ORIGINAL PAPER

Characterization of Free and Bound Lipids among Four CornGenotypes as Affected by Drying and Storage Temperatures

Karamatollah Rezaei • Trust Beta • Carl J. Bern •

Pamela J. White

Received: 7 July 2011 / Revised: 9 December 2011 / Accepted: 2 January 2012 / Published online: 21 January 2012

� AOCS 2012

Abstract For whole grains, the most sensitive compo-

nents to the environmental changes are the lipids. In the

current study, the effects of drying temperature (27 and

93 �C) and storage temperature (4 and 27 �C) on the fatty

acid (FA) levels and lipid classes of endosperm lipids on

four selected corn genotypes were investigated during a

12-month storage period. Storage temperature indicated

greater impact on the FA composition than did the drying

temperature. The ratio of saturated to unsaturated fatty

acids, which was around 0.20–0.22 levels in the free lipid

(FL) fractions of all corn types studied here, did not change

significantly due to the drying and storage temperatures.

However, in the bound lipid (BL) fractions, it was changed

by a change in the drying and storage temperature in some

of the corn types. Some changes were also found in the

lipid classes within the FL and BL fractions of the studied

corn samples. No lysophosphatidylcholine (LPC) was

found in the FL fractions. In the BL fractions of two of the

corn samples, the level of free fatty acids (FFA) increased

more likely due to the deterioration of LPC. The results

of the current study indicated a possible migration of

triglycerides and FFA between the FL and BL fractions due

to drying and storage at higher temperatures.

Keywords Bound lipids � Corn � Drying � Endosperm �Free lipids � Post-harvest � Storage

Introduction

The food industry relies on a consistent supply of high-

quality grains to maintain product properties and to avoid

consequent changes in the processing parameters. To fulfill

this need, grain producers must follow certain post-harvest

drying and storage procedures so that grains can retain a

consistent and high level of quality. For whole grains, the

most sensitive components to the environmental changes

are the lipids. The impact of drying and storage conditions

on the lipid components of the grain can in turn influence

the processing properties of the grains, which may also

adversely impact the consumer acceptance of the end

products. In particular, the free lipids (FL) and the bound

lipids (BL) inside the grains can undergo certain changes

that influence the functionality and the sensory attributes of

the final products. For example, aging corn and sorghum

grains at 50 and 60 �C decreased the amounts of pericarp

removed from these grains, resulting in cooking losses [1].

In general, FL include those lipid components freely

available on the starch surfaces and can be easily removed

using simple extraction procedures such as Soxhlet and

maceration. The BL include lipids integrated in the starch

structure whose removal requires extra efforts to release

them from starch. FL are composed of triglycerides (TG),

glycolipids, phospholipids and free fatty acids (FFA) while

K. Rezaei (&)

Department of Food Science, Engineering and Technology,

University of Tehran, 31587-77871 Karaj, Iran

e-mail: krezaee@ut.ac.ir

T. Beta

Department of Food Science, University of Manitoba,

Winnipeg, MB R3T 2N2, Canada

C. J. Bern

Department of Agriculture and Biosystems Engineering,

Iowa State University, Ames, IA 50011, USA

P. J. White

Department of Food Science and Human Nutrition and Center

for Crops Utilization Research, Iowa State University,

Ames, IA 50011-1061, USA

123

J Am Oil Chem Soc (2012) 89:1201–1210

DOI 10.1007/s11746-012-2011-4

BL mainly include lysophospholipids and FFA integrated in

the starch structure [2]. The percentage of total lipids

extracted from samples may be different due to the drying

and storage temperatures and also due to the drying and

storage periods and also the extraction period. Despite the

obvious importance of the impact of the lipid changes on

the quality of corn, there are no reports in the literature that

quantify FL and BL in the endosperm of different corn

genotypes and provide their changes due to drying and

storage conditions. The corn mutants, waxy and high-

amylose, along with normal corn genotypes, are widely

used in the industry; thus, knowledge about the lipid

changes in these corn types during drying and storage is

needed to fully understand their possible impacts on the

processing of corn grains in food applications. Formation

of amylose–lipid inclusion complexes [3] and amylose–

lipid–protein component interactions have demonstrated to

contribute the endosperm functional properties [4, 5].

Chemical shifts of 13C NMR in lipid–amylose inclusion

complexes were different for different fatty acids (FA)

indicating different binding properties between amylose

and different FA [3]. Zhang and Hamaker [4] and Zhang

et al. [5] showed that water absorption and the relative

solubilities of sorghum starch were affected by the type of

FFA present. They also reported the formation of three-way

structures among starch, protein and FFA, and indicated

that FFA and starch contributions in these three-component

interactions were of the highest importance to starch func-

tionality. In a study on the corn starches from different

sources, Liu et al. [6] showed that the gelatinization

behaviors of corn starches were dependent on the ratio of

amylose/amylopectin as well as on their moisture and lipid

contents. Therefore, since the amylose content, type of FA

and their concentrations are different among different corn

types, the objective of this study was to determine the

impact of drying and storage temperatures and their com-

bined effects on the FA compositions as well as the lipid

classes of the FL and BL components of the endosperm

lipids in waxy, high-amylose and normal corn genotypes.

