32.29 ± 1.06 d 1.78 ± 0.03 e 3.06 ± 0.18 a

Yolo 87.66 ± 18.60 de 30.61 ± 7.68 cde 59.42 ± 12.67 abc 12.22 ± 1.85 cdef 10.86 ± 1.49 b 6.40 ± 1.13 cd 44.20 ± 3.44 ab 16.77 ± 2.91 bcd 34.10 ± 3.06 d 4.85 ± 1.23 bcd 0.86 ± 0.39 d

LSD

Butte 96.53 ± 3.49 b 31.43 ± 1.15 c 53.56 ± 1.21 abc 13.20 ± 0.69 a 11.02 ± 0.53 a 5.45 ± 0.03 ab 34.92 ± 3.61 ab 12.28 ± 0.37 ab 36.29 ± 2.09 b 19.43 ± 1.90 a 2.05 ± 0.13 b

Fresno 86.94 ± 3.27 b 42.21 ± 0.65 bc 53.47 ± 0.52 abc 12.03 ± 0.29 a 10.86 ± 0.58 a 4.93 ± 0.09 b 23.71 ± 2.99 bc 13.96 ± 0.40 ab 42.24 ± 0.77 b 2.17 ± 0.04 c 0.00 ± 0.00 d

Kern 173.53 ± 44.35 a 52.91 ± 13.88 b 64.10 ± 17.00 ab 9.52 ± 4.25 a 8.78 ± 2.37 abc 7.69 ± 2.13 a 43.97 ± 13.56 a 11.82 ± 2.56 b 66.23 ± 13.38 a 6.94 ± 1.87 b 0.00 ± 0.00 d

Merced 63.14 ± 2.94 b 30.88 ± 1.71 c 38.31 ± 1.61 c 11.07 ± 0.83 a 5.89 ± 0.37 c 6.14 ± 0.38 ab 20.70 ± 2.45 bc 14.06 ± 0.63 ab 31.09 ± 0.52 b 18.05 ± 0.54 a 1.93 ± 0.06 b

San Joaquin 97.40 ± 2.75 b 101.86 ± 2.42 a 68.44 ± 0.41 a 13.40 ± 0.42 a 8.77 ± 0.08 abc 4.81 ± 0.05 b 24.83 ± 1.18 bc 14.97 ± 0.08 a 28.33 ± 0.55 b 6.78 ± 2.05 b 2.97 ± 0.17 a

Stanislaus 70.83 ± 3.13 b 40.79 ± 2.06 bc 43.27 ± 2.76 c 9.74 ± 0.44 a 9.96 ± 0.60 ab 4.60 ± 0.26 b 19.21 ± 0.69 bc 12.66 ± 0.59 ab 40.79 ± 1.52 b 5.07 ± 0.57 bc 0.60 ± 0.04 c

Tulare 70.98 ± 1.45 b 32.68 ± 1.81 c 45.32 ± 2.25 bc 9.47 ± 0.36 a 7.24 ± 0.47 bc 5.61 ± 0.29 ab 17.94 ± 0.61 c 12.07 ± 0.45 ab 43.23 ± 1.15 b 16.69 ± 0.25 a 1.83 ± 0.22 b

LSD

Butte 144.17 ± 11.25 a 40.92 ± 0.97 bc 68.67 ± 3.94 a 14.34 ± 0.58 a 13.64 ± 0.27 ab 8.30 ± 0.52 ab 60.23 ± 3.00 cd 18.19 ± 0.55 abc 49.86 ± 2.81 b 9.27 ± 1.65 a 0.57 ± 0.07 c

Fresno 91.72 ± 2.55 b 34.50 ± 0.66 c 51.98 ± 2.03 b 7.22 ± 0.39 c 9.20 ± 0.37 cd 5.07 ± 0.42 bc 45.99 ± 3.77 cd 15.87 ± 0.41 c 57.48 ± 2.12 ab 5.05 ± 0.19 bc 0.87 ± 0.05 c

Kern 162.95 ± 17.77 a 46.15 ± 1.73 b 62.06 ± 3.64 a 9.99 ± 0.81 bc 8.91 ± 1.58 de 7.21 ± 0.17 abc 65.74 ± 8.20 bc 16.88 ± 0.57 b 48.76 ± 2.51 b 5.71 ± 0.92 b 0.85 ± 0.14 c

Merced 139.99 ± 29.92 a 10.45 ± 0.54 d 22.30 ± 3.54 c 7.81 ± 1.30 c 9.02 ± 0.94 cd 10.17 ± 3.93 a 84.52 ± 17.60 b 7.18 ± 1.60 d 33.56 ± 4.66 c 4.76 ± 0.62 bcd 1.43 ± 0.11 b

San Joaquin 182.57 ± 7.84 a 39.71 ± 3.50 bc 65.11 ± 2.03 a 12.83 ± 1.88 ab 15.17 ± 0.76 a 8.15 ± 0.30 ab 115.60 ± 1.12 a 19.27 ± 1.31 ab 62.31 ± 5.28 a 2.63 ± 0.23 d 2.54 ± 0.14 a

Stanislaus 57.19 ± 4.41 b 4.81 ± 1.10 d 9.22 ± 0.34 d 3.48 ± 0.98 d 5.73 ± 1.65 e 3.47 ± 0.28 c 36.24 ± 7.44 d 3.04 ± 0.94 e 26.14 ± 7.29 c 2.81 ± 0.27 cd 0.74 ± 0.37 c

Tulare 179.33 ± 6.86 a 110.44 ± 4.55 a 64.70 ± 3.94 a 8.47 ± 0.32 c 11.98 ± 0.64 bc 5.56 ± 0.31 bc 122.57 ± 3.76 a 20.56 ± 0.82 a 34.42 ± 1.32 c 4.81 ± 0.10 bcd 0.05 ± 0.00 d

LSD

0.363.8515.25

2.26 0.49

Tyr Cys

12.29 2.36 0.75

Padre

3.81 3.48

Price

42.66 6.93 8.97 3.03

2.934.9519.4516.1449.94

3.00 4.47

2.44

Nonpareil

50.35 19.19 21.97 3.82

24.12 2.87 12.34

Asn Asp Glu Ser Gln Pro

3.1116.27

Gly Arg Ala

13.42 5.43

81

7

aValues in bold are significant (r � 0.273 and r � -0.273) based on critical values table for Pearson’s correlation coefficients (n = 58, p � 0.05)

Table 23. Correlation coefficients among free amino acids of select almond seeds a

Asn Asp Glu Ser Gln Gly His Arg Thr Ala Pro Tyr Val Met Cys Ile Leu Phe Lys

Asn 1.000Asp 0.449 1.000Glu 0.584 0.732 1.000Ser 0.265 0.433 0.737 1.000Gln 0.560 0.254 0.547 0.447 1.000Gly 0.454 -0.010 0.372 0.467 0.358 1.000His 0.626 0.402 0.452 0.399 0.352 0.601 1.000Arg 0.678 0.359 0.309 0.076 0.496 0.164 0.374 1.000Thr 0.726 0.436 0.599 0.275 0.545 0.466 0.569 0.602 1.000Ala 0.484 0.554 0.836 0.689 0.471 0.371 0.418 0.356 0.637 1.000Pro 0.320 -0.245 0.101 -0.052 0.482 0.153 0.128 0.179 0.262 0.120 1.000Tyr -0.192 0.115 0.107 0.174 -0.015 -0.138 0.021 -0.150 -0.142 0.131 0.019 1.000Val 0.370 0.502 0.745 0.545 0.333 0.232 0.299 0.269 0.542 0.800 0.069 -0.071 1.000Met -0.022 -0.123 -0.076 0.064 0.023 0.168 0.001 0.183 -0.108 -0.052 -0.097 -0.293 0.009 1.000Cys -0.132 -0.037 0.020 0.195 0.004 0.044 0.123 -0.019 -0.177 0.049 0.009 0.397 -0.051 0.185 1.000Ile -0.230 -0.221 -0.068 0.004 0.217 -0.075 -0.486 0.057 -0.013 0.011 0.123 -0.183 0.060 0.369 -0.181 1.000

Leu -0.208 0.063 0.103 0.277 -0.211 0.093 0.056 -0.439 -0.119 0.115 -0.232 0.141 0.069 -0.316 0.253 -0.376 1.000Phe 0.022 0.096 0.444 0.497 0.351 0.401 0.120 0.063 0.330 0.633 0.081 -0.131 0.605 0.185 0.036 0.354 0.175 1.000Lys 0.326 0.297 0.572 0.383 0.428 0.210 0.271 0.383 0.570 0.713 0.280 0.001 0.745 -0.020 -0.106 0.219 -0.060 0.632 1.000

82

Electrophoresis Analysis

SDS-PAGE in the presence of 2% �-ME electrophoretic profiles of almond and

macadamia nut seed proteins are shown in Figures 7A and 8A.

Almond. SDS-PAGE analysis of almond seed proteins stained with Coomassie revealed

a range of polypeptides between 10-80 kDa with the 2 major subunits of AMP (estimated MWs

of 42-46 and 20-22 kDa) present in all tested almond seeds. Polypeptide profiles of tested

almond seeds were consistent with profiles previously reported for cultivar Nonpareil (170) and

almond genotypes (49). In select almond seeds, several prominent polypeptides were present

between 22 and 42 kDa. For instance, cultivar Mission (Figure 7A, labeled 3A-J) regardless of

growth location and/or harvest year has a prominent polypeptide with an estimated MW of 37-39

kDa that is not consistently present in the other tested cultivars. Several dominate polypeptides

are also present between 25 and 27 kDa in select almond seeds (i.e. 4E and 4J in Figure 7A).

Macadamia nut. Cultivar variation was also observed in Coomassie and silver stained

SDS-PAGE profiles of macadamia nut cultivars (Figure 8, A and B). The polypeptide profile of

macadamia nut proteins reveal a range of polypeptides between 6-80 kDa with several major

polypeptides with estimated MWs of 48-51, 20-21, 17-18, 14-15, and 6-10 kDa (Figure 8A).

Cultivar profiles differed mainly in the low molecular weight peptides, in particular the 22 kDa

polypeptide in Blue Diamond variety and the 17-18 kDa polypeptide in cultivars Keaau and

Mauka (Figure 8, A and B). The predominate polypeptide at 48-51 kDa stained with the

glycoprotein staining procedure, indicating the polypeptide is a glycoprotein (Figure 8C). All

tested macadamia nut cultivars appear to contain this glycoprotein. A glycoprotein is a protein

that attaches to a carbohydrate usually during a co- or post-translational modification. The

carbohydrate group may assist in the folding or improved stability of the protein. Glycoproteins

in several tree nuts, the 27 and 55 kDa polypeptides in almonds (236) and the 48 kDa

polypeptide in hazelnut (237), have also been identified.

ELISA

Almond. In order to assess immunoreactivity of almond seed proteins an anti-AMP mAb

(4C10)-based sandwich ELISA was developed to standardize an AMP standard curve and

previously standardized pAb-based inhibition ELISAs (137, 138, 194) for detection of AMP and

total soluble almond proteins were used.

83

The ELISA results suggested the major commercial almond cultivar Nonpareil Supreme

(the source of proteins for antibody production) had ~52.8% AMP (mAb 4C10 sandwich ELISA)

and ~63.7% AMP (rabbit anti-AMP inhibition ELISA). Immunoreactivity of AMP (assessed by

mAb 4C10 sandwich ELISA) was influenced by cultivar, with cultivars Butte, Carmel,

Nonpareil, and Monterey ranged from 49.1-69.8%, while cultivars Mission, Padre, Price, and

Sonora had a lower range of 22.2-29.6% (Table 24). Similar immunoreactivities were found

using the rabbit anti-AMP inhibition ELISA, 58.4-88.8% for cultivars Butte, Carmel, Nonpareil,

and Monterey and 28.3-46.1% for cultivars Mission, Padre, Price, and Sonora (Table 24).

