MEASUREMENT OF SOLIDS IN TOMATO PASTE AND THE ...

MEASUREMENT OF SOLIDS IN TOMATO PASTE AND THE COMPOSITION

AND PROPERTIES OF THE SOLUBLE SOLIDS FRACTION

A Thesis

Presented to

The Faculty of Graduate Studies

of

The University of Guelph

by

SAHAR JAZAERI

In partial fulfilment of requirements

for the degree of

Master of Science

March, 2009

©Sahar Jazaeri, 2009

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ABSTRACT

MEASUREMENT OF SOLIDS IN TOMATO PASTE AND THE

COMPOSITION AND PROPERTIES OF THE SOLUBLE SOLIDS FRACTION

SAHAR JAZAERI Advisor: University of Guelph, March 2009 Dr.Yukio Kakuda

This thesis is an evaluation of two analytical procedures in the determination

of total, soluble solids and insoluble solids in tomato paste.

The microwave oven method was compared to the vacuum oven method. The

vacuum oven method measures each solids fraction in the paste directly while

the microwave method measures the total solids directly but employs an

equation to calculate soluble and insoluble solids. The microwave method was

faster and less labour intensive but gave small but significantly higher values for

total (%) and insoluble solids (%) and lower values for soluble solids. These

small differences although significant may not be important at the production

level and therefore the microwave method is recommended for use by the

industry. Additionally the soluble solids fraction showed to contain large amounts

of lycopene-rich particles held in suspension. These particles were destabilized

with pectinase treatment causing their precipitation. The presence of nitrogen in

the soluble fraction and the loss of stability following pectinase treatment

suggests that the particle is a pectin-polypeptide-lycopene complex.

ACKNOWLEDGEMENTS

I am in debt to my advisor, Dr. Yukio Kakuda for his scientific advises, support

and patience through my study. I am also grateful to my advisory committee, Dr.

Gopinadhan Paliyath and Dr. Steve Gismondi for their constructive guidance.

It is an opportunity to thank Dr.Milena Corredig and Dr.Massimo Marcone for

their encouragements and providing new source of information and insight

through this investigation.

I specially thanks my friends Saeed Rahimi Yazdi , Azadeh (Rose) Namvar

and Azadeh Koushan who helped me through my research by sharing their

academic knowledge and friendship.

Also I would like to express my great gratitude to Douglas Wigle who shared

his experience and knowledge through all parts of this research.

Finally, I would like to thank my family, my mother ,Mina Kazemi Kourdestani,

and sisters, Khandan , Sadaf and my brother in law Farzam for their love,

support.