Materials and Methods

Corn Types and Treatments

The effects of drying temperature (27 and 93 �C) and

storage temperature (4 and 27 �C for 12 months) and also

combined effects of drying (at 93 �C) and storage (at 27 �C

for 12 months) were studied using two genetically paired

corn types, normal-1 (Pioneer 3335) and waxy (Pioneer

33A63). For comparison purposes, a second genetic pair,

normal-2 (B73 9 Oh43) and high-amylose (B73ae 9

Oh43ae), were also investigated for the combined effects of

drying at 93 �C and storage at 27 �C for 12 months. Nor-

mal-1 and waxy corn seeds, both from the same genetic

parents, were obtained from Pioneer Hi-Bred International

(Johnston, IA). Normal-2 and high-amylose corn seeds

were from the same genetic parents and were kindly pro-

vided by Dr. David Glover, formerly with Purdue Univer-

sity. All corn types were grown at the Iowa State University

Agricultural Experiment Station (Ames, IA) plots during

the same year and combine-harvested at about 21% mois-

ture level. All moistures are on a %wet basis. Control

samples were dried at 27 �C and maintained under refri-

gerated conditions for 12 months prior to the analysis.

According to the Megazyme amylose/amylopectin assay kit

[7], the amylose contents of these four corn genotypes were

20.4% for normal-1, 20.9% for normal-2, 1.9% for waxy

and 42.2% for high-amylose.

To study the drying effect, freshly harvested corn was

dried at two different temperatures selected from the

temperature conditions commonly used by corn producers.

The seeds from each corn type were divided into two parts.

One part was dried with natural air in screened-bottom

trays by circulating ambient air (about 27 �C) through the

chamber at a rate of 2.2 m3/min/Mg until corn moisture

was reduced to about 12%. Moisture loss was determined

by weighing the corn before and after drying. The second

part was dried by using an oven dryer. In this case, corn

was placed on screened-bottom trays to a depth of

approximately 5 cm and positioned in the chamber. Air

at 93 �C was forced through the chamber at a rate of

89 m3/min/Mg until corn moisture was reduced to about

10%. The higher flow rate for the latter was to allow the

rapid removal of moisture accumulated due to higher

temperature and to avoid torching the sample. The hot

grain was rapidly cooled by using forced ambient air at a

rate of 2.2 m3/min/Mg. The samples were maintained at

4 �C until analyzed for FL and BL about a year later.

To investigate the effect of storage temperature, part of

the corn samples dried at 93 �C and part of the normal-1

and waxy corn samples dried at 27 �C were stored (for

12 months) at 27 �C and the other half was stored (for

12 months) at 4 �C. The time of 12-month storage was

selected to correspond with the likely maximum time for

storage at a farm or other grain-storage facility. The con-

tainers were opened periodically and the grain was stirred

during storage to maintain aerobic conditions.

Germ Removal and Milling

Corn kernels were soaked in distilled water at 37 �C for

24 h prior to manual removal of the germ. The degermed

corn kernels were then partly dried overnight in an oven at

60–65 �C and milled into flour with a Retsch mill

(Brinkmann GmbH and Co. KG, Germany) equipped with

1202 J Am Oil Chem Soc (2012) 89:1201–1210

123

a 0.5 mm (pore size) sieve. Moisture levels of the milled

endosperm were measured in four replicates by using the

AACC Method 44-19 [8] and incorporated in the results.

Extraction of Free Lipids

In order to extract the FL fractions, flour samples (*1.50 g

each) were placed in polypropylene centrifuge tubes and

using the methods reported by Man and Cauberghe [9]

and Conforti, Harris and Rinehart [10] 15 mL of water-

saturated butanol (WSB) was added to each tube, shaken

for 10 min at 200 rpm and centrifuged for 1 min at

2,2609g by using a centrifuge model Centra CL2 (Inter-

national Equipment Company, Needham Heights, MA).

The supernatant was placed into a 250-mL round-bottom

collecting flask and the flour residue was re-extracted twice

more as just described, with the supernatant being added to

the collecting flask. Finally, using a 15 mL solution of

dichloromethane (DM):methanol (MH) (2:1, vol/vol), the

residue was quickly washed and transferred into a Maxi-

spinTM filter tube (Alltech Associates, Inc., Deerfield, IL)

made of polyvinylidene fluoride (PVDF) equipped with

membrane filters (with a pore size of 0.45 lm) for the

removal of the residue and centrifuged for 5 min (at

2,2609g). The supernatant was placed in a 250-mL round-

bottom collecting flask and the flour residue was

re-extracted twice more as just described with the super-

natant added again to the collecting flask. Finally, using

15 mL of a solution of DM:MH (2:1, vol/vol), the residue

was quickly rinsed and transferred into a Maxi-spinTM filter

tube made of PVDF equipped with membrane filters

(Alltech Associates Inc., Deerfield, IL) with a pore size of

0.45 lm for the removal of the residue and centrifuged for

5 min (at 2,2609g). The supernatant organic phase was

again added to the collecting flask. Methanol was used to

remove the interstitial WSB and non-polar lipids from the

residue [10]. All of the WSB and DM:MH (2:1, vol/vol)

phases collected in the 250-mL flask were rotary-evapo-

rated at reduced pressure (*5 mmHg) and 40 �C until

dried. The flasks were then washed and their contents were

quantitatively transferred into capped tubes by using

DM:MH (2:1). After homogenization and centrifugation to

precipitate any suspended flour, aliquots were taken for FA

and lipid-class analysis.