Consistent with the average AMP immunoreactivity (74.0 ± 14.0%) reported in almond cultivars

Carmel, Mission, Neplus, Nonpareil, and Peerless using rabbit anti-AMP inhibition ELISA

(139). Geographic location and cultivar x county interaction influenced the immunoreactivity of

AMP in tested almond cultivars. The mAb 4C10 sandwich ELISA found AMP immunoreactivity

was higher in South Valley grown almond seeds (~63.2%) and in majority of the tested cultivars

from Butte County (cultivars Butte, Mission, Padre, and Price) and Kern County (cultivars Butte,

Carmel, Mission, and Nonpareil) (Table 25). The increase in AMP immunoreactivity for

cultivars Butte, Mission, and Padre from Butte County and cultivars Carmel and Mission from

Kern County was also detected using rabbit anti-AMP inhibition ELISA. Similarly, both ELISAs

detected a decrease in AMP immunoreactivity for cultivars Butte, Mission, and Padre grown in

San Joaquin County (Table 25). Influence of harvest year on immunoreactivity of AMP in tested

almond cultivars was not significant (Table 24).

Total soluble protein immunoreactivity of tested almond seeds (assessed by rabbit anti-

total soluble almond protein inhibition ELISA) varied by cultivar (Table 24). Judged against the

major commercial almond cultivar Nonpareil Supreme (100%), the immunoreactivity of tested

almond cultivars was 120.4% for Butte, 133.6% for Carmel, 114.1% for Mission, 147.8% for

Monterey, 112.0% for Nonpareil, 88.1% for Padre, 84.8% for Price, and 54.8% for Sonora

(Table 24). Geographic location also influenced total protein immunoreactivity of tested almond

cultivars, with noteworthy increases in cultivars grown in Butte County (cultivars Butte,

Mission, and Padre) and Fresno County (cultivars Carmel, Mission, and Nonpareil). Similarly,

decreased immunoreactivity was detected in cultivars Carmel, Mission, and Nonpareil grown in

Colusa County (Table 25). Harvest year was not found to significantly influence the total soluble

protein immunoreactivity of tested almond seed samples (Table 24).

84

Figure 7. Polypeptide profile and AMP antigenicity of select almond cultivars assessed by (A) SDS-PAGE stained with Coomassie stain; protein load = 30�g, (B) Western blot probed with mAb 4C10; protein load = 30�g, (C) Dot blot probed with mAb 4C10; protein load = 500 ng.

85

1

Figure 8. SDS-PAGE analysis of macadamia nut cultivars. A. Coomassie stain; protein load = 30 �g. B. Silver stain; protein load = 10 �g. C. Glycoprotein stain; protein load = 200 �g. D. Western blot; protein load = 30 �g. Primary Ab = Protein G purified rabbit anti- macadamia nut IgG (1:16,000 v/v). Secondary Ab = HRP-labeled goat anti-rabbit pAb (1:40,000 v/v).

A B D C

86

Table 24. Effect of cultivar, geographic location, and harvest year on the immunoreactivity of select almond cultivars assessed by ELISAa

aAlmond proteins were assessed by bmAb 4C10 sandwich ELISA, crabbit anti-AMP inhibition ELISA and danti-total soluble almond proteins pAb inhibition ELISA and all IC50 values are expressed as ng protein per 1 ml (ng/ml). Data are expressed as mean ± SEM (n = 3). For each column, means with different letters are significantly different (p � 0.05) according to Fisher’s LSD test. AMP (%) = (AMP IC50 value / IC50 value for test sample) x 100.

Cultivar

Butte 28.47 ± 1.64 c 91.09 ± 5.35 ef 48.99 ± 2.31 cd

Carmel 27.48 ± 1.59 c 66.27 ± 2.71 f 44.12 ± 2.30 cd

Mission 85.52 ± 8.46 a 207.68 ± 17.98 a 51.69 ± 5.04 cd

Monterey 33.36 ± 3.81 c 94.53 ± 4.07 ef 39.88 ± 4.01 d

Nonpareil 39.09 ± 2.55 c 100.74 ± 5.88 de 52.64 ± 2.18 c

Padre 64.81 ± 4.18 b 127.78 ± 12.67 cd 66.93 ± 5.18 b

Price 77.29 ± 10.08 ab 156.53 ± 10.59 bc 69.54 ± 3.77 b

Sonora 86.51 ± 5.16 a 184.83 ± 5.75 ab 107.49 ± 5.63 a

LSD

County

Butte 48.78 ± 5.69 abc 89.16 ± 10.48 cde 69.76 ± 7.61 a

Colusa 56.46 ± 7.41 a 116.43 ± 19.12 abc 69.78 ± 5.01 a

Fresno 25.38 ± 2.67 d 80.50 ± 2.67 de 36.35 ± 1.37 d

Glenn 51.23 ± 13.94 ab 137.72 ± 25.08 a 45.99 ± 7.09 cd

Kern 24.59 ± 2.88 d 71.18 ± 11.88 de 47.98 ± 7.40 cd

Madera 37.72 ± 6.36 bcd 120.13 ± 32.84 ab 65.38 ± 6.88 a

Merced 27.04 ± 1.12 d 71.89 ± 8.09 de 44.93 ± 1.84 cd

Sacramento 25.63 ± 2.55 d 58.79 ± 1.93 e 36.77 ± 2.45 d

San Joaquin 34.91 ± 5.39 d 66.67 ± 4.90 de 61.91 ± 4.12 ab

Stanislaus 30.65 ± 4.19 d 91.41 ± 10.45 bcd 47.78 ± 4.46 cd

Tulare 36.84 ± 4.52 cd 67.65 ± 9.53 de 50.36 ± 7.20 bc

Yolo 32.51 ± 2.86 d 90.28 ± 8.45 bcd 43.34 ± 2.35 cd

LSD

Region

North Valley 37.62 ± 3.00 a 87.35 ± 6.13 a 48.11 ± 2.70 a

South Valley 30.33 ± 1.63 b 81.49 ± 4.55 a 48.79 ± 2.03 a

LSD

Year

2003-2004 32.03 ± 6.34 a 101.83 ± 11.05 a 50.45 ± 2.95 a

2005-2006 35.85 ± 4.33 a 101.88 ± 9.02 a 48.16 ± 4.92 a

LSD

Nonpareil Supreme 36.33 ± 5.32 92.32 ± 3.21 58.98 ± 2.79

AMP 19.18 ± 1.25 58.85 ± 2.34

14.64 28.23 12.87

4C10 mAbb

Rabbit anti-AMP

pAbc

Rabbit anti-

almond pAbd

15.90 31.42 11.80

13.61 30.93 12.16

6.4214.496.22

ND

87

Table 25. Interaction of cultivar and geographic location on the immunoreactivity of select almond cultivars assessed by ELISAa

Cultivar County

Butte 17.04 ± 1.26 b 66.97 ± 6.41 cd 41.92 ± 3.43 b

Fresno 25.77 ± 2.95 ab 73.47 ± 6.19 cd 49.93 ± 4.19 ab

Kern 24.82 ± 2.37 ab 93.56 ± 9.64 bcd 46.79 ± 6.40 ab

Madera 25.25 ± 3.07 ab 88.34 ± 11.40 cd 53.56 ± 11.65 ab

Merced 27.66 ± 2.83 ab 104.04 ± 7.78 bc 39.05 ± 7.53 b

Sacramento 27.55 ± 0.72 ab 55.05 ± 0.76 d 42.41 ± 2.81 ab

San Joaquin 34.47 ± 3.92 a 148.55 ± 14.04 a 63.64 ± 11.37 a

Stanislaus 27.73 ± 2.88 ab 81.47 ± 7.10 cd 53.06 ± 9.06 ab

Tulare 36.91 ± 2.20 a 128.71 ± 14.30 ab 44.78 ± 4.75 ab

Yolo 37.53 ± 8.50 a 97.33 ± 23.09 bc 52.06 ± 9.23 ab

LSD

Butte 44.38 ± 10.84 a 66.81 ± 1.82 cd 68.38 ± 15.33 a

Colusa 46.10 ± 8.41 a 75.86 ± 6.57 bc 68.03 ± 10.51 a

Fresno 20.01 ± 2.26 c 77.82 ± 3.29 bc 35.52 ± 2.05 cd

Glenn 31.51 ± 8.57 bc 82.62 ± 6.84 b 34.09 ± 2.23 cd

Kern 21.86 ± 5.41 c 45.82 ± 6.03 g 50.44 ± 14.72 bc

Madera 23.95 ± 3.05 c 47.50 ± 7.78 efg 53.77 ± 7.18 ab

Merced 28.81 ± 0.42 bc 55.42 ± 5.29 defg 42.00 ± 2.14 bcd

Sacramento 20.09 ± 1.79 c 60.91 ± 2.40 def 29.77 ± 2.35 d

San Joaquin 27.62 ± 2.92 bc 59.27 ± 4.85 def 55.55 ± 3.93 ab

Stanislaus 31.47 ± 6.71 bc 53.54 ± 3.71 defg 41.53 ± 9.29 bcd

Tulare 27.14 ± 2.06 bc 46.98 ± 3.85 fg 35.68 ± 6.40 cd

Yolo 36.40 ± 4.40 ab 117.37 ± 7.77 a 49.21 ± 3.15 bc

LSD

Butte 39.55 ± 1.55 de 93.28 ± 5.13 e 46.19 ± 13.80 b

Colusa 76.55 ± 17.27 c 178.91 ± 9.62 c 58.26 ± 6.48 ab

Fresno 72.52 ± 13.17 cd 171.34 ± 10.75 c 31.37 ± 6.14 b

Glenn 81.84 ± 6.53 c 169.21 ± 7.50 c 31.62 ± 6.33 b

Kern 32.75 ± 11.02 e 131.66 ± 11.39 d 28.61 ± 6.10 b

Merced 128.43 ± 9.44 b 316.67 ± 21.22 b 54.72 ± 0.73 ab

San Joaquin 168.17 ± 13.00 a 402.28 ± 17.86 a 53.06 ± 1.99 ab

Stanislaus 84.94 ± 10.54 c 202.89 ± 10.07 c 80.70 ± 14.57 a

LSD

14.05 39.46 21.66

Carmel

4C10 mAbb

Rabbit anti-AMP

pAbc

Rabbit anti-

almond pAbd

12.17 13.45 16.63

Butte

Mission

33.79 36.53 33.33

aAlmond proteins were assessed by bmAb 4C10 sandwich ELISA, crabbit anti-AMP inhibition ELISA and danti-total soluble almond proteins pAb inhibition ELISA and all IC50 values are expressed as ng protein per 1 ml (ng/ml). Data are expressed as mean ± SEM (n = 3). For each column, means with different letters are significantly different (p � 0.05) according to Fisher’s LSD test.

88

aAlmond proteins were assessed by bmAb 4C10 sandwich ELISA, crabbit anti-AMP inhibition ELISA and danti-total soluble almond proteins pAb inhibition ELISA and all IC50 values are expressed as ng protein per 1 ml (ng/ml). Data are expressed as mean ± SEM (n = 3). For each column, means with different letters are significantly different (p � 0.05) according to Fisher’s LSD test.