To my lovely family, Mina, Khandan and Sadaf

i

Table of Contents Page

Chapter 1

MEASUREMENT OF SOLIDS IN TOMATO PASTE

1.1 Introduction 1

1.2 Literature Review 8

1.2.1 Tomato 8

1.2.2 Tomato Paste 9

1.2.3 Tomato Paste Processing 10

1.2.4 Total Solids 12

1.2.4.1 Total Solids: AOAC Method 13

1.2.4.1.a Vacuum Oven Method of AOAC 13

1.2.4.1.D Microwave Oven Method of AOAC 15

1.2.4.2 Total Solids: Canadian Method 16

1.2.4.3 Total Solids by NTSS: United State Method 17

1.2.5 Water Soluble Solids 19

1.2.5.1 Soluble Solids: AOAC Method 19

1.2.5.2 Soluble Solids: Formula Method 21

1.2.6 Water Insoluble Solids 22

1.2.6.1 Water Insoluble Solids: AOAC Method 22

1.2.6.2 Water Insoluble Solids: Formula Method 22

1.3 Experimental 25

1.3.1 Material and Equipment 25

ii

1.3.2 Methods 26

1.3.2.1 Vacuum Oven Methods 26

1.3.2.1.a Total Solids (Vacuum Oven) 26

1.3.2.1.a.b Sample Preparation 26

1.3.2.1.a.c Procedure 27

1.3.2.1 .b Water Insoluble Solids (Vacuum Oven) 28

1.3.2.1.b.a Sample Preparation 29

1.3.2.1.b.b Procedure 29

1.3.2.1 .c Water Soluble Solids (Vacuum Oven) 31

1.3.2.1.c.a Sample Preparation 31

1.3.2.1.c.b Procedure 31

1.3.2.2 Microwave Oven Method 32

1.3.2.2.a Total Solid (Microwave Oven) 32

1.3.2.2.a.b Sample Preparation 33

1.3.2.2.a.c Procedure 33

1.3.2.2.b Water Insoluble Solid (Microwave Oven) 34

1.3.2.2.C Solids in Supernatant Fraction

(Microwave) 34

1.3.2.2.c.a Sample Preparation 34

1.3.2.2.c.b Procedure 35

1.3.2.2.d Water Soluble Solid (Microwave Oven) 35

1.4 Results 37

1.4.1 Vacuum Oven Method 37

1.4.1.1 Total Solids (vacuum Oven) 37

iii

1.4.1.2 Water Insoluble Solids (Vacuum Oven) 37

1.4.1.3 Water Soluble Solids (Vacuum Oven) 38

1.4.2 Microwave Oven Method 38

1.4.2.1 Total Solid (Microwave Oven) 39

1.4.2.2 Solids in the Soluble Supernatant (Microwave Oven) 39

1.4.2.3 Water Insoluble Solids (Microwave Oven) 40

1.4.2.4 Soluble Solids (Microwave Oven) 40

1.4.3 Comparison of Methods (Microwave vs. Vacuum oven) 41

1.5 Statistical Analysis 42

1.5.1 Repeatability (Vacuum and Microwave Oven) 42

1.5.1.1 Total Solids (Vacuum Oven) 42

1.5.1.2 Water Insoluble Solids (Vacuum Oven) 43

1.5.1.3 Water Soluble Solid (Vacuum Oven) 43

1.5.1.4 Total solids (Microwave Oven) 43

1.5.1.5 Solids in the Supernatant Fraction (Microwave Oven) ....43

1.5.2 Comparison of Methods (Vacuum vs. Microwave Oven) 43

1.5.2.1 Total Solids (Vacuum vs. Microwave oven) 43

1.5.2.1.a Equality of the Methods (Total Solids) 43

1.5.2.1 .b Regression Equation of the Methods

(Total Solids) 44

1.5.2.1.C Regression of Exact Equality (Total Solids) 44

1.5.2.1.d Average Error Between Methods (Total Solids) 45

1.5.2.2 Water Insoluble Solids (Vacuum vs. Microwave oven) ....46

iv

1.5.2.2.a Equality of the methods

(Water Insoluble Solids) 46

1.5.2.2.D Regression Equation of the Methods (Water Insoluble Solids) 46

1.5.2.2.c The Regression of Exact Equality (Water Insoluble Solids) 46

1.5.2.2.d Average Error Between Methods

(Water Insoluble Solids) 47

1.5.2.3 Water Soluble Solid (Microwave vs. Vacuum oven) 47

1.5.2.3.a Equality of the Methods (Water Soluble Solids) 47

1.5.2.3.b Regression Equation of the Methods (Water Soluble Solids) 48

1.5.2.3.C Regression of Exact Equality (Water Soluble Solids) 48

1.5.2.3.d Average Difference of the Methods (Water Insoluble Solids) 49

1.6 Discussion 50

V

Page

Chapter 2

THE COMPOSITION AND PROPERTIES OF THE SOLUBLE SOLIDS