Extraction of Bound Lipids

To extract the BL, the residue from the extraction of the FL

was digested with Thermamyl (Novozymes North America

Inc., Franklinton, NC) at 85 �C for 90 min [11] followed

by extraction with DM:MH (2:1, vol/vol). To separate the

organic and water phases and to remove the digested flour,

Maxi-spinTM filter tubes (Alltech Associates Inc.) made of

PVDF equipped with membrane filters with a pore size of

0.45 lm were used (similar to the previous stage). The

digested materials along with the water suspending them

were transferred into Maxi-spinTM tubes with no mem-

brane filters while washing with 25 mL DM:MH (2:1, vol/

vol). The tubes were closed tightly and shaken vigorously

for a total of 3.0 min. The contents were transferred onto

the membrane filter inside a second Maxi-spinTM tube

(with filter) and centrifuged at 2,2609g for 3.0 min for

phase separation. About two-thirds of the solvent mixture

was recovered in the organic (lower) phase composed of

dichloromethane with some methanol and the rest stayed

with water phase. After separation, the organic phase was

dried by passing it through a silica filter loaded with 3.0 g

anhydrous sodium sulfate. To improve the lipid recovery,

extraction of flour residue was continued for a second and a

third time by using 12 mL dichloromethane alone at each

stage while the original water phase containing some

methanol was retained with the flours. The organic phases

from the second and the third extraction stages were dried,

as was the case with ‘‘Extraction of Free Lipids’’ for FL

extraction, and combined with the organic phase collected

in the first stage. After centrifugation for 3.0 min at

2,2609g to precipitate any suspended flour, the organic

phases were combined, fully homogenized and aliquots

were taken for FA and lipid-class analysis.

Fatty Acid Analysis of the Free and Bound Lipid

Extracts

To prepare the samples for FA analysis, the volume of each

extract was reduced under a gentle flow of nitrogen gas and

an aliquot was then converted into FA methyl esters

according to Method Ce 2-66 from American Oil Chem-

ists’ Society [12]. The semi-quantitative FA analyses of the

extracted FL and BL were performed using a gas chro-

matograph (GC) (Hewlett Packard series II model 5890)

equipped with an SP-2330 fused-silica column (15 m 9

0.25 mm 9 0.2 lm; Supelco, Bellefonte, PA), an auto-

sampler and a flame ionization detector (FID). Agilent

Chemstation software was used for the control of the

equipment and also for the data acquisition and data pro-

cessing. Oven temperature programming started from

40 �C and increased to 220 �C at 10 �C/min, where it was

held for 7.0 min. Both the injection port and the detector

were set at 230 �C. Duplicate injections were performed

for each sample. A mixture of external FA methyl ester

standards was used to identify the chromatographic peaks

and then the relative area counts of C16:0 (palmitic acid),

C18:0 (stearic acid), C18:1 (oleic acid), C18:2 (linoleic acid)

and C18:3 (linolenic acid) were reported as semi-quantita-

tive relative FA levels. The retention times for the FA

methyl esters of the above FA were obtained at 4.44, 5.51,

J Am Oil Chem Soc (2012) 89:1201–1210 1203

123

5.83, 6.38 and 7.09 min, respectively. Five major FA

(palmitic, stearic, oleic, linoleic and linolenic) account for

over 99% of the total FA found in corn oil [13, 14].

Therefore, in this study only these FA will be discussed.

Although the most accurate data for absolute FA methyl

ester composition are obtained by determining GC

response factors for each FA methyl ester and calculating

the amounts based on the size of the peak in relation to the

response, it is a common practice [15, 16] to report relative

area % of the key FA in vegetable oils, such as corn and

soybean oils, in this fashion. Total lipid concentrations of

the kernels (as ground flour) were measured by using the

AACC Method 30-25 [17].

Analysis of Lipid Classes

Changes in the lipid classes were monitored using an

HPLC system (HP 1100, Agilent Technologies, Santa

Clara, CA) equipped with a evaporative light scattering

detector operated at 65 �C and a gas (N2) flow rate of

1.78 L/min. Separation of the sample components was

performed on a Pholipidec LC column (25 cm 9 4.6 mm)

from Astec (Whippany, NJ). Sample injection was per-

formed using the autosampler assembled into the HPLC

system. Agilent Chemstation software was used for the

control of the equipment and also for the data acquisition

and data processing. A gradient elution programming

(Table 1) was used to elute the samples. Phosphatidyleth-

anolamine (PE), phosphatidylinositol (PI), lysophosphati-

dylcholine (LPC), lysophosphatidylethanolamine (LPE)

purchased from Sigma-Aldrich Co. (St. Louis, MO) and

also triolein (as a representative for semi-quantitative TG

analysis), dilinolein (as a representative for semi-quanti-

tative diglyceride analysis) and oleic acid (as a represen-

tative for semi-quantitative FFA) were used as external

standards to identify the peaks on the HPLC chromatogram

and also to estimate their quantities. Relative response

factors obtained in this study for the above compounds

were 18.6 for triolein, 20.0 for dilinolein, 27.2 for oleic

acid, 20.1 for PE, 15.9 for LPE, 5.6 for PI and 1.0 for LPC.

Statistical Analysis

In the current study, each variable was studied separately

and a completely randomized design was considered for

each treatment. The General Linear Model (GLM) proce-

dure of the Statistical Analysis System (SAS) release 8.2

(SAS Institute, Inc., Cary, NC) was used for data analysis.

All of the extractions were carried out on duplicate aliquots

of the flours. Means and least significant differences (LSD,

95% confidence level) were determined to evaluate the

differences in the FA compositions of FL and BL fractions

among the various treated corn types.

Results and Discussion

Characterization of the Free and Bound Lipids

from Different Corn Types After Drying at 27 �C

and Storage for 12 Months at 4 �C (Control Samples)

Regardless of drying and storage conditions, palmitic and

stearic acids were found at greater levels in the BL frac-

tions than in the FL fractions of all corn type (Tables 2, 3).

On the other hand, oleic, linoleic and linolenic acids were

at greater levels in the FL fractions than in the BL fractions

for each corn type. Genotypes may differ with respect to

the distribution of the FA between FL and BL, based on

their starch characteristics. For example, complexation and

dissociation behaviors of starch molecules depend partly

on the degree of polymerization of amylose and on the

branch chain lengths of amylopectin [2, 18, 19]. The ratios

of saturated to unsaturated (S/U) FA among the different

corn types of this study are also shown in Tables 2 and 3.