Table 25 continued. Interaction of cultivar and geographic location on the immunoreactivity of select almond cultivars assessed by ELISAa

Cultivar County

Butte 53.17 ± 5.03 abc 111.50 ± 6.81 c 71.14 ± 7.25 ab

Colusa 66.82 ± 9.82 ab 157.00 ± 11.77 b 71.53 ± 3.44 ab

Fresno 33.44 ± 4.02 defg 84.51 ± 4.30 d 37.60 ± 1.58 d

Glenn 70.94 ± 22.57 a 192.82 ± 7.89 a 57.88 ± 10.26 bc

Kern 27.33 ± 2.16 fg 96.54 ± 5.10 cd 45.51 ± 7.13 cd

Madera 51.49 ± 1.86 bcd 192.75 ± 7.64 a 76.98 ± 7.09 a

Merced 25.27 ± 1.72 g 88.35 ± 5.28 cd 47.86 ± 1.93 cd

Sacramento 31.17 ± 4.08 efg 56.68 ± 2.98 e 43.76 ± 2.79 d

San Joaquin 49.48 ± 12.11 bcde 81.47 ± 2.42 d 74.64 ± 2.28 a

Stanislaus 30.38 ± 5.33 efg 84.51 ± 4.30 d 49.86 ± 5.21 cd

Tulare 46.54 ± 1.95 cdef 88.32 ± 3.52 cd 65.04 ± 1.61 ab

Yolo 30.57 ± 3.67 efg 76.74 ± 7.03 de 40.40 ± 2.47 d

LSD

Butte 51.98 ± 10.72 cd 81.19 ± 5.89 bc 56.33 ± 6.01 bc

Fresno 72.67 ± 12.28 b 114.45 ± 18.24 abc 81.10 ± 8.18 ab

Kern 70.23 ± 3.07 bc 118.12 ± 21.36 abc 58.29 ± 2.67 bc

Merced 92.23 ± 2.78 a 163.67 ± 17.54 ab 107.31 ± 17.26 a

San Joaquin 74.29 ± 3.69 ab 164.35 ± 66.06 ab 60.51 ± 3.04 bc

Stanislaus 41.14 ± 1.29 d 71.86 ± 4.05 c 43.65 ± 4.31 c

Tulare 51.14 ± 2.08 cd 180.80 ± 15.11 a 61.29 ± 11.17 bc

LSD

Butte 50.99 ± 4.50 bc 154.13 ± 2.95 bc 77.99 ± 2.61 a

Fresno 43.71 ± 0.71 bc 210.50 ± 35.07 a 79.67 ± 6.96 a

Kern 96.01 ± 6.28 ab 203.57 ± 5.08 ab 77.30 ± 10.78 a

Merced 77.43 ± 18.02 bc 133.29 ± 28.86 cd 69.94 ± 12.16 ab

San Joaquin 90.62 ± 28.04 b 162.93 ± 12.60 abc 49.56 ± 5.68 bc

Stanislaus 149.56 ± 33.88 a 145.10 ± 12.66 c 83.79 ± 2.60 a

Tulare 32.70 ± 3.37 c 86.20 ± 5.75 d 48.54 ± 0.47 c

LSD 53.67 55.03 21.06

19.40 23.93 13.76

19.33 84.24 26.32

Rabbit anti-AMP

pAbc

Rabbit anti-

almond pAbd

Price

Nonpareil

Padre

4C10 mAbb

89

Macadamia nut. A competitive inhibition ELISA with good sensitivity and a wide

detection range (5-1,000 ng/ml) was developed and optimized for coating buffer (Figure 9),

antigen coating concentration (Figure 10), and Protein G purified rabbit anti-macadamia nut IgG

dilution (Table 26). Of the 4 tested coating buffers, plates coated with macadamia nut proteins

diluted in PBS gave a superior protein binding signal (Figure 9). Coating of macadamia nut

proteins above 125 ng/well did not significantly increase the binding signal (Figure 9 and 10),

therefore plates were coated at this concentration. The IC50 values obtained from diluting Protein

G purified rabbit anti-macadamia nut IgG from 1:8,000 to 1:80,000 (v/v) were not significantly

different (Table 27), however diluting the pAb 1:25,000 (v/v) obtained the lowest IC50 value

while also providing a strong signal after 20 min of color development. Therefore, the optimized

ELISA conditions included coating 125 ng/well of total soluble macadamia nut proteins diluted

in PBS buffer and diluting Protein G purified rabbit anti-macadamia nut IgG to 1:25,000 (v/v). A

representative standard curve from the optimized assay is depicted in Figure 11.

Figure 9. Checkerboard titration for optimization of coating buffer in non-competitive ELISA. Protein G purified rabbit anti-macadamia nut IgG dilution was 1:4,000 v/v. Data are expressed as mean ± SEM (n = 4).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

0 50 100 150 200 250 300 350 400 450 500

Ab

sorb

ance

405

nm

Coated Macadamia nut protein (ng)

BSB

Citrate

Carbonate

PBS

90

Table 26. IgG purified macadamia nut pAb titer optimizationa

aData are expressed as mean ± SEM (n = 4)

344.5 ± 105.0

102.6 ± 18.0

133.4 ± 19.3

72.6 ± 1.3

54.3 ± 2.4

52.2 ± 5.1

24.1 ± 1.0

34.5 ± 1.9

40.1 ± 3.7

55.4 ± 5.3

56.6 ± 4.3

81.9 ± 11.1

8

Macadamia

pAb dilution

( x 1000)

IC50 value (ng/ml)

4

5

6

80

100

LSD 57.9

10

20

25

30

40

50

Figure 10. Checkerboard titration using PBS coating buffer in non-competitive ELISA to optimize macadamia nut protein (antigen) coating concentration. Protein G purified rabbit anti-macadamia nut IgG dilutions are indicated in figure legend (X = 1,000). Data are expressed as mean ± SEM (n = 4).

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 50 100 150 200 250 300 350 400 450 500

Ab

sorb

an

ce 4

05 n

m

Coated Macadamia nut protein (ng) in PSB (pH 7.2)

500

1X

2X

4X

8X

16X

32X

64X

128X

256X

512X

91

The average IC50 value for the major macadamia nut commercial variety (Blue Diamond)

was 21.75 ± 0.94 ng/ml (mean ± SEM, n= 70). The Blue Diamond variety was used as the

reference as this variety was the source of protein (antigen) for rabbit pAb production.

Immunoreactivity of Blue Diamond variety and cultivars Keauhou, Purvis, and Kakea was

similar, however immunoreactivity of Trader Joe’s variety and cultivars Keaau and Mauka was

significantly lower (34.3%, 52.4%, and 37.5%, respectively) (Figure 12).

Figure 11. Representative competitive inhibition ELISA standard curve for macadamia nut. The IC50 value (mean ± SEM) for soluble macadamia nut protein was 21.75 ± 0.94 ng/ml (n = 70).

Figure 12. Immunoreactivity (%) of macadamia nut varieties assessed by inhibition ELISA (n= 4). * = significantly different compared to control (Blue Diamond, p � 0.05, LSD = 23.18).

0

20

40

60

80

100

120

140

Blue

Diamond

Trader

Joe's

#246

'Keauhou'

#294

'Purvis'

#508

'Kakea'

#660

'Keaau'

#741

'Mauka'

% Im

mu

no

rea

cti

vit

y

Macadamia nut seed varieties

** *

92

Western and Dot blots

Almond. Western blots probed with mAb 4C10 detected a significant variation in AMP

antigenicity among the tested almond seeds (Figure 7B). Similar to the reported variation

observed between almond genotypes probed with mAb 4C10 (49). The intensity of the AMP

polypeptide (63 kDa) recognized by mAb 4C10 varied by cultivar and geographic location. Dot

blotting experiments further confirmed the variation, as shown by the difference in signal

intensities (Figure 7C).

Macadamia nut. Western blotting analysis identified nine sets of immunoreactive

polypeptides with estimated MWs of 94-96, 59-62, 48-51, 37-39, 31-33, 26-28, 20-21, 17-19 and

6-10 kDa (Figure 8D) among which 48-50 and 17-19 kDa polypeptides exhibited the strongest

reactivity. Significant differences with respect to band intensity in the polypeptide masses of 6-

10 kDa and 20-21 kDa were seen among the tested macadamia nut seed samples. Polypeptides

59-62, 48-51, 37-39, 31-33, 26-28, and 17-19 kDa did not appear to show significant variations

in antigenicity among the the tested macadamia nut seed samples.

Cross-reactivity Studies

Determining antibody cross-reactivity is of importance as foods containing almond and

macadamia nut seeds may also contain other tree nuts, oilseeds, legumes, cereals, or additional

ingredients which if recognized by the antibody could cause problems with accurate detection

and labeling.

Almond. The anti-AMP mAb 4C10 was analyzed for cross-reactivity using Western

blotting, Dot blotting, and ELISA (Figure 13, A-C). Western and Dot blotting assays did not

detect any cross-reactivity between mAb 4C10 and 17 seed proteins. Similarly, the sandwich

ELISA only detected a range of 0.00-0.15% cross-reactivity for 51 foods/ingredients (Figure

13C).

Macadamia nut. Protein G purified rabbit anti-macadamia nut IgG was also analyzed for

cross-reactivity using Western blotting (Figure 14) and inhibition ELISA (Table 27). Western

blotting analysis found cross-reactivity of Protein G purified rabbit anti-macadamia nut IgG with

majority of the 62 tested samples. However, inhibition ELISA found only 0.00-0.11% cross-

reactivity with 68 different foods/ingredients and 1.46% cross-reactivity cinnamon (Table 27).

93

Figure 13. Cross-reactivity of mAb 4C10 with select foods/ingredients assessed by (A) Western blot; protein load = 20 �g, (B) Dot blot; protein load = 500 ng for almond and 2 �g for test samples, and (C) sandwich ELISA; values are % cross-reactivity and are expressed as mean ± SEM (n = 3).

94

1

Figure 14. Cross-reactivity of select foods/ingredients with Protein G purified rabbit anti-macadamia nut IgG (1:16,000 v/v) assessed by Western blotting. Protein loads: 10 �g macadamia and 30 �g for all test samples.

95

Table 27. Cross-reactivity of select foods/ingredients with Protein G purified rabbit anti-macadamia nut IgG assessed by inhibition ELISAa

aData are reported as mean ± SEM (n = 3).

Sample Sample

Almond 0.001 ± 0.000 Poppy Seed 0.012 ± 0.000Brazil Nut 0.003 ± 0.002 Barley 0.002 ± 0.000

Cashew 0.000 ± 0.000 Corn 0.005 ± 0.001Hazelnut 0.002 ± 0.001 Wheat 0.005 ± 0.000

Pecan 0.000 ± 0.000 Egg White 0.007 ± 0.001Pistachio 0.001 ± 0.001 Egg Yolk 0.001 ± 0.000Pinenut 0.006 ± 0.001 Raisin 0.007 ± 0.001Walnut 0.016 ± 0.002 Cocoa 0.006 ± 0.000

Inca Peanut 0.001 ± 0.001 Non-fat Dry Milk 0.002 ± 0.000Spanish Peanut 0.000 ± 0.000 Vanilla Icecream 0.005 ± 0.001Virginia Peanut 0.005 ± 0.001 Rice 0.000 ± 0.000

Black Bean 0.007 ± 0.000 Spinach 0.002 ± 0.000Black-eye Pea 0.005 ± 0.001 Cauliflower 0.001 ± 0.000

Chickpea 0.003 ± 0.003 Sesame 0.002 ± 0.000Cow Pea 0.004 ± 0.004 Pepitas 0.000 ± 0.000

Fava Bean 0.008 ± 0.004 Black Pepper 0.008 ± 0.001Green Lentil 0.008 ± 0.002 Cardamom 0.015 ± 0.001Green Pea 0.009 ± 0.002 Cinnamon 1.458 ± 0.989

Red Kidney 0.014 ± 0.000 Nutmeg 0.078 ± 0.001Horse Bean 0.026 ± 0.001 Cherries 0.014 ± 0.001Lima Bean 0.005 ± 0.002 Oats 0.014 ± 0.002

Lupine Seed 0.006 ± 0.000 Millet 0.016 ± 0.001Moth Bean 0.004 ± 0.001 Sorghum 0.016 ± 0.001Mung Bean 0.004 ± 0.002 Salt 0.024 ± 0.002Navy Bean 0.003 ± 0.001 Sugar 0.012 ± 0.001Pigeon Pea 0.003 ± 0.000 Baking Powder 0.006 ± 0.000Pinto Bean 0.005 ± 0.002 Vanilla Extract 0.110 ± 0.012

Soybean 0.003 ± 0.001 Chocolate Chip Cookie 0.009 ± 0.001Tepary Bean 0.005 ± 0.000 Coconut 0.006 ± 0.000

Urd 0.024 ± 0.003 Milk Chocolate 0.012 ± 0.001Val 0.000 ± 0.000 White Chocolate 0.002 ± 0.000

Winged Bean 0.000 ± 0.000 Dark Chocolate 0.002 ± 0.000Flaxseed 0.000 ± 0.000 Trail Mix 0.000 ± 0.000

Sunflower 0.000 ± 0.000 All-purpose Wheat Flour 0.002 ± 0.001Amaranth 0.000 ± 0.000

%

Crossreactivity

%

Crossreactivity

LSD = 0.334

96

Stability Studies

Almond and macadamia nut seeds are often subjected to a variety of processing

treatments (i.e. roasting and blanching) which may alter the protein structure. The structural

changes, as a result of processing treatments, may alter protein detection by destroying epitopes,

exposing previously hidden epitopes and/or creating new epitopes (197). Therefore, antigenic

stability of almond and macadamia nut seed proteins exposed to various processing treatments

(pH exposure and thermal processing) was assessed using immunoassays.