FRACTION

2.1 Introduction 52

2.2 Literature Review 53

2.2.1 Tomato Composition 53

2.2.1.1 Solids in Tomato 53

2.2.1.2 Tomato Chromoplasts 57

2.2.2 Lycopene 59

2.2.2.1 Lycopene in Tomato 59

2.2.2.2 Lycopene Structure 60

2.2.2.3 Lycopene Isomers in Tomato 60

2.2.2.4 Lycopene and Health Benefits 61

2.2.2.5 Lycopene Extraction by Enzyme 61

2.2.2.6 Lycopene and Tomato Processing 62

2.2.2.7 Lycopene and Temperature 62

2.2.2.8 Lycopene and Storage 63

2.2.2.9 Lycopene and Illumination 63

2.2.2.10 Lycopene in Different Food Systems 64

2.2.2.11 Lycopene and its Bioavailability in Processed Tomato 64

2.2.2.12 Lycopene Rich Granules in Tomato Juice 66

vi

2.2.2.13 Lycopene, Protein and Different Elements (Ca, Mg,

P and N) in Various Fractions of Tomato Juice 67

2.2.2.14 Stabilization of Lycopene 69

2.2.2.14.a Encapsulation of All-Trans- Lycopene by

Cyclodextrins 69

2.2.2.14.b Lycopene Coating with Protein 70

2.2.2.14.c Nano-encapsulation of Lycopene by Casein 70

2.2.2.14.d Pectin and lycopene in Tomato and

Tomato Products 71

2.2.3 Pectin 72

2.2.3.1 Peptide-Pectin Interaction and Gelation Behavior of

Plant Cell Wall Pectin 73

2.2.3.2 Pectin-Protein Interaction in Tomato Products 74

2.3 Experimental 76

2.3.1 Sample Preparation 76

2.3.1.1 Paste (Diluted) 77

2.3.1.2 Soluble Solids (Diluted) 77

2.3.1.3 Soluble Solids (Dried) 77

2.3.1.4 Paste (Dried) 78

2.3.1.5 Soluble Solids (Dilute-Dialysis) 78

2.3.1.6 Soluble Solids (Dried-Dialysis) 78

2.3.2 Total Solid and Total Soluble Solids 78

2.3.3 Soluble Solids Dry Weight (1s t Centrifugation) 78

2.3.4 Pectin Determination 79

2.3.4.1 Material and Equipment 79 vii

2.3.4.2 Methods 79

2.3.5 Lycopene Determination 80

2.3.5.1 Material and Equipment 81

2.3.5.2 Methods 81

2.3.6 Nitrogen Determination 82


2.3.6.2 Methods 82

2.3.7 Gel Electrophoresis (SDS-PAGE) 83


2.3.7.2 Methods 83

2.3.8 Fatty Acid Composition 84


2.3.8.2 Methods 85

2.3.9 Enzymatic Treatment of Soluble Solids 85


2.3.9.2 Methods 86

2.3.10 Ions Determination (Ca+2, Fe +2, Mg +2, K+, Na +, P-5) by

ICP-OES 88


2.3.10.2 Methods 89

2.3.11 Transmission Electron Microscopy (TEM) Analysis 89


2.3.12.2 Methods 89

2.4 Results 91

viii

2.4.1 Schematic Diagram of Sample Replications 91

2.4.2 Total Solids and Total Soluble Solids 92

2.4.3 Soluble Solid Dry Weight (1s t centrifugation) 92

2.4.4 Pectin Determination 92

2.4.5 Lycopene Determination 93

2.4.6 Nitrogen Determination 94

2.4.7 Gel Electrophoresis (SDS-PAGE) 94

2.4.8 Fatty Acids Determination 96

2.4.9 Enzymatic Treatment of Soluble Solids 96

2.4.10 Ions Determination (Ca+2, Fe +2, Mg +2, K+, Na +, P-5) bylCP-OES 98

2.4.11 Transmission Electron Microscopy (TEM) Analysis 99

2.6 Discussion 103

2.7 Conclusion 109

2.8 Future Study 112

References 114

IX

List of Tables Page

Table 1.1 Percent Total Solids (%TS) in Tomato Pastes by

Vacuum Oven 37

Table 1.2 Percent Water Insoluble Solids (%WIS) in Tomato Pastes by

Vacuum oven. Mean of Three Determinations (%)

± Standard Deviation 38

Table 1.3 Percent Water Soluble Solids (%SS) in Tomato Pastes by

Vacuum Oven. Mean of Three Determinations (%)

± Standard Deviation 38

Table 1.4 Percent Total Solids (%TS) in Tomato Pastes by Microwave.

Mean of Three Determinations (%) ±Standard Deviation 39

Table 1.5 Percent Solids in the Supernatant Fraction (%SSF) in Tomato

Pastes by Microwave. Mean of Three Determinations (%)