S/U ratios of 0.20–0.22 are found in the FL fractions of

control samples. On the other hand, such a ratio varies

within 0.31–0.85 levels in the BL fractions. This indicates

that the FL and BL among the different corn samples of

this study are unique in their S/U ratios, which can be used

for further processing purposes of grains depending on the

type of application they are considered for.

In the FL fractions, the percentages of palmitic and

stearic acids were not different among all corn types

(Tables 2, 3). However, oleic acid differed among all

types, with high-amylose being the greatest (26.0%),

Table 1 Gradient elution programming used in this study for the

separation of lipid classes on a Pholipidec LC column

(25 cm 9 4.6 mm)

Step Time A (%) B (%) C (%) D (%) Flow rate

(mL/min)

1 0 70 30 0 0 0.05

2 2 70 30 0 0 1.1

3 4 100 0 0 0 1.1

4 8 100 0 0 0 1.1

5 9 70 0 0 30 1.1

6 13 50 0 0 50 1.1

7 17 0 0 50 50 1.1

8 19 0 0 60 40 1.1

9 21 0 0 0 100 1.1

10 24 0 0 0 100 1.1

11 27 100 0 0 0 1.1

12 29 70 30 0 0 0.05

A chloroform, B hexane, C chloroform:methanol:water:ammonia

(50:48:1:1, vol/vol) and D methanol

1204 J Am Oil Chem Soc (2012) 89:1201–1210

123

followed by normal-2 (21.0%), normal-1 (19.0%), and

waxy (17.5%) corn types, respectively. Linoleic acid per-

centages were similar in normal-1 (60.0%), waxy (60.4%)

and normal-2 (58.2%) corn types and lower in high-amy-

lose (52.7%). Several reports describe the FA composition

of corn oil [13, 14, 20, 21]; however, no data report

exclusively on the FA composition of the FL fractions of

the lipids from the endosperm. In general, the ranges of FA

found in the FL fractions of the endosperm from the corn

types in our study agree with those of Morrison [13] for

Table 2 Fatty acid levels of the free lipid (FL) and bound lipid (BL) extracts from normal-1 and waxy corn types dried and stored under

different temperature conditions

Sample

description

Drying

temperature

(�C)

Storage

temperature

(�C)

Lipidsw Fatty acid levelsx (%) S/Uy

16:0 18:0 18:1 18:2 18:3

Normal-1 27 4 FL 15.6 ± 0.6g 2.0 ± 0.1e 19.0 ± 0.5e 60.0 ± 0.9ab 3.5 ± 0.3bc 0.21

Normal-1 27 27 FL 13.8 ± 0.1ghi 2.1 ± 0.0e 22.3 ± 0.1a 58.3 ± 0.0ab 3.5 ± 0.0bc 0.21

Normal-1 93 4 FL 15.2 ± 0.2ghi 2.0 ± 0.0e 21.0 ± 0.1b 58.1 ± 0.3b 3.6 ± 0.1b 0.21

Normal-1 93 27 FL 13.5 ± 0.3hi 2.1 ± 0.0e 20.3 ± 0.1bc 60.4 ± 0.2a 3.7 ± 0.0b 0.21

Waxy 27 4 FL 15.5 ± 0.2gh 2.6 ± 0.2de 17.5 ± 0.1f 60.4 ± 0.1ab 4.0 ± 0.0ab 0.22

Waxy 27 27 FL 13.9 ± 0.0ghi 2.1 ± 0.1e 19.9 ± 0.2cd 60.5 ± 0.4a 3.6 ± 0.0b 0.21

Waxy 93 4 FL 14.7 ± 0.1ghi 2.3 ± 0.1e 19.2 ± 0.1de 60.0 ± 0.1ab 3.8 ± 0.0ab 0.22

Waxy 93 27 FL 13.4 ± 0.1i 2.2 ± 0.2e 20.6 ± 0.4bc 60.1 ± 0.5ab 3.7 ± 0.1b 0.21

Normal-1 27 4 BL 41.5 ± 0.9b 5.1 ± 0.1bc 10.8 ± 0.0ij 40.6 ± 0.6e 2.0 ± 0.1de 0.39

Normal-1 27 27 BL 48.6 ± 0.3a 2.8 ± 0.0de 10.4 ± 0.1j 36.7 ± 0.1f 1.5 ± 0.0e 0.38

Normal-1 93 4 BL 39.1 ± 0.4c 3.6 ± 0.0cde 11.3 ± 0.3i 43.9 ± 0.1d 2.2 ± 0.0de 0.33

Normal-1 93 27 BL 48.0 ± 0.3a 3.1 ± 0.1de 10.2 ± 0.1j 37.2 ± 0.1f 1.5 ± 0.0e 0.38

Waxy 27 4 BL 22.7 ± 2.8f 6.6 ± 0.7b 16.3 ± 1.0g 49.9 ± 0.0c 4.6 ± 1.1a 0.31

Waxy 27 27 BL 29.1 ± 1.2e 10.3 ± 3.4a 14.2 ± 0.6h 43.8 ± 3.3d 2.7 ± 0.7cd 0.43

Waxy 93 4 BL 33.0 ± 1.6d 8.5 ± 0.0a 14.0 ± 0.1h 40.0 ± 1.7e 4.5 ± 0.3a 0.41

Waxy 93 27 BL 32.5 ± 0.3d 4.5 ± 0.4dc 14.5 ± 0.3h 45.8 ± 1.0d 2.6 ± 0.0cd 0.32

w In each column, means with the same letter are not significantly different (P [ 0.05). Duplicate extractions were performed on each sample

type with a given treatmentx The designations 16:0, 18:0, 18:1, 18:2 and 18:3 represent palmitic, stearic, oleic, linoleic and linolenic acids, respectively. The semi-

quantitative data reported here are the relative area percentages of the peaks representing different fatty acids identified on the GC

chromatogramsy S/U represents the ratio of total saturated to total unsaturated fatty acids for each treatment