Almond. Regardless of the thermal processing treatment, the 63 kDa AMP polypeptide

retained its antigenicity (Figure 15). The stability of almond seed proteins, assessed by rabbit

pAb-based inhibition ELISA, to various thermal processing treatments and/or -irradiation is

reported (139, 140, 196-198). Exposure to pH 3 and 13 caused loss of detection by mAb 4C10 in

Western blotting (Figure 16, B and E), however the mAb-based sandwich ELISA weakly

detected the polypeptide in pH treated samples that were neutralized prior to analysis. A decrease

in AMP antigenicity as a result of acid pH exposure has been reported (140, 167).

Macadamia nut. Assessing the stability of macadamia nut proteins to thermal processing

found that dry heat methods, microwaving and dry roasting, had the most impact on decreasing

the antigenicity of these proteins (Figure 17). However regardless of the thermal processing

treatment, macadamia nut proteins retained their antigenicity and were detected by the

immunoassays. Western blotting detected macadamia nut proteins in all tested pH exposed

samples (Figure 18, B and E), however antigenicity was significantly decreased in proteins

exposed to pH 1, 3 and 5 that were not neutralized prior to analysis (Figure 18B). Inhibition

ELISA also detected macadamia nut proteins in the tested pH exposed samples (Figure 18, C and

F), however a significant decrease in the detection of proteins exposed to pH 1, 3 and 13 (not

neutralized) and exposed to pH 1 and 13 (neutralized) was observed. Differences in protein

solubility could explain the variation observed between pH exposed samples neutralized prior to

analysis and those not neutralized, as the proteins in samples not neutralized prior to analysis

may have precipitated under the normal assay conditions.

Spiking Studies

Processed foods containing almond and macadamia nut seeds may also contain other

ingredients that may interfere or alter detection of almond and macadamia nut proteins.

Therefore, foods/ingredients commonly found in processed foods containing almond and

97

macadamia nut seeds were spiked with BSB extracted proteins or defatted flour and recovery

was assessed by ELISA.

Almond. Assuming ~60% AMP in the BSB protein extract, the assay was able to detect

10 �g almond seed protein (or 6 �g AMP) in 100 mg food matrix (equivalent to 100 ppm) in the

majority (10 out of 16) of tested food matrices (Table 28). The assay also detected defatted

almond seed flour at 100 ppm (equivalent to 100 �g defatted almond seed flour per 100 g food

matrix) in 6 of the 10 tested food matrices (Table 28). Four commercial products with almond

seeds as one of the declared ingredients were found to contain 4.1-26.3% almond seed (w/w) on

as is basis (Table 30).

Macadamia nut. The rabbit anti-macadamia nut inhibition ELISA successfully detected

1 �g macadamia nut protein in 100 mg (equivalent to 10 ppm) in all 13 tested food matrices and

as low as 1 ppm macadamia nut protein in 5 of the 13 tested food matrices. The assay also

detected 10 �g of defatted macadamia nut flour in 100 mg (10 ppm) in 6 of the 8 tested food

matrices (Table 29). Two commercial products with macadamia nut seeds as one of the declared

ingredients were found to contain 22.6-34.0% macadamia nut seed (w/w) on as is basis (Table

30).

CONCLUSION

Both almond and macadamia nut seed samples differed significantly in physical

characteristics, chemical composition, and protein antigenicity. Growth location and harvest year

also significantly affected physical characteristics, chemical composition, and protein

antigenicity of the tested almond cultivars.

The sensitive immunoassays developed in this study successfully detected almond and

macadamia nut proteins at concentrations applicable for the food industry and may contribute to

safer consumer products.

98

Figure 15. Effect of thermal processing on antigenicity of AMP assessed by (A) SDS-PAGE stained with Ponceu S stain; protein load = 30�g, (B) Western blot probed with mAb 4C10; protein load = 30�g, (C) mAb 4C10 sandwich ELISA; values are % immunoreactivity [(IC50 of processed sample/IC50 of unprocessed sample) x 100] and are expressed as mean ± SEM (n = 3, p � 0.05).

99

Figure 16. Effect of pH on the antigenicity of AMP assessed by (A) SDS-PAGE stained with Ponceu S stain and (D) Coomassie stain; protein load = 20 �g, (B and E) Western blot probed with mAb 4C10; protein load = 20 �g, and (C and F) mAb 4C10 sandwich ELISA, values are % immunoreactivity [(IC50 of processed sample/IC50 of unprocessed sample) x 100] and are expressed as mean ± SEM (n = 3, p � 0.05). A, B, C: Defatted almond seed flour exposed to desired pH value and neutralized prior to analysis. D, E, F: Defatted almond seed flour exposed to desired pH value and analyzed directly (not neutralized).

100

1

Figure 17. Effect of thermal processing on immunoreactivity of macadamia nut proteins assessed by ELISA and Dot blot, (A) SDS-PAGE stained with Coomassie stain; protein load = 30 �g, and (B) Western blot probed with Protein G purified rabbit anti-macadamia nut IgG (1:16,000 v/v); protein load = 30 �g. ELISA values are % immunoreactivity [(IC50 of processed sample/IC50 of unprocessed sample) x 100] and are expressed as mean ± SEM (n = 3, p � 0.05).

Mean ± SEM Mean ± SEMUP Unprocessed 100.00 ± 0.00 100.00 ± 0.00B1 Blanch 100 °C, 1 min 100.91 ± 6.97 96.62 ± 1.22B2 Blanch 100 °C, 4 min 117.65 ± 17.25 74.43 ± 0.59B3 Blanch 100 °C, 7 min 105.14 ± 4.20 80.71 ± 4.75B4 Blanch 100 °C, 10 min 108.93 ± 14.16 82.99 ± 7.47A1 Autoclave 191 °C, 5 min 105.49 ± 18.35 93.37 ± 2.72A2 Autoclave 191 °C, 10 min 70.04 ± 8.23 91.58 ± 1.03A3 Autoclave 191 °C, 20 min 69.95 ± 10.08 95.46 ± 1.19A4 Autoclave 191 °C, 30 min 48.31 ± 6.94 82.06 ± 0.79M1 Microwave 500W, 1 min 103.42 ± 3.40 89.02 ± 1.83M2 Microwave 500W, 2 min 73.57 ± 7.01 73.22 ± 1.47M3 Microwave 1000W, 1 min 77.88 ± 10.48 76.92 ± 0.94M4 Microwave 1000W, 2 min 50.84 ± 2.77 58.86 ± 0.42R1 Dry Roast 140 °C, 20 min 71.74 ± 5.12 59.69 ± 1.69R2 Dry Roast 140 °C, 30 min 7.14 ± 0.61 24.10 ± 1.50R3 Dry Roast 170 °C, 15 min 68.70 ± 3.14 66.43 ± 0.19R4 Dry Roast 170 °C, 20 min 8.18 ± 1.14 25.11 ± 1.75R5 Dry Roast 200 °C, 10 min 82.65 ± 7.79 53.79 ± 0.49R6 Dry Roast 200 °C, 15 min 2.86 ± 0.28 13.74 ± 3.01

ELISA

LSD = 24.14

Dot blot

LSD = 6.95

Sample Code Thermal Treatment

101

2

Figure 18. Effect of pH on immunoreactivity of macadamia nut proteins assessed by (A and D) SDS-PAGE stained with Coomassie stain; protein load = 20 �g, (B and E) Western blot probed with Protein G purified rabbit anti-macadamia nut IgG (1:16,000 v/v); protein load = 20 �g, and (C and F) inhibition ELISA, values are % immunoreactivity [(IC50 of processed sample/IC50 of unprocessed sample) x 100] and are expressed as mean ± SEM (n = 3, p � 0.05). A, B, C: Defatted macadamia nut flour exposed to desired pH value and analyzed directly (not neutralized). D, E, F: Defatted macadamia nut flour exposed to desired pH value and neutralized prior to analysis.

A

B

C

D

E

F pH Mean ± SEM pH Mean ± SEMBSB 100.00 ± 0.00 BSB 100.00 ± 0.00

1 0.01 ± 0.00 1 0.07 ± 0.003 0.06 ± 0.01 3 106.05 ± 11.685 63.14 ± 7.42 5 117.25 ± 10.197 98.26 ± 8.85 7 94.08 ± 4.339 93.47 ± 2.68 9 113.18 ± 5.43

11 74.65 ± 4.57 11 64.19 ± 4.5913 0.01 ± 0.00 13 2.88 ± 0.35

LSD = 15.27 LSD = 19.41

102

Table 28. Detection of AMP in 100 mg food matrices spiked with almond seed protein extract or defatted almond seed floura

aTheoretical yield of soluble almond seed protein and AMP in 100 mg defatted flour is estimated to be ~20% and 10-11%, respectively. Data are reported as mean ± SEM (n = 3, p � 0.05). ND = not determined.

Almond Extract 10% 0.00 ± 0.00 10.26 ± 0.62 0.40 ± 0.06 0.02 ± 0.00Coconut 0.00 ± 0.00 3.20 ± 0.21 0.40 ± 0.01 0.05 ± 0.00 8.15 ± 0.63 0.47 ± 0.03 0.07 ± 0.01

Dark Chocolate 0.01 ± 0.01 0.47 ± 0.04 0.09 ± 0.01 0.01 ± 0.00 0.23 ± 0.04 0.04 ± 0.01 0.02 ± 0.01Green Beans 0.19 ± 0.04 9.50 ± 2.25 0.62 ± 0.04 0.19 ± 0.01 1.07 ± 0.07 0.39 ± 0.01 0.20 ± 0.03

Honey Bunches of Oats Cereal 0.03 ± 0.00 7.72 ± 0.42 0.54 ± 0.01 0.09 ± 0.01 8.37 ± 0.26 1.23 ± 0.06 0.17 ± 0.00Milk Chocolate 0.09 ± 0.05 1.29 ± 0.08 0.18 ± 0.05 0.00 ± 0.00 1.68 ± 0.16 0.20 ± 0.02 0.03 ± 0.02

Mounds Chocolate Candy Bar 0.03 ± 0.00 0.97 ± 0.18 0.14 ± 0.05 0.03 ± 0.02 0.89 ± 0.04 0.13 ± 0.01 0.03 ± 0.00Salt, 100 mg 0.00 ± 0.00 3.49 ± 0.49 0.04 ± 0.00 0.00 ± 0.00Salt, 100 mg 0.00 ± 0.00 2.81 ± 0.30 0.31 ± 0.03 0.01 ± 0.00

Sugar, 100 mg 0.00 ± 0.00 6.83 ± 0.47 0.00 ± 0.00 0.00 ± 0.00Sugar, 10 mg 0.00 ± 0.00 6.91 ± 0.21 0.34 ± 0.05 0.02 ± 0.00

Trail Mix 0.00 ± 0.00 5.52 ± 0.49 0.58 ± 0.04 0.06 ± 0.02 24.55 ± 1.09 2.11 ± 0.19 0.22 ± 0.01Vanilla Extract 10% 0.00 ± 0.00 0.01 ± 0.00 0.00 ± 0.00 0.00 ± 0.00Vanilla Ice Cream 0.00 ± 0.00 7.75 ± 1.16 0.85 ± 0.05 0.07 ± 0.01 15.79 ± 0.54 2.02 ± 0.17 0.20 ± 0.04

Wheat Flour 0.02 ± 0.00 8.34 ± 0.59 0.94 ± 0.03 0.07 ± 0.02 26.9 ± 3.08 0.79 ± 0.02 0.13 ± 0.01White Chocolate 0.07 ± 0.01 6.73 ± 0.36 0.87 ± 0.06 0.08 ± 0.01 17.49 ± 2.44 2.55 ± 0.20 0.29 ± 0.02

LSD

ND ND ND

0.05 2.07 0.11 0.03 3.83 0.30 0.05

ND ND NDND ND ND

ND ND NDND ND ND

10 μg 1 μg 0.1 μg

Spiked with defatted almond seed

100 μg 10 μg 1 μgFood Matrices Unspiked

Spiked with almond seed protein

ND ND ND

103

1 Table 29. Detection of macadamia nut protein in 100 mg of food matrices spiked with macadamia nut protein extract or defatted macadamia nut floura

aTheoretical yield of soluble macadamia nut protein in 100 mg defatted flour is estimated to be ~14%. Data are reported as mean ± SEM (n = 3). ND = not determined.