±Standard Deviation 39

Table 1.6 Percent Water Insoluble Solids (WIS) in Tomato Pastes by

Equation 3 (Microwave Oven) 40

x

Table 1.7 Percent Soluble Solids (%SS) by Difference Between Total

and Insoluble Solids (Microwave Oven) 40

Table 1.8 Comparison of Mean Values of % Total, % Water Insoluble and

% Water Soluble Solids Measured by Vacuum and Microwave

Methods 41

Table 1.9 Total Solids, t-Test: Paired Means 44

Table 1.10 Water Insoluble Solids,t-Test: Paired Means 46

Table 1.11 Water Insoluble Solids,t-Test: Paired Means 48

Table 2.1 Organic Acids in Fresh and Processed Tomato 54

Table 2.2 Free Amino Acids in Pastes Made from Red Tomatoes and Amino

Acid Composition of Water-Soluble Proteins in Tomato Juice and

Tomato Plastids 56

Table 2.3 Fatty Acid Composition of Tomato Seed Oil (%) from the Hot Break

Process and Fatty Acid Composition of Tomato Plastids 57

XI

Table 2.4 Distribution of Protein, Lycopene, and Ca, Mg, P and N Among

Tomato Juice Fractions 68

Table 2.5 Combination of Enzymes in Their Optimum Conditions 87

Table 2.6 Total and Soluble Solids Content of Tomato Paste 92

Table 2.7 Solids in Soluble Solids Fraction (1s t centrifugation) 92

Table 2.8 Pectin Content in Paste and Soluble Solid (ng/g dry wt) 93

Table 2.9 Lycopene Content in Paste and Soluble Solid (|ig/g dry weight)....93

Table 2.10 Nitrogen Content of Soluble Solid, Paste and Dialyzed

Soluble Solid (% dry wt.) 94

Table 2.11 Ions in Filtrated, Dialyzed and Original Soluble Solids 99

xii

List of Figures Page

Figure 1.1 Mean Composition of Tomato Fruit 9

Figure 1.2 Simplified Flow Diagram for the Manufacture of Tomato Paste 11

Figure 1.3 Flow Chart for Solids Analysis by the Vacuum

and Microwave Oven Method 25

Figure 1.4 The Regression of Exact Equality Between Vacuum and Microwave

Oven Method in Total Solid Determination 45


Oven Method in Water Insoluble Solid Determination 47


Oven Method in Water Soluble Solid Determination 49

Figure 2.1 The Basic Structure of Lycopene 60

Figure 2.2 Structural Formula for Partly Methylated Poly-Galacturonic 72

Figure 2.3 Schematic Illustration of the Egg-Box Model 73

Xlll

Figure 2.4 Suspected Schematic Model of Pectin-Protein

Interaction in Tomato Products 75

Figure 2.5 Schematic Illustration of Sample Preparation and

Related Measurement 76

Figure 2.6 Enzymatic Treatment of Soluble Solids in Terms of

Concentration, Temperature and pH 87

Figure 2.7 Combinations of Enzymes in Their Optimum Conditions 88

Figure 2.8 Schematic Illustration of Sample Replications 91

Figure 2.9 SDS-PAGE of Tomato Paste and Its Different Fraction

in Gel Cross Linking of 18% and 12.5% 95

Figure 2.10 Enzyme Treatment of Soluble Solids 97

Figure 2.11 Ultracentrifuged Soluble Solid Samples Showing Three Distinct

Layers and an Ultracentrifuged Sample After 1h 100

xiv

Figure 2.12 Transmission Electron Micrograph of Soluble Solids 101

Figure 2.13 Transmission Electron Micrograph of Top Layer After

Ultracentrifugation 101

Figure 2.14 Transmission Electron Micrograph of Middle Layer After


Figure 2.15 Transmission Electron Micrograph of Bottom Layer After


xv

Chapter 1

MEASUREMENT OF SOLIDS IN TOMATO PASTE

1.1 Introduction

Tomatoes are part of the solanacea family which includes many other familiar

food products such as paprika, chili pepper, potato and eggplants. Tomatoes

were not accepted as a food until the mid 19th century but since that time there

has been a steady increase in production to the point where in 1979 tomatoes

were ranked third in the world behind grapes (ranked first) and citrus fruits

(ranked second) (Heutink, 1986). In those early days, the nutritional value of

tomatoes was given less attention than the processing conditions required to

produce such products as ketchup, tomato juice and pasta sauce. The first legal

classification of tomatoes was given in 1887 when a U.S. tariff law imposed a

duty on vegetables and included tomatoes in this category. This ambiguity

persisted until 1893 when the U.S Supreme Court settled the controversy by

declaring tomato a vegetable based on its common application in culinary

practices (Nix v Hedden 1893). This decision demonstrates the importance of

commerce over science since the scientific definition still categorizes tomato as a

fruit.

The two main approaches to evaluate tomato product quality are the

quantitative and qualitative examination of their solids content. The quantitative

determination of solids can be used to estimate the potential yield of final

product, the effects of the growing season and the variations between varieties.

Furthermore the solids content can be used to determine if they comply with

1

standards such as the U.S.A standards of identity for tomato pulp (puree) and

paste.