Table 3 Combined effects of drying and storage temperatures on the fatty acid levels of the free lipid (FL) and bound lipid (BL) extracts from

normal-2 and high-amylose corn types

Sample

description

Drying

temperature

(�C)

Storage

temperature

(�C)

Lipidsw Fatty acid levelsx (%) S/Uy

16:0 18:0 18:1 18:2 18:3

Normal-2 27 4 FL 13.9 ± 0.1c 2.6 ± 0.5dc 21.0 ± 0.3b 58.1 ± 0.7a 4.4 ± 0.1c 0.20

Normal-2 93 27 FL 14.2 ± 0.2c 2.3 ± 0.0d 20.4 ± 0.1b 57.9 ± 0.1a 5.2 ± 0.0a 0.20

High-Amylose 27 4 FL 14.0 ± 0.0c 2.5 ± 0.0d 26.0 ± 0.0a 52.7 ± 0.0b 4.7 ± 0.0b 0.20

High-Amylose 93 27 FL 13.8 ± 0.0c 2.6 ± 0.0dc 26.9 ± 0.0a 51.9 ± 0.0b 4.9 ± 0.0b 0.20

Normal-2 27 4 BL 42.4 ± 1.9a 3.6 ± 0.7bc 12.0 ± 0.1d 39.6 ± 1.3d 2.4 ± 0.1d 0.85

Normal-2 93 27 BL 40.6 ± 1.9a 3.8 ± 0.2b 12.9 ± 0.9d 40.3 ± 1.3d 2.5 ± 0.1d 0.80

High-Amylose 27 4 BL 40.0 ± 3.3a 4.1 ± 0.2b 16.1 ± 0.3c 37.7 ± 2.7d 2.1 ± 0.1e 0.79

High-Amylose 93 27 BL 22.9 ± 0.0b 8.1 ± 0.7a 17.0 ± 0.0c 47.3 ± 0.7c 4.8 ± 0.0b 0.45

w In each column, means with the same letter are not significantly different (P [ 0.05). Duplicate extractions were performed on each sample

type with a given treatmentx The designations 16:0, 18:0, 18:1, 18:2 and 18:3 represent palmitic, stearic, oleic, linoleic and linolenic acids, respectively. The semi-

quantitative data reported here are the relative area percentages of the peaks representing different fatty acids identified herey S/U represents the ratio of total saturated to total unsaturated fatty acids

J Am Oil Chem Soc (2012) 89:1201–1210 1205

123

combined amounts of FL and BL, except for the linolenic

acid, which was slightly greater in our study. According to

Morrison [13], total endosperm lipids were composed of

11–22% palmitic acid, 2–3% stearic acid, 17–39% oleic

acid, 34–63% linoleic acid and 1–3% linolenic acid.

A typical commercial corn oil coming from the germ,

rather than the endosperm studied here, would likely have

FA composition falling within this range, with a typical

composition being 11.6% palmitic acid, 1.8% stearic acid,

25.2% oleic acid, 59.7% linoleic acid and 0.8% linolenic

acid [14].

In the BL fractions of the control sample, waxy corn had

greater percentages of linoleic and linolenic acids and less

palmitic acid than did the other corn types, which showed

similar levels for these three FA (Tables 2, 3). Stearic acid

was less in normal-2 than in waxy corn. Oleic acid per-

centages occurred in the following order: waxy = high

amylose [ normal-2 [ normal-1. Kitahara et al. [22]

reported compositions of 31.6, 2.4, 14.5, 48.5 and 3.0% (w/w,

respectively) for palmitic, stearic, oleic, linoleic and lino-

lenic acids in the BL fraction of normal corn starch, where

other than linolenic acid the levels of FA are somewhat

different from those presented in Tables 2, 3. However,

mean values reported by White and Weber [14] of 37%

palmitic acid, 3% stearic acid, 11% oleic acid, 46% linoleic

acid and 3% linolenic acid in the BL fraction of non-waxy

corn starches are fairly close to the values obtained in this

study for both normal corn types dried at 27 �C. Also, for

the saturated FA, the complexation behavior of amylose

with FFA (in addition to other parameters) also depends on

the degree of polymerization and chain length of amylose

[19], which are also different for the various genotypes of a

given cereal [3] and for different levels of amylose con-

centration as was the case in the current study (20.4% for

normal-1, 20.9% for normal-2, 1.9% for waxy and 42.2%

for high-amylose). Starch granules may have different

sizes, shapes and structures, depending upon the botanical

origins, and amylose and amylopectin can vary in molec-

ular weight and structure accordingly [3]. Furthermore, the

complexation capacities of unsaturated FA (oleic, linoleic

and linolenic acids) with starch pastes differed from those

of saturated ones and within the saturated FA (caprylic,

lauric, myristic, palmitic and stearic acids) [19]. Also, for

the saturated FA, the complexation behavior was depen-

dent on the FA chain length: as the chain length increased,

the complexation started at a lower FA concentration [19].

Moreover, a higher degree of complexation was observed

with the unsaturated FA than with the saturated FA, and the

complexation level was increased with an increase in the

number of double bonds [4, 5, 19]. Degree of unsaturation

and the geometric configuration (cis or trans) of FFA also

can play an important role in the binding properties of FFA

to amylase [4, 5]. Therefore, the differences observed in

the FA levels of different corn genotypes as observed in

this study are reasonable. The triacylglycerol does not form

complexes with starch [19]. Total lipid concentrations

of the ground flours were measured at 3.1 ± 0.1% (w/w)

for normal-1 and normal-2, 3.4 ± 0.1% for waxy and

4.4 ± 0.1% for high-amylose corn types. The moisture

levels of the flours were within 7.5–9.0%.