10 μg 1 μg 0.1 μg 100 μg 10 μg 1 μg

Chocolate Chip Cookie 0.14 ± 0.02 8.99 ± 0.33 0.55 ± 0.04 0.15 ± 0.00 8.65 ± 1.88 0.73 ± 0.04 0.08 ± 0.00Coconut 0.07 ± 0.01 9.89 ± 0.28 1.52 ± 0.16 0.05 ± 0.01 8.90 ± 0.87 1.92 ± 0.07 0.36 ± 0.04

Dark Chocolate 0.02 ± 0.00 2.68 ± 0.38 0.25 ± 0.00 0.03 ± 0.00 2.61 ± 0.29 0.16 ± 0.00 0.03 ± 0.00Milk Chocolate 0.34 ± 0.04 11.07 ± 1.43 0.98 ± 0.07 0.13 ± 0.01 18.51 ± 0.83 1.37 ± 0.20 0.30 ± 0.02

White Chocolate 0.03 ± 0.00 5.40 ± 0.56 0.40 ± 0.05 0.06 ± 0.00 19.81 ± 1.20 0.93 ± 0.01 0.04 ± 0.01Trail Mix 0.01 ± 0.00 10.02 ± 1.09 0.86 ± 0.05 0.04 ± 0.00 9.20 ± 0.34 0.22 ± 0.02 0.04 ± 0.00

Wheat Flour 0.04 ± 0.01 9.05 ± 0.33 1.20 ± 0.08 0.15 ± 0.01 25.56 ± 2.63 1.64 ± 0.14 0.37 ± 0.07Vanilla Icecream 0.12 ± 0.03 15.74 ± 0.77 1.25 ± 0.02 0.21 ± 0.01 14.40 ± 1.23 1.54 ± 0.32 0.16 ± 0.02

Salt (100 mg) 0.01 ± 0.00 4.55 ± 0.80 0.17 ± 0.02 0.01 ± 0.00 ND ND NDSalt (10 mg) 0.01 ± 0.00 10.12 ± 0.16 1.01 ± 0.09 0.07 ± 0.01 ND ND ND

Sugar (100 mg) 0.00 ± 0.00 6.75 ± 0.07 0.08 ± 0.00 0.00 ± 0.00 ND ND NDSugar (10 mg) 0.00 ± 0.00 8.70 ± 1.23 0.96 ± 0.04 0.06 ± 0.00 ND ND ND

Vanilla Extract (10%) 0.03 ± 0.00 9.66 ± 0.95 0.88 ± 0.02 0.10 ± 0.00 ND ND NDLSD (p = 0.05) 0.05 2.24 0.19 0.02 4.11 0.43 0.08

Unspiked

Spiked with macadamia nut

protein extract

Spiked with defatted macadamia

nut flourFood Matrices

104

Commercial Foods containing

Almond Mean ± SEM

Almond Joy candy bar 26.34 ± 1.06Almond Praline ice cream 12.68 ± 1.01

Honey Bunches of Oats with slivered almonds breakfast cereal 25.83 ± 7.47

Green beans with slivered almonds 4.11 ± 0.29Commercial Foods containing

Macadamia nut Mean ± SEM

Chocolate chip and macadamia nut cookie

22.58 ± 0.45

Godiva chocolate bar with macadamia nut and coconut 33.95 ± 3.12

aData are reported as gram per 100 gram edible portion and are expressed as mean ± SEM (n = 4).

Table 30. Detection of almond and macadamia nut in commercially prepared foods using ELISAa

105

Sum of Squares df

Mean Square F Sig.

Cultivar Between Groups 1.399 7 0.200 12.102 0.000Within Groups 3.535 214 0.017

Total 4.935 221

County Between Groups 0.316 11 0.029 1.773 0.070Within Groups 1.506 93 0.016

Total 1.822 104

Region Between Groups 0.004 1 0.004 0.198 0.657Within Groups 1.819 103 0.018

Total 1.823 104

Harvest year Between Groups 0.082 1 0.082 3.278 0.077Within Groups 1.076 43 0.025

Total 1.158 44

cv. Butte x county Between Groups 44.971 9 4.997 1385.218 0.000Within Groups 0.115 32 0.004

Total 45.087 41

cv. Carmel x county Between Groups 66.364 11 6.033 3656.726 0.000Within Groups 0.064 39 0.002

Total 66.428 50

cv. Mission x county Between Groups 35.665 7 5.095 1189.454 0.000Within Groups 0.081 19 0.004

Total 35.746 26

cv. Nonpareil x county Between Groups 74.880 11 6.807 885.310 0.000Within Groups 0.323 42 0.008

Total 75.203 53

cv. Padre x county Between Groups 25.839 6 4.307 5927.549 0.000Within Groups 0.010 14 0.001

Total 25.849 20

cv. Price x county Between Groups 20.751 6 3.458 3217.790 0.000Within Groups 0.015 14 0.001

Total 20.766 20

Seed Weight

APPENDIX A

ANOVA Tables

Tables 9 and 10. Individual seed weight of select almond seeds

106

Sum of Squares df

Mean Square F Sig.


Total 5018.453 739


Total 1519.183 349


Total 1519.183 349


Total 1075.060 149


Total 3099.457 139


Total 4460.211 169


Total 1939.688 89


Total 4708.512 149


Total 1472.991 69


Total 1641.289 69

Seed Length

Tables 9 and 10. Seed length of select almond seeds

107

Sum of Squares df

Mean Square F Sig.


Total 665.078 739


Total 345.776 349


Total 345.776 349


Total 119.066 149


Total 1714.152 139


Total 2077.746 169


Total 1186.109 89


Total 2335.954 149


Total 885.775 69


Total 832.368 69

Seed Width

Tables 9 and 10. Seed width of select almond seeds

108

Sum of Squares df

Mean Square F Sig.


Total 698.033 739


Total 176.865 349


Total 176.865 349


Total 139.696 149


Total 1310.242 139


Total 1467.318 169


Total 904.756 89


Total 1493.371 149


Total 684.024 69


Total 575.313 69

Seed Thickness

Tables 9 and 10. Seed thickness of select almond seeds

109

Sum of Squares df

Mean Square F Sig.


Total 97.131 221


Total 39.080 104


Total 43.438 104


Total 28.467 44


Total 20.773 41


Total 22.773 50


Total 23.623 26


Total 17.372 53


Total 4.696 20


Total 7.407 20

Moisture content

Tables 9 and 10. Moisture content of select almond seeds

110

Sum of Squares df

Mean Square F Sig.


Total 4113.833 221


Total 1925.007 104


Total 1925.007 104


Total 1068.399 44


Total 645.672 41


Total 854.523 50


Total 172.972 26


Total 1475.149 53


Total 299.399 20


Total 599.392 20

Lipid content

Tables 9 and 10. Lipid content of select almond seeds

111

Sum of Squares df

Mean Square F Sig.


Total 1035.931 221


Total 425.823 104


Total 425.823 104


Total 184.154 44


Total 128.731 41


Total 226.563 50


Total 89.060 26


Total 252.025 53


Total 51.890 20


Total 237.669 20

Protein content

Tables 9 and 10. Protein content of select almond seeds

112

Sum of Squares df

Mean Square F Sig.


Total 7.067 168


Total 4.773 84


Total 4.773 87


Total 2.077 38


Total 0.935 33


Total 3.131 40


Total 0.348 18


Total 1.651 43


Total 0.094 13


Total 0.899 13

Ash content

Tables 9 and 10. Ash content of select almond seeds

113

Sum of Squares df

Mean Square F Sig.


Total 196.565 221


Total 109.556 104


Total 109.556 104


Total 31.340 44


Total 26.093 41


Total 34.929 50


Total 17.854 26


Total 74.634 53


Total 21.845 20


Total 12.090 20

Total soluble sugar content

Tables 9 and 10. Total soluble sugar content of select almond seeds

114

Sum of Squares df

Mean Square F Sig.


Total 0.772 221


Total 0.327 104


Total 0.327 104


Total 0.186 44


Total 0.157 41


Total 0.129 50


Total 0.065 26


Total 0.198 53


Total 0.033 20


Total 0.072 20

Tannin content

Tables 9 and 10. Tannin content of select almond seeds

115

Sum of Squares df

Mean Square F Sig.

Seed weight Between Groups 2.323 6 0.387 10.386 0.000Within Groups 0.522 14 0.037

Total 2.844 20

Seed diameter Between Groups 19.159 6 3.193 2.073 0.069Within Groups 97.057 63 1.541

Total 116.217 69

Seed thickness Between Groups 18.763 6 3.127 2.482 0.032Within Groups 79.362 63 1.260

Total 98.125 69

Moisture Between Groups 2.116 6 3.53E-01 158.412 0.000Within Groups 3.12E-02 14 2.23E-03

Total 2.147 20

Lipid Between Groups 79.025 6 13.171 2.350 0.088Within Groups 78.481 14 5.606

Total 157.506 20

Protein Between Groups 5.139 6 8.57E-01 1.744 0.183Within Groups 6.876 14 4.91E-01

Total 12.016 20

Ash Between Groups 3.09E-01 6 5.15E-02 60.437 0.000Within Groups 1.19E-02 14 8.53E-04

Total 3.21E-01 20

Sugars Between Groups 25.393 6 4.232 282.739 0.000Within Groups 2.10E-01 14 1.50E-02

Total 25.602 20

Tannins Between Groups 2.34E-04 6 3.90E-05 1.299 0.320Within Groups 4.20E-04 14 3.00E-05

Total 6.55E-04 20


Table 11. Physical characteristics and chemical composition of macadamia nut seeds

116

Sum of Squares df

Mean Square F Sig.

Sum of Squares df

Mean Square F Sig.