Effect of Drying Temperature on the Fatty Acid Levels

of Free and Bound Lipids in Normal-1 and Waxy Corn

Types During a 12-Month Storage at 4 �C

In the FL fractions of both normal-1 and waxy corn types,

oleic acid was the only FA that was statistically different

when the corn was dried at 93 �C, rather than at 27 �C, and

stored for 12 months at 4 �C (Table 2). In the BL fraction

of normal-1 corn, only palmitic and linoleic acids differed

between the two drying temperatures. In the BL fraction of

waxy corn, the percentages of all FA, except for linolenic

acid, differed with a change in the drying temperature.

A decline of 12.2% in the combined amounts of oleic and

linoleic concentrations with drying at 93 �C might be

related to the sensitivity of unsaturated bonds in these FA

to the heat. A similar decline in linolenic acid concentra-

tion would have been expected, but that change did not

occur. Corresponding to these changes, an increase in the

combined percentages of palmitic and stearic acids

occurred with an increase in drying temperature. Such

changes were not observed in the FL. These findings are

consistent with the S/U ratios of FL and BL fractions

among all corn types studied here (Tables 2, 3). In the FL

fractions, the S/U ratio did not change by a change in the

drying conditions. But, in the BL fractions, distinct changes

in the S/U ratios are observed in the normal-1 and waxy

corn types as drying temperature is changed.

By analyzing many corn samples at different tempera-

tures, Lagrandeur et al. [23] demonstrated that both lipid

and carbohydrate metabolisms could occur in corn at tem-

peratures above 13 �C. After storage of corn for less than a

day, Black et al. [24] reported an increase in the percentage

of palmitic acid (from *12% at -25 �C to * over 40% at

160 �C) in the extracted oil. Total extractable amounts of oil

also dropped from *5.5% (w/w) to as low as 1.3% with

increased temperature suggesting increased binding of FA

at higher temperatures, and increased preferential binding

of oleic and linoleic acids resulting in an increase in the

relative percentage of palmitic acid in the extracted oil.

Tang and Copeland [19] showed that the complexation of

amylose and FFA was dependent on the type of FFA and

that for a given FFA a minimum concentration level was

required for the complexation to occur.

1206 J Am Oil Chem Soc (2012) 89:1201–1210

123

Effect of Storage Temperature on the Fatty Acid Levels

of Free and Bound Lipids in Normal-1 and Waxy Corn

Types

Compared with the samples stored at 4 �C, those stored at

27 �C resulted in increases in the oleic acid concentrations

of FL fractions (Table 2). In the BL fraction of normal-1,

there was an increase in palmitic acid and a decrease in

stearic and linoleic acids. The increase in the relative

percentage of palmitic acid accounted for a total relative

reduction of 7.1% in the remaining C18 FA (18:0, 18:1,

18:2 and 18:3). In the waxy corn, all the FA in the BL

fraction changed due to a change in the storage tempera-

ture. A decrease of 10.1% in the overall concentrations of

oleic, linoleic and linolenic acids, corresponding to a

10.1% increase in palmitic and stearic acids, might be

related to the greater sensitivities of these more-unsaturated

FA during the storage at higher temperatures. In the case of

waxy corn, a higher storage temperature resulted in a

decline in the percentage of linolenic acid in the BL below

that of the FL. In agreement with these results, Black et al.

[24] observed an increase in palmitic acid percentage

during storage of milled corn fractions. Christensen [25]

reported that with an increase in storage temperature, the

fat acidity value of corn also increased. Keitel [26, 27]

reported both oxidative and hydrolytic reactions in kernels

dried at 140–180 �C and stored for 6 months at 20 �C, with

oxidation mainly affected by atmospheric air and hydro-

lysis occurring in the fresh samples stored in an oxygen-

free atmosphere. The oxidation was more pronounced for

corn meal than for whole corn. Storage can cause the

liberation of FFA from the triacylglycerol backbones [4, 5].

In agreement with that, Tremazi and Saeed [28] reported

appreciable changes in the acid value, thiocyanogen value

and color of corn oil from the stored grain (for 1 year).

Lipid deterioration involves hydrolysis more than oxida-

tion [29]. Changes in the storage temperature in the current

study resulted in slight changes in the S/U ratios of the BL

fraction (Tables 2, 3). Black et al. [24] noted that oxidation

of unsaturated FA can also reduce the total extractable oil.

Vertucci [30] verified such deterioration with DSC ther-

mograms of corn stored for 22 years, where an increase in

the enthalpy of lipid transition was observed with an

increase in the storage time. He used seeds of similar

genetic background and assumed that the physical prop-

erties of seed lipids harvested in 1958 were the same as

those of the seeds harvested in 1980. Hydrolytic reactions

occurring during grain storage [26, 27, 29] lead to a

reduction in the total amount of FA left on the triacyl-

glycerol backbones. Also, since polyunsaturated FA dete-

riorate more rapidly with oxidation, new proportions in the

FA profiles would be expected (as was the case in the

current study).

Combined Effects of Drying and Storage Temperatures

on the Fatty Acid Levels of Free and Bound Lipids

in Corn Types

The combined effects of drying and storage temperatures

were studied using all four corn types, with comparisons

made between the corn dried at 27 �C and stored for

12 months at 4 �C and that dried at 93 �C and stored for

12 months at 27 �C (Tables 2, 3). Only minor, but some-

times significant, changes occurred in the FL fractions at

the higher drying and storage temperatures. Other than

linolenic acid, which increased slightly in normal-2

(Table 3), the only FA that changed due to a difference in

storage temperature was oleic acid in all corn types

(Tables 2, 3).

In the BL fraction, however, more substantial changes

occurred among the FA concentrations due to the combined

effects of drying and storage temperatures (Tables 2, 3).

Compared to corn dried at 27 �C and stored at 4 �C (i.e., the

control sample), nearly all the FA for normal-1, waxy and

high-amylose corn types dried at 93 �C and stored at 27 �C

were not only statistically different, but the differences were

large in many cases. In particular, sizable differences

occurred for waxy and high-amylose corn types. These

changes were similar to those for the normal-1 and waxy

corn types when they were dried at 27 �C and stored at

27 �C compared with drying at 27 �C and storage at 4 �C,

where, for example, linoleic and linolenic acids in the BL

generally decreased by substantial amounts. Due to such

increase in the drying and storage temperatures, palmitic

acid increased in the BL fractions of both normal-1 and

waxy corn types, but the levels of linoleic acid decreased in

both corn types. In waxy corn, increases in palmitic and

stearic acids corresponded well with the total decreases in

all unsaturated C18 FA. Also, contrary to what observed

with waxy corn, palmitic acid concentration decreased in

high-amylose while the concentrations of all other FA

increased. Combined effects of drying and storage tem-

peratures on the S/U ratio were mainly found in normal-2

and high-amylose corn types.

An additional comparison of the BL in normal-1 dried at

93 �C and stored at 27 �C with the BL from the corn dried

at 93 �C and stored at 4 �C showed many substantial

differences, such as an increase of 8.9% in palmitic acid

with corresponding decreases in oleic and linoleic acids

(Table 2). Fewer and smaller differences in FA levels

occurred for waxy corn due to a difference in storage

temperature, but some changes were significant. The final

comparison of normal-1 and waxy corns dried at 27 �C and

stored at 27 �C with the corns dried at 93 �C and stored at

27 �C revealed no differences for the normal-1 corn, and

differences only in palmitic and stearic acids for the waxy

corn. These data indicate that storage temperature had a

J Am Oil Chem Soc (2012) 89:1201–1210 1207

123

greater influence on the FA composition of the samples

than did differences in the drying temperature.

Changes in the Levels of Lipid Classes in the Free

Lipid and Bound Lipid Fractions

The results for the changes in the lipid classes among the

different samples of this study are shown in Tables 4 and 5.

For normal-1, normal-2 and high-amylose samples dried at

27 �C and stored for 12 months at 4 �C, majority of TG,

PE, PI and LPE was detected in the FL and little or none

was found in the BL fractions (Tables 4, 5). On the other

hand, LPC was exclusively found in the BL fractions of

normal-1, normal-2 and high-amylose corn types and none

was found in the FL and BL fractions of the waxy samples

nor was it found in the FL fractions of normal-1, normal-2

and high-amylose corn samples. TG indicated higher

contribution (76.6%) among all the lipid classes in the BL

fraction of waxy samples, while that of the FL was as low

as 26.8%. FFA were found at the highest levels (42.3%) in

the FL of the waxy corn samples. PE was found in both FL

and BL fractions of waxy corn samples. Drying at 93 �C

resulted in a major reduction in PE contribution and cor-

responding increase in the TG and PI plus LPE levels in the

FL fraction of normal-1. TG and PE maintained their levels

inside the FL fraction of normal-1 corn during the drying at

27 �C and storage at 27 �C for 12 months. However, dry-

ing at 93 �C and combined drying at 93 �C and storage at

27 �C resulted in significant changes in the levels of TG

and PE in the FL fraction of normal-1 corn (Table 4). The

higher storage temperature (27 vs. 4 �C) also resulted in

loss of TG in the BL fraction. Some differences were also

observed in the levels of the above components due to the

combined drying and storage temperatures (Table 4).

For waxy corn, the above treatments resulted in no

significant changes among TG levels in the FL fraction.

Storage temperature (with or without the accelerated dry-

ing at 93 �C) resulted in significant changes in the PE

levels in the FL fraction of waxy corn indicating a greater

effect of storage temperature than drying temperature.

In the high-amylose corn, TG level was reduced in the FL

fraction (from 56.7 to 38.6%) and increased in the BL

fraction (from 2.5 to 10.1%) due to the combined effects of

drying at 93 �C and storage at 27 �C. This indicates a

possible migration of TG from the FL into the BL due to

the above treatment. As a consequence, contribution of PE

was improved in the FL fraction (from 15.5 to 38.2%). TG

were also found in the BL fraction of normal-2 samples.

Table 4 Changes in the levels of lipid classes in the free lipid (FL) and bound lipid (BL) extracts from normal-1 and waxy corn types dried and

stored under different temperature conditions

Sample

description

Drying

temperature

(�C)

Storage

temperature

(�C)

Lipidsw Lipid classes (%)x

TG DG FFA PE PI ? LPE LPC

Normal-1 27 4 FL 49.3 ± 2.6c 3.0 ± 0.3b 14.5 ± 0.4cd 30.2 ± 3.5c 3.1 ± 1.6c 0.0 ± 0.0d

Normal-1 27 27 FL 54.5 ± 2.0bc 2.0 ± 0.3b 9.4 ± 0.1de 29.3 ± 2.0cd 4.9 ± 0.4ab 0.0 ± 0.0d

Waxy 27 4 FL 26.8 ± 2.5e 2.5 ± 0.8b 42.3 ± 1.0a 25.1 ± 0.3de 3.4 ± 0.4bc 0.0 ± 0.0d