His Between Groups 59.046 7 8.435 9.637 0.000 29.722 11 2.702 3.500 0.000Within Groups 187.311 214 0.875 71.793 93 0.772

Total 246.357 221 101.515 104Thr Between Groups 1.099 7 0.157 1.979 0.059 3.981 11 0.362 4.844 0.000

Within Groups 16.975 214 0.079 6.949 93 0.075Total 18.074 221 10.930 104

Val Between Groups 5.358 7 0.765 6.115 0.000 8.635 11 0.785 9.352 0.000Within Groups 26.788 214 0.125 7.806 93 0.084

Total 32.146 221 16.441 104Met Between Groups 10.081 7 1.440 1.072 0.383 7.876 11 0.716 9.020 0.000


Ile Between Groups 3.824 7 0.546 5.154 0.000 5.777 11 0.525 11.040 0.000Within Groups 22.679 214 0.106 4.424 93 0.048

Total 26.503 221 10.202 104Leu Between Groups 11.945 7 1.706 6.462 0.000 14.634 11 1.330 7.560 0.000


Phe Between Groups 27.199 7 3.886 4.895 0.000 81.412 11 7.401 26.501 0.000Within Groups 169.878 214 0.794 25.973 93 0.279

Total 197.077 221 107.385 104Lys Between Groups 2.955 7 0.422 5.923 0.000 4.213 11 0.383 13.022 0.000


Trp Between Groups 6.322 7 0.903 6.207 0.000 4.032 11 0.367 4.288 0.000Within Groups 31.135 214 0.145 7.949 93 0.085

Total 37.457 221 11.981 104

Cultivars

Total amino acids

County

Table 12. Cultivar, geographic location, and harvest year variation on the essential amino acid composition of select almond seeds

117

Sum of Squares df

Mean Square F Sig.

Sum of Squares df

Mean Square F Sig.













Trp Between Groups 1.994 1 1.994 20.557 0.000 0.134 1 0.134 1.901 0.175Within Groups 9.987 103 0.097 3.020 43 0.070

Total 11.981 104 3.153 44

Harvest year

Total amino acids

Region

Table 12 continued. Cultivar, geographic location, and harvest year variation on the essential amino acid composition of select almond seeds

118

Sum of Squares df

Mean Square F Sig.

Sum of Squares df

Mean Square F Sig.

Asx Between Groups 78.444 7 11.206 3.887 0.001 159.115 11 14.465 8.249 0.000Within Groups 617.025 214 2.883 163.073 93 1.753

Total 695.469 221 322.189 104Glx Between Groups 268.007 7 38.287 5.157 0.000 327.305 11 29.755 3.953 0.000


Ser Between Groups 1.187 7 0.170 1.630 0.128 1.944 11 0.177 2.152 0.024Within Groups 22.259 214 0.104 7.634 93 0.082

Total 23.446 221 9.578 104Gly Between Groups 13.587 7 1.941 6.965 0.000 8.299 11 0.754 2.223 0.019


Arg Between Groups 94.927 7 13.561 7.601 0.000 72.921 11 6.629 5.566 0.000Within Groups 381.817 214 1.784 110.768 93 1.191

Total 476.744 221 183.689 104Ala Between Groups 58.320 7 8.331 39.238 0.000 8.983 11 0.817 5.636 0.000


Pro Between Groups 28.430 7 4.061 2.784 0.009 91.786 11 8.344 9.541 0.000Within Groups 312.241 214 1.459 81.336 93 0.875

Total 340.671 221 173.122 104Tyr Between Groups 18.022 7 2.575 8.239 0.000 16.635 11 1.512 9.072 0.000


Cys Between Groups 0.460 7 0.066 8.637 0.000 0.213 11 0.019 4.447 0.000Within Groups 1.629 214 0.008 0.405 93 0.004

Total 2.090 221 0.618 104

Total amino acids

Cultivar County

Table 13. Cultivar, geographic location, and harvest year variation on the non-essential amino acid composition of select almond seeds

119

Sum of Squares df

Mean Square F Sig.

Sum of Squares df

Mean Square F Sig.

Asx Between Groups 2.076 1 2.076 0.668 0.416 46.161 1 46.161 29.975 0.000Within Groups 320.112 103 3.108 66.220 43 1.540

Total 322.189 104 112.381 44Glx Between Groups 16.665 1 16.665 1.698 0.196 209.139 1 209.139 47.756 0.000


Ser Between Groups 0.000 1 0.000 0.000 1.000 0.083 1 0.083 1.348 0.252Within Groups 9.578 103 0.093 2.643 43 0.061










Total 0.618 104 0.425 44

Total amino acids

Region Harvest year

Table 13 continued. Cultivar, geographic location, and harvest year variation on the non-essential amino acid composition of select almond seeds

120

Sum of Squares df

Mean Square F Sig.

Sum of Squares df

Mean Square F Sig.

Sum of Squares df

Mean Square F Sig.

His Between Groups 35.220 9 3.913 10.527 0.000 56.325 11 5.120 306.429 0.000 29.487 7 4.212 7.404 0.000Within Groups 11.896 32 0.372 0.652 39 0.017 10.809 19 0.569

Total 47.116 41 56.977 50 40.297 26Thr Between Groups 1.075 9 0.119 15.640 0.000 2.942 11 0.267 22.751 0.000 2.794 7 0.399 30.131 0.000

Within Groups 0.244 32 0.008 0.459 39 0.012 0.252 19 0.013Total 1.319 41 3.401 50 3.046 26

Val Between Groups 5.708 9 0.634 17.609 0.000 5.063 11 0.460 11.447 0.000 4.634 7 0.662 9.885 0.000Within Groups 1.152 32 0.036 1.568 39 0.040 1.272 19 0.067

Total 6.860 41 6.631 50 5.906 26Met Between Groups 2.134 9 0.237 3.671 0.003 9.935 11 0.903 45.512 0.000 18.762 7 2.680 409.819 0.000


Ile Between Groups 5.611 9 0.623 32.676 0.000 3.190 11 0.290 7.982 0.000 1.723 7 0.246 2.750 0.038Within Groups 0.611 32 0.019 1.417 39 0.036 1.701 19 0.090

Total 6.221 41 4.607 50 3.425 26Leu Between Groups 10.272 9 1.141 15.975 0.000 8.359 11 0.760 3.760 0.001 7.926 7 1.132 7.067 0.000


Phe Between Groups 26.262 9 2.918 2.811 0.015 40.817 11 3.711 13.529 0.000 13.286 7 1.898 51.966 0.000Within Groups 33.216 32 1.038 10.697 39 0.274 0.694 19 0.037

Total 59.478 41 51.514 50 13.980 26Lys Between Groups 2.288 9 0.254 8.808 0.000 2.027 11 0.184 8.935 0.000 0.650 7 0.093 3.094 0.024


Trp Between Groups 5.825 9 0.647 12.525 0.000 6.969 11 0.634 23.229 0.000 1.826 7 0.261 65.403 0.000Within Groups 1.654 32 0.052 1.064 39 0.027 0.076 19 0.004

Total 7.479 41 8.033 50 1.902 26

Total amino acids

cv. Butte x county cv. Carmel x county cv. Mission x county

Table 14. Interaction of cultivar and geographic location on the essential amino acid composition of select almond seeds

121

Sum of Squares df

Mean Square F Sig.

Sum of Squares df

Mean Square F Sig.

Sum of Squares df

Mean Square F Sig.













Trp Between Groups 4.069 11 0.370 19.012 0.000 8.150 6 1.358 1246.224 0.000 6.844 6 1.141 198.557 0.000Within Groups 0.817 42 0.019 0.015 14 0.001 0.080 14 0.006

Total 4.887 53 8.166 20 6.925 20

Total amino acids

cv. Nonpareil x county cv. Padre x county cv. Price x county

Table 14 continued. Interaction of cultivar and geographic location on the essential amino acid composition of select almond seeds

122

Sum of Squares df

Mean Square F Sig.

Sum of Squares df

Mean Square F Sig.

Sum of Squares df

Mean Square F Sig.

Asx Between Groups 79.289 9 8.810 20.484 0.000 90.616 11 8.238 14.785 0.000 66.119 7 9.446 5.105 0.002Within Groups 13.763 32 0.430 21.729 39 0.557 35.156 19 1.850

Total 93.052 41 112.345 50 101.275 26Glx Between Groups 257.378 9 28.598 15.417 0.000 396.307 11 36.028 32.450 0.000 65.564 7 9.366 1.722 0.164


Ser Between Groups 2.212 9 0.246 16.121 0.000 3.623 11 0.329 13.253 0.000 1.922 7 0.275 13.121 0.000Within Groups 0.488 32 0.015 0.969 39 0.025 0.398 19 0.021

Total 2.700 41 4.593 50 2.320 26Gly Between Groups 8.725 9 0.969 19.910 0.000 14.687 11 1.335 30.795 0.000 7.577 7 1.082 17.026 0.000


Arg Between Groups 81.134 9 9.015 15.220 0.000 84.435 11 7.676 44.332 0.000 37.586 7 5.369 3.760 0.010Within Groups 18.953 32 0.592 6.753 39 0.173 27.135 19 1.428

Total 100.087 41 91.188 50 64.721 26Ala Between Groups 6.609 9 0.734 7.117 0.000 9.479 11 0.862 15.420 0.000 2.902 7 0.415 20.756 0.000


Pro Between Groups 39.187 9 4.354 3.291 0.006 54.775 11 4.980 16.999 0.000 29.776 7 4.254 15.919 0.000Within Groups 42.343 32 1.323 11.424 39 0.293 5.077 19 0.267

Total 81.530 41 66.199 50 34.854 26Tyr Between Groups 0.891 9 0.099 0.793 0.625 16.720 11 1.520 9.781 0.000 43.383 7 6.198 151.154 0.000


Cys Between Groups 0.225 9 0.025 5.729 0.000 0.383 11 0.035 47.788 0.000 0.294 7 0.042 4.020 0.007Within Groups 0.140 32 0.004 0.028 39 0.001 0.199 19 0.010

Total 0.365 41 0.411 50 0.493 26

Total amino acids


Table 15. Interaction of cultivar and geographic location on the non-essential amino acid composition of select almond seeds

123

Sum of Squares df

Mean Square F Sig.

Sum of Squares df

Mean Square F Sig.

Sum of Squares df

Mean Square F Sig.

Asx Between Groups 152.896 11 13.900 9.391 0.001 63.568 6 10.595 597.517 0.000 61.439 6 10.240 3032.561 0.000Within Groups 62.163 42 1.480 0.248 14 0.018 0.047 14 0.003

Total 215.058 53 63.816 20 61.486 20Glx Between Groups 502.053 11 45.641 12.602 0.000 91.033 6 15.172 357.496 0.000 73.484 6 12.247 1336.511 0.000


Ser Between Groups 3.923 11 0.357 13.063 0.128 6.936 6 1.156 2933.940 0.000 1.766 6 0.294 385.515 0.000Within Groups 1.147 42 0.027 0.006 14 0.000 0.011 14 0.001










Total 0.314 53 0.282 20 0.134 20

Total amino acids


Table 15 continued. Interaction of cultivar and geographic location on the non-essential amino acid composition of select almond seeds

124

Sum of Squares df

Mean Square F Sig.

Asx Between Groups 3.010 6 0.502 4.195 0.013Within Groups 1.674 14 0.120

Total 4.683 20Glx Between Groups 60.872 6 10.145 24.257 0.000

Within Groups 5.856 14 0.418Total 66.727 20

Ser Between Groups 2.692 6 0.449 7.287 0.000Within Groups 0.862 14 0.062

Total 3.554 20Gly Between Groups 3.470 6 0.578 15.619 0.000


His Between Groups 0.534 6 0.089 2.513 0.073Within Groups 0.496 14 0.035

Total 1.030 20Arg Between Groups 6.750 6 1.125 3.680 0.021


Thr Between Groups 4.996 6 0.833 11.197 0.000Within Groups 1.041 14 0.074

Total 6.038 20Ala Between Groups 1.010 6 0.168 1.392 0.285


Total amino acids

Table 16. Amino acid composition of select macadamia nut seeds

125

Sum of Squares df

Mean Square F Sig.

Pro Between Groups 5.311 6 0.885 3.911 0.017Within Groups 3.168 14 0.226

Total 8.480 20Tyr Between Groups 1.507 6 0.251 1.155 0.383


Val Between Groups 1.354 6 0.226 27.413 0.000Within Groups 0.115 14 0.008

Total 1.469 20Met Between Groups 0.479 6 0.080 10.429 0.000


Cys Between Groups 0.222 6 0.037 2.659 0.062Within Groups 0.194 14 0.014

Total 0.416 20Ile Between Groups 0.474 6 0.079 6.675 0.000


Leu Between Groups 1.158 6 0.193 4.119 0.014Within Groups 0.656 14 0.047

Total 1.814 20Phe Between Groups 0.792 6 0.132 4.585 0.009


Lys Between Groups 0.948 6 0.158 3.683 0.021Within Groups 0.600 14 0.043

Total 1.548 20Trp Between Groups 0.347 6 0.058 14.899 0.000


Total amino acids

Table 16 continued. Amino acid composition of select macadamia nut seeds

126

Sum of Squares df

Mean Square F Sig.

Sum of Squares df

Mean Square F Sig.













Free amino acids

Cultivar County

Table 19. Cultivar, geographic location, and harvest year variation on the free (essential) amino acid composition of select almond seeds

127

Sum of Squares df

Mean Square F Sig.

Sum of Squares df

Mean Square F Sig.













Harvest year

Free amino acids

Region

Table 19 continued. Cultivar, geographic location, and harvest year variation on the free (essential) amino acid composition of select almond seeds

128

Sum of Squares df

Mean Square F Sig.

Sum of Squares df

Mean Square F Sig.

Asn Between Groups 151020.626 7 21574.375 7.051 0.000 208633.747 11 18966.704 6.090 0.000Within Groups 654761.962 214 3059.635 289617.911 93 3114.171

Total 805782.589 221 498251.658 104Asp Between Groups 5562.319 7 794.617 1.393 0.210 9501.092 11 863.736 1.986 0.038


Glu Between Groups 5158.113 7 736.873 2.154 0.040 12688.980 11 1153.544 4.335 0.000Within Groups 73223.125 214 342.164 24748.948 93 266.118

Total 78381.238 221 37437.928 104Ser Between Groups 280.282 7 40.040 3.412 0.002 463.567 11 42.142 4.079 0.000


Gln Between Groups 391.054 7 55.865 5.072 0.000 580.927 11 52.812 6.685 0.000Within Groups 2356.937 214 11.014 734.687 93 7.900










Total 384.907 221 189.250 104

Free amino acids

Cultivar County

Table 20. Cultivar, geographic location, and harvest year variation on the free (non-essential) amino acid composition of select almond seeds

129

Sum of Squares df

Mean Square F Sig.

Sum of Squares df

Mean Square F Sig.

Asn Between Groups 18930.330 1 18930.330 4.068 0.046 49626.988 1 49626.988 26.589 0.000Within Groups 479321.330 103 4653.610 80257.792 43 1866.460

Total 498251.660 104 129884.780 44Asp Between Groups 3.350 1 3.350 0.007 0.934 6390.569 1 6390.569 58.996 0.000


Glu Between Groups 2594.550 1 2594.550 7.670 0.007 1037.144 1 1037.144 7.532 0.009Within Groups 34843.380 103 338.290 5920.852 43 137.694

Total 37437.930 104 6957.996 44Ser Between Groups 236.460 1 236.460 20.508 0.000 2.841 1 2.841 0.340 0.563


Gln Between Groups 276.930 1 276.930 27.610 0.000 4.138 1 4.138 0.403 0.529Within Groups 1038.680 103 10.030 441.491 43 10.267










Total 189.250 104 96.614 44

Harvest year

Free amino acids

Region

Table 20 continued. Cultivar, geographic location, and harvest year variation on the free (non-essential) amino acid composition of select almond seeds

130

Sum of Squares df

Mean Square F Sig.

Sum of Squares df

Mean Square F Sig.

Sum of Squares df

Mean Square F Sig.













Free amino acids

cv. Butte x county cv. Carmel x county cv. Mission x countyTable 21. Interaction of cultivar and geographic location on the free (essential) amino acid composition of select almond seeds

131

Sum of Squares df

Mean Square F Sig.

Sum of Squares df

Mean Square F Sig.

Sum of Squares df

Mean Square F Sig.













Free amino acids


Table 21 continued. Interaction of cultivar and geographic location on the free (essential) amino acid composition of select almond seeds

132

Sum of Squares df

Mean Square F Sig.

Sum of Squares df

Mean Square F Sig.

Sum of Squares df

Mean Square F Sig.

Asn Between Groups 92638.935 9 10293.215 8.831 0.000 317363.107 11 28851.192 31.775 0.000 4339.499 7 619.928 0.327 0.932Within Groups 37297.063 32 1165.533 35411.919 39 907.998 35975.351 19 1893.440

Total 129935.998 41 352775.026 50 40314.849 26Asp Between Groups 32685.905 9 3631.767 52.017 0.000 28638.449 11 2603.495 27.001 0.000 3272.309 7 467.473 3.859 0.009


Glu Between Groups 10224.551 9 1136.061 6.349 0.000 15198.314 11 1381.665 15.493 0.000 3348.058 7 478.294 5.468 0.002Within Groups 5726.162 32 178.943 3478.070 39 89.181 1661.893 19 87.468

Total 15950.713 41 18676.384 50 5009.950 26Ser Between Groups 130.013 9 14.446 2.124 0.057 387.105 11 35.191 4.069 0.000 203.791 7 29.113 6.682 0.000


Gln Between Groups 217.253 9 24.139 1.996 0.073 493.495 11 44.863 15.536 0.000 181.831 7 25.976 8.459 0.000Within Groups 387.047 32 12.095 112.618 39 2.888 58.343 19 3.071










Total 118.534 41 123.900 50 29.829 26

Free amino acids


Table 22. Interaction of cultivar and geographic location on the free (non-essential) amino acid composition of select almond seeds

133

Sum of Squares df

Mean Square F Sig.

Sum of Squares df

Mean Square F Sig.

Sum of Squares df

Mean Square F Sig.

Asn Between Groups 92239.777 11 8385.434 5.880 0.000 44667.206 6 7444.534 8.608 0.001 42207.438 6 7034.573 11.151 0.000Within Groups 59893.111 42 1426.026 12107.317 14 864.808 8831.593 14 630.828

Total 152132.887 53 56774.523 20 51039.031 20Asp Between Groups 8863.876 11 805.807 3.891 0.001 11661.431 6 1943.572 21.518 0.000 21625.017 6 3604.169 216.751 0.000


Glu Between Groups 9666.915 11 878.810 3.237 0.003 2725.661 6 454.277 3.463 0.026 11540.278 6 1923.380 68.881 0.000Within Groups 11402.826 42 271.496 1836.756 14 131.197 390.928 14 27.923

Total 21069.741 53 4562.417 20 11931.206 20Ser Between Groups 441.518 11 40.138 4.879 0.000 60.984 6 10.164 1.197 0.363 388.874 6 64.812 20.309 0.000


Gln Between Groups 413.893 11 37.627 4.613 0.000 173.521 6 28.920 9.696 0.000 199.464 6 33.244 10.656 0.000Within Groups 342.593 42 8.157 41.756 14 2.983 43.678 14 3.120










Total 66.543 53 24.609 20 13.405 20

Free amino acids


Table 22 continued. Interaction of cultivar and geographic location on the free (non-essential) amino acid composition of select almond seeds

134

4C10 mAb sandwich ELISA

Sum of

Squares df

Mean

Square F Sig.


Total 238620.855 221


Total 28619.390 104


Total 28619.390 104

Harvest Year Between Groups 157.598 1 157.598 0.267 0.608Within Groups 25424.834 43 591.275

Total 25582.431 44Sum of

Squares df

Mean

Square F Sig.


Total 951278.073 221


Total 148742.918 104


Total 148742.920 104


Total 94457.508 44Sum of

Squares df

Mean

Square F Sig.


Total 95652.800 221


Total 29034.884 104


Total 29034.880 104


Total 19681.608 44

Rabbit anti-AMP inhibition

ELISA

Rabbit anti-almond inhibition

ELISA

Table 24. Effect of cultivar, geographic location, and harvest year on the immunoreactivity of select almond cultivars assessed by ELISA

135

ELISA cv. Butte x countySum of Squares df Mean Square F Sig.

4C10 mAb Between Groups 14550.175 9 1616.686 15.919 0.000Within Groups 3249.914 32 101.560

Total 17800.089 41

R anti-AMP pAb Between Groups 41859.623 9 4651.069 5.804 0.000Within Groups 25645.209 32 801.413

Total 67504.832 41

R anti-almond pAb Between Groups 2321.193 9 257.910 1.068 0.412Within Groups 7729.421 32 241.544

Total 10050.615 41

ELISAcv. Carmel x

countySum of Squares df Mean Square F Sig.


Total 24254.811 50


Total 124898.917 50


Total 17991.806 50

ELISA cv. Mission x



Total 92011.324 26


Total 474679.619 26


Total 17907.453 26

Table 25. Interaction of cultivar and geographic location on the immunoreactivity of select almond cultivars assessed by ELISA

136

ELISA cv. Nonpareil x



Total 21357.495 53


Total 105629.511 53


Total 13636.222 53

ELISA cv. Padre x



Total 14643.313 20


Total 72745.189 20


Total 15063.402 20

ELISA cv. Price x countySum of Squares df Mean Square F Sig.


Total 62989.005 20


Total 88878.258 20


Total 11408.724 20

Table 25 continued. Interaction of cultivar and geographic location on the immunoreactivity of select almond cultivars assessed by ELISA

137

mAb 4C10 Cross-reactivity

Sum of Squares df

Mean Square F Sig.

Between Groups 6.77E-02 50 1.35E-03 176.289 0.000Within Groups 3.92E-04 51 7.68E-06

Total 6.81E-02 101


Sum of Squares df

Mean Square F Sig.

Between Groups 14022.366 6 2337.061 9.154 0.000Within Groups 5361.384 21 255.304

Total 19383.750 27

Macadamia pAb Cross-reactivity

Sum of Squares df

Mean Square F Sig.

Between Groups 4.176 68 6.14E-02 2.167 0.001Within Groups 1.955 69 2.83E-02

Total 6.131 137

Processed almond samples

Sum of Squares df

Mean Square F Sig.


Total 18547.538 38

Figure 9. Immunoreactivity (%) of macadamia nut varieties assessed by inhibition ELISA.

Figure 10. Cross-reactivity of mAb 4C10 with select foods/ingredients assessed by sandwich ELISA (C).

Table 27. Cross-reactivity of select foods/ingredients with Protein G purified rabbit anti-macadamia nut IgG assessed by inhibition ELISA

Figure 12. Effect of thermal processing on antigenticity of AMP assessed by (C) mAb 4C10 sandwich ELISA.

138

pH exposed almond flour (neutralized)

Sum of Squares df

Mean Square F Sig.


Total 33008.077 15

pH exposed almond flour (not

neutralized)Sum of Squares df

Mean Square F Sig.


Total 46072.288 23

Figure 13. Effect of pH on the antigenicity of AMP assessed by (C and F) mAb 4C10 sandwich ELISA. C: Defatted almond seed flour exposed to desired pH value and neutralized prior to analysis. F: Defatted almond seed flour exposed to desired pH value and analyzed directly (not neutralized). Figure 14. Effect of thermal processing on immunoreactivity of macadamia nut proteins assessed by ELISA and Dot blot.

Sum of Squares df

Mean Square F Sig.

ELISA Between Groups 90048.507 18 5002.695 17.035 0.000Within Groups 16739.717 57 293.679

Total 106788.223 75

Dot blot Between Groups 47702.256 18 2650.125 109.686 0.000Within Groups 1377.175 57 24.161

Total 49079.432 75

Processed macadamia nut samples

139

pH exposed macadamia nut flour

(not neutralized)Sum of Squares df

Mean Square F Sig.