Waxy 27 27 FL 32.0 ± 0.2e 1.9 ± 0.1b 24.2 ± 2.3b 36.2 ± 0.1b 5.8 ± 2.1a 0.0 ± 0.0d

Normal-1 93 4 FL 57.2 ± 0.4b 4.2 ± 0.2b 12.5 ± 0.5de 20.3 ± 1.1f 6.2 ± 0.4a 0.0 ± 0.0d

Normal-1 93 27 FL 38.9 ± 1.2d 2.1 ± 0.1b 8.2 ± 0.4def 47.3 ± 0.8a 3.6 ± 0.1bc 0.0 ± 0.0d

Waxy 93 4 FL 31.4 ± 1.3e 2.6 ± 0.5b 39.5 ± 0.7a 22.7 ± 0.1ef 3.7 ± 1.3d 0.0 ± 0.0d

Waxy 93 27 FL 28.3 ± 3.6e 1.5 ± 0.1b 22.4 ± 0.1bc 44.6 ± 3.7a 3.23 ± 0.0bc 0.0 ± 0.0d

Normal-1 27 4 BL 3.6 ± 0.5fg 25.3 ± 6.5a 10.4 ± 14.7de 0.3 ± 0.1h 0.0 ± 0.0d 60.5 ± 7.8b

Normal-1 27 27 BL 0.0 ± 0.0g 0.0 ± 0.0b 13.3 ± 0.6de 0.0 ± 0.0h 0.0 ± 0.0d 86.8 ± 0.6a

Waxy 27 4 BL 76.6 ± 7.8a 2.0 ± 1.4b 11.0 ± 0.3de 10.4 ± 5.0g 0.0 ± 0.0d 0.0 ± 0.0d

Waxy 27 27 BL 72.0 ± 2.7a 3.2 ± 1.8b 0.0 ± 0.0f 21.7 ± 0.8ef 0.0 ± 0.0d 3.2 ± 1.7d

Normal-1 93 4 BL 2.3 ± 0.3fg 28.1 ± 11.8a 24.8 ± 5.9b 0.2 ± 0.1h 0.0 ± 0.0d 44.8 ± 5.6c

Normal-1 93 27 BL 0.5 ± 0.0g 0.0 ± 0.0b 12.1 ± 0.1de 0.2 ± 0.2h 0.2 ± 0.2h 87.2 ± 0.4a

Waxy 93 4 BL 77.1 ± 7.8a 2.4 ± 1.5b 8.6 ± 2.2de 11.9 ± 4.1g 0.0 ± 0.0d 0.0 ± 0.0d

Waxy 93 27 BL 7.5 ± 1.9f 0.0 ± 0.0b 5.8 ± 0.3ef 0.0 ± 0.0h 0.0 ± 0.0d 86.7 ± 2.3a

w In each column, means with the same letter are not significantly different (P [ 0.05). Duplicate extractions were performed on each sample

type with a given treatmentx The designations TG, DG, FA, PE, PI, LPE and LPC represent triglycerides, diglycerides, fatty acids, phosphatidyl ethanolamine, phosphatidyl

inositol, lysophosphatidyl ethanolamine, lysophosphatidyl choline, respectively. The semi-quantitative data reported here are estimated based on

the use of relative response factors determined for triolein (18.6), diolein (20.0) and oleic acid (27.2) as representatives for triglycerides,

diglycerides and free fatty acids, respectively, and PE (20.1), PI (5.6), LPE (15.9) and LPC (1.0)

1208 J Am Oil Chem Soc (2012) 89:1201–1210

123

According to the results of this study (Table 5), deterio-

ration of LPC component in the BL fractions of normal-2

and high-amylose corn samples resulted in the increase in

the FFA levels of these two corn samples due to the

combined effects of drying at 93 �C and storage at 27 �C.

Higher drying temperature also resulted in a higher FFA

level in the BL fraction of normal-1 corn sample (Table 4).

Conclusions

The current study showed that FA levels of FL and BL in

corn endosperm were affected by post-harvest processing

(drying and storage) temperature. Oleic, linoleic and lino-

lenic acids were greater in the FL fractions than in the BL

fractions for all corn types except for the waxy corn. Palmitic

acid was greater in the BL fraction than in the FL fraction for

each corn type. The S/U ratios in the FL fractions of the

studied corn types were within 0.20–0.22 levels but, those of

BL fractions were as high as 0.80. However, changes in the

drying and storage temperatures impacted only the S/U

ratios of the BL fraction indicating that the internal activities

of the kernels has to be minimized by reducing drying and/or

storage temperature in order to maintain the functional

properties of the grains in the final product. TG and PE were

among the lipid classes that contributed the most in the FL

fractions of normal-2 and high-amylose corn samples. LPC

was exclusively found in the BL fractions of normal-1,

normal-2 and high-amylose corn types. Drying at 93 �C and

combined drying at 93 �C and storage at 27 �C for

12 months resulted in significant changes in the TG and PE

levels in the FL fraction of normal-1. LPC was not found in

the FL fractions, but, it was found in the BL fractions of

normal-1, normal-2 and high-amylose corn samples. BL

fraction from waxy corn did not indicate any LPC at all.

Losses in the FFA were found in the BL fraction of waxy

corn but it was increased in the BL fractions of normal-2 and

high-amylose. There is a need to further correlate these

findings with the sensory attributes of resulting food prod-

ucts made from corn and to determine ways to dry and store

corn to achieve the best quality products.

Acknowledgments This project was supported by the National

Research Initiative of the USDA Cooperative State Research, Edu-

cation and Extension Service, Grant Number 2001-35503-10030.

Partial assistance from the Research Council of School of Agriculture

and Natural Resources of the University of Tehran is also appreciated.

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