Total 48290.159 27

pH exposed macadamia nut flour

(neutralized)Sum of Squares df

Mean Square F Sig.


Total 56341.862 29

Figure 15. Effect of pH on immunoreactivity of macadamia nut proteins assessed by (C and F) inhibition ELISA. C: Defatted macadamia nut flour exposed to desired pH value and analyzed directly (not neutralized). F: Defatted macadamia nut flour exposed to desired pH value and neutralized prior to analysis.

140

Sum of Squares df

Mean Square F Sig.

Unspiked Between Groups 1.16E-01 15 7.74E-03 9.590 0.000Within Groups 2.58E-02 32 8.08E-04

Total 1.42E-01 47

10 �g extract Between Groups 509.361 15 33.957 21.614 0.000Within Groups 50.274 32 1.571

Total 559.635 47

1 �g extract Between Groups 4.454 15 2.97E-01 67.892 0.000Within Groups 1.40E-01 32 4.37E-03

Total 4.594 47

0.1 �g extract Between Groups 1.56E-01 15 1.04E-02 15.194 0.000Within Groups 2.19E-02 32 6.84E-04

Total 1.93E-01 47

100 �g flour Between Groups 2778.873 9 308.764 58.467 0.000Within Groups 105.619 20 5.281

Total 2884.492 29

10 �g flour Between Groups 22.624 9 2.514 76.416 0.000Within Groups 6.58E-01 20 3.29E-02

Total 23.282 29

1 �g flour Between Groups 2.26E-01 9 2.51E-02 12.448 0.000Within Groups 4.04E-02 20 2.02E-03

Total 2.66E-01 29

Almond mAb 4C10 ELISA

Table 28. Detection of AMP in foods spiked with almond seed protein extract or defatted almond seed flour

141

Sum of Squares df

Mean Square F Sig.

Unspiked Between Groups 3.22E-01 12 2.69E-02 35.436 0.000Within Groups 1.97E-02 26 7.58E-04

Total 3.42E-01 38

10 �g extract Between Groups 388.790 12 32.399 18.285 0.000Within Groups 46.071 26 1.772

Total 434.860 38

1 �g extract Between Groups 7.246 12 6.04E-01 49.476 0.000Within Groups 3.17E-01 26 1.22E-02

Total 7.563 38

0.1 �g extract Between Groups 3.97E-01 12 3.31E-02 250.864 0.000Within Groups 3.43E-03 26 1.32E-04

Total 4.01E-01 38

100 �g flour Between Groups 1179.097 7 168.442 29.885 0.000Within Groups 90.180 16 5.636

Total 1269.277 23

10 �g flour Between Groups 9.174 7 1.311 20.944 0.000Within Groups 1.001 16 6.26E-02

Total 10.175 23

1 �g flour Between Groups 4.76E-01 7 6.80E-02 28.793 0.000Within Groups 3.78E-02 16 2.36E-03

Total 5.14E-01 23

Macadamia pAb ELISA

Table 29. Detection of macadamia nut protein in foods spiked with macadamia nut protein extract or defatted macadamia nut flour

142

APPENDIX B

Animal Research Approval

145

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BIOGRAPHICAL SKETCH

EDUCATION Doctor of Philosophy, Food Science May 2003-August 2008 Florida State University, Tallahassee, Florida Master of Science, Nutrition & Food Science August 2001-May 2003 Florida State University, Tallahassee, Florida Bachelor of Science, Nutrition & Food Science August 1997-April 2001 Florida State University, Tallahassee, Florida RESEARCH EXPERIENCE Food Science Research Laboratory August 2001-April 2008 Florida State University, Tallahassee, Florida Toxicology Laboratory Fall 2000 Florida Department of Agriculture, Tallahassee, Florida TEACHING EXPERIENCE Graduate Teaching Assistant August 2001-April 2008 Florida State University, Tallahassee, Florida Total of 17 semesters teaching approximately 800 students Courses taught:

- Foods (FOS 3026) - Wellness/Risk Reduction (HSC 4711) - Food Science Laboratory (FOS 4114C) - Foods Laboratory (FOS 3026L) - Food and Society (HUN 2125)

WORK EXPERIENCE Johnny G. Certified Spinning Instructor March 2004-August 2008 Women’s World Aerobic Fitness and Weight Loss Center Tallahassee, Florida Nutrition Instructor January 2003-July 2006 Women’s World Aerobic Fitness and Weight Loss Center Tallahassee, Florida PUBLICATIONS

- Shridhar K. Sathe, Erin K. Monaghan, Harshal H. Kshrisagar, Mahesh Venkatachalam. (2008) Chemical Composition of Edible Nut Seeds and its Implications in Human Health. In: Shahidi, F. and Alasalvar, C. (Eds.), Tree Nut Nutraceuticals and Phytochemicals. CRC Press Inc., Boca Raton, FL. ISBN: 0849337356, In press.

- Enzyme Linked ImmunoSorbent Assay (ELISA) for Sulfur-Rich Protein (SRP) in Soybean (Glycine max L.) and Certain Other Edible Plant Seeds. Erin K. Monaghan, Mahesh Venkatachalam, Margaret Seavy, Kenneth H. Roux, and Shridhar K. Sathe, Journal of

Agricultural and Food Chemistry, 2008, Volume 56, Issue 3, pp. 765-777.

165

- Effects of Processing on Antigenic Stability of Cashew Nut (Anacardium occidentale L.) Proteins. Mahesh Venkatachalam, Erin K. Monaghan, Harshal H. Kshrisagar, Kenneth H. Roux, and Shridhar K. Sathe. Journal of Agricultural and Food Chemistry, In Press.

- A Sensitive Enzyme Linked Immuno-Sorbent Assay (ELISA) for Macadamia (Macadamia

integrifolia) Nut Detection. Erin K. Monaghan, Kenneth H. Roux, and Shridhar K. Sathe. In

preparation.

- Chemical Composition of Almond (Prunus dulcis L.) Cultivars Grown in California. Erin K.

Monaghan, Kenneth H. Roux, and Shridhar K. Sathe. In preparation. INSTITUTE OF FOOD TECHNOLOGISTS (IFT) PRESENTATIONS - Competitive Inhibition Enzyme Linked Immuno-Sorbent Assay (ELISA) for Brazil

(Bertholletia excelsa) Nut Detection. Girdhari M. Sharma, Erin K. Monaghan, Kenneth H. Roux, and Shridhar K. Sathe. New Orleans, Louisiana, June 28-July 1, 2008, Accepted into

Food Safety & Toxicology Division Student Paper Competition - A Sensitive and Specific Monoclonal Antibody (mAb)-based Immunoassay for Detection of a

Stable 63 kDa Almond Major Protein (AMP) Polypeptide in Almonds (Prunus dulcis L.), Abstract #193-29. Erin K. Monaghan, Kenneth H. Roux and Shridhar K. Sathe. Chicago, Illinois, July 28-August 2, 2007, Accepted into Food Safety & Toxicology Division Student

Paper Competition - A Sensitive Enzyme Linked Immuno-Sorbent Assay (ELISA) for Macadamia (Macadamia

integrifolia) Nut Detection, Abstract #187-32. Erin K. Monaghan, Kenneth H. Roux and Shridhar K. Sathe. Chicago, Illinois, July 28-August 2, 2007

- Immunoaffinity Purification of SRP-like Proteins from Plant Seeds using Anti-Soybean

(Glycine max L.) Sulfur-Rich Protein Rabbit Polyclonal Antibodies (pAbs), Abstract #007-19. Erin K. Monaghan, Kenneth H. Roux and Shridhar K. Sathe. Chicago, Illinois, July 28-August 2, 2007

- Effects of Environmental Factors on Almond (Prunus dulcis L.) Fatty Acid Composition,

Abstract #188-28. Shridhar K. Sathe, Navindra Seeram, Erin K. Monaghan, Harshal H. Kshirsagar, David Heber, and Karen Lapsley. Chicago, Illinois, July 28-August 2, 2007

- Monoclonal Antibody (mAb)-Based Immunoassay for Quantification of the Major Storage

Protein (AMP) in California Grown Almonds (Prunus dulcis L.), Abstract #091-14. Erin K.

Monaghan, Jason Robotham, Kenneth H. Roux and Shridhar K. Sathe. Orlando, Florida, June 24-28, 2006, Orally presented as part of ‘Food Allergens Pavilion’

- Monoclonal Antibody (mAb)-Based Immunoassay to Assess the Impact of Thermal Processing

on Cashew Nut (Anacardium occidentale L.) Allergens Ana o 1, Ana o 2, and Ana o 3, Abstract #091-13. Erin K. Monaghan, Mahesh Venkatachalam, Harshal H. Kshirsagar, Jason Robotham, Kenneth H. Roux and Shridhar K. Sathe. Orlando, Florida, June 24-28, 2006,

Orally presented as part of ‘Food Allergens Pavilion’

- Chemical Composition of Macadamia (Macadamia intergrifolia) Cultivars Grown in Hawaii, Abstract #039F-26. Erin K. Monaghan, Kenneth H. Roux and Shridhar K. Sathe. Orlando, Florida, June 24-28, 2006

166

- Chemical Composition of Almond (Prunus dulcis L.) Cultivars Grown in Various California Counties, Abstract #18C-27. Erin K. Monaghan and Shridhar K. Sathe. New Orleans, Louisiana, July 15-20, 2005

- Chemical Composition of Tree Nuts, Abstract #67C-22. Mahesh Venkatachalam, Erin K.

Monaghan, Harshal H. Kshirsagar, and Shridhar K. Sathe. Las Vegas, Nevada, July 12-16, 2004

- Detection of Cross Reactive Proteins in various Plant Seeds to the Sulfur-Rich Protein in

Soybeans (Glycine max L.), Abstract #60B-6. Erin K. Monaghan, Mahesh Venkatachalam, Kenneth H. Roux, and Shridhar K. Sathe. Chicago, Illinois, July 12-16, 2003

- Enzyme Linked ImmunoSorbent Assay (ELISA) for Sulfur-Rich Protein (SRP) in Soybeans

(Glycine max L.), Abstract #30C-12. Erin K. Monaghan, Mahesh Venkatachalam, Suzanne S. Teuber, Kenneth H. Roux, and Shirdhar K. Sathe. Anaheim, California, June 30-July 3, 2002

PROFESSIONAL DEVELOPMENT ACTIVITIES

- Trained/Supervised undergraduate recruits and junior colleagues, 2006-2008

- Developed Nutrition & Health chapter as part of the Palm Beach Law Enforcement Academy fitness manual, November 2007

- Attended “Assessing the Effects of Food Processing on Allergenic Potential Workshop” in Estoril, Portugal. Organized by ILSI Health and Environmental Sciences Institute (HESI), June 2006

- Volunteer for College of Human Science’s Centennial Celebration, Spring 2006 - Presenter for College of Human Science’s Research & Creativity Day, February 2005 & 2006

- Nutrition, Food and Exercise Science Representative for the Graduate Student Advisory Council (GSAC), 2003-2006

- Certificate in College Teaching from the Department of Educational Leadership and Policy Studies, 2003-2005

- Developed Nutrition & Health manual for Women’s World Aerobic Fitness and Weight Loss Center, Spring 2004

- Judge for the 2004 Capital Regional Science & Engineering Fair, February 2004

- Teaching Certificate from the Program for Instructional Excellence (PIE), August 2003 - Member of Institute of Food Technologists, Fall 2000-present HONORS AND AWARDS

- Awarded 1st place in the Food Safety & Toxicology Division Student Paper Competition, IFT National Conference, Chicago, Illinois, July 28-August 2, 2007

- Outstanding Teaching Assistant Award nominee, Spring 2003 and Spring 2004 - Department of Nutrition, Food and Exercise Science speaker for College of Human Science’s

Student Convocation, Spring 2001

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