In the industry, the attribute that is relied upon the most to assess quality is

the flow characteristics of the finished products. Like the solids content, the flow

characteristics are dependent on the growing conditions, variety of tomato and

production practices. Based on these relationships, it seems that the solids

measurements can be used to estimate the quality of tomato products.

Acknowledging the importance of solids content of tomato and tomato products,

the government agencies proceeded to establish standards to determine solids

(total, water soluble and water insoluble solids) and used these standards to rank

tomato products. Since most tomato products such as ketchup, pizza and pasta

sauce utilize tomato paste as their main ingredient, the determination of the

solids content of paste has become an important factor not only for the paste

manufacturer but also for the users of paste. Because of the importance of solids

on tomato product quality, regulations on solids content of paste products and

their determination have been implemented not only by government but also by

nonprofit scientific organizations. For example, the Association of Official

Analytical Chemist (AOAC) has developed recognized methods, which have

been referenced and used internationally.

In general there are three different solids in paste that can be measured: total

solids, soluble solids and insoluble solids. It is apparent that total solids are the

sum of the soluble solids and insoluble solids. Total solids are the most

recognized of the three solids as they play a major role in commerce. However,

2

the definition of total solids (in paste) in the U.S is different from that in Canada.

The Canadian government describes pastes as a product with a certain amount

(> 20%) of salt free solids determined by vacuum oven. The sources of salt that

have to be deducted from the measured total solids are both native and added

(Health Protection Branch Ottawa 1981). In the U.S regulations, the native salt is

not eliminated and the terminology "Natural Tomato Soluble Solids" is used in

place of total solids. The Food and Drug Administration (USDA 2000-1) defines

tomato paste as a product not containing less than 24.0% 'Natural Tomato

Soluble Solids' and is determined by refractometry according to the AOAC

method (AOAC 2000).

All Official Methods of Analysis of the AOAC require an inter-laboratory

collaborative study prior to approval. These standardization procedures result in

a reproducible method that is precise and accurate when performed exactly as

outlined. The AOAC approved method for total solids (AOAC 1980) utilizes

vacuum oven drying to remove water but has been criticized as being too

elaborate and time consuming and an alternative procedure should be found.

One such procedure was the microwave oven method. A collaborative study

among 14 laboratories was organized to compare the microwave oven method to

the vacuum oven method. The findings of the collaborative study resulted in the

approval of the microwave oven method as an alternative for the previously

approved vacuum oven method (Chin 1985). However, the microwave oven

method has been shown to produce values higher than the vacuum oven when

3

scrutinizing unpublished industry data and some internal documents from tomato

processing companies.

Various explanations have been put forward to identify the reasons for the

lack of agreement between the results produced by microwave oven and vacuum

oven. Some authors have attributed the differences to the use of dissimilar

microwave models (the first employed while developing the method and the

second when doing the comparison test), failure of the technician to conduct the

assigned procedures properly and failure of the vacuum oven to attain the

conditions specified in the official method.

Even though some researchers have expressed concerns over the

discrepancy between the two methods, the ease with which the microwave

procedure can be performed compared to the vacuum oven procedure has

established the microwave as the preferred method in the industry.

Most of our information on the two methods has been gleaned from total

solids data that were determined with non-standardized procedures and older

equipment. There haven't been any recent investigations comparing the two

methods employing the recommended procedures and modification or taking into

account potential sources of error. One objective of this research was to compare

the microwave and vacuum oven methods and addressing those limiting factors

that were observed in the early studies.

In addition to total solid which has been the main focus of most researchers,

the total insoluble solids fraction is also important as it is the major component

that determines the consistency of many tomato products and serves as a source

4

of many important nutrients. However, the low levels of insoluble solids in

comparison with the soluble solids make them difficult to measure accurately.

The official method of analysis requires several washing steps with hot water

followed by filtration and drying in a vacuum oven (AOAC 2000). This method

measures the insoluble solid directly. However, due to the extensive time of

analysis and the multiple steps, this procedure is not popular.

A second approach that can be used to determine water insoluble solids is an

indirect procedure that employs a model to calculate the water insoluble solids.

In this procedure the total solids and the solids in the soluble fraction are

determined experimentally and used in an empirically derived equation (Bohart

1940). This formula method (indirect) has a big advantage over the vacuum oven

method in terms of greatly reducing the workload and significantly shortening the

analysis time. Although this method has been recommended by the National

Canners Association (Lamb 1977) and adopted and used by the tomato

processors, very little academic attention has been given to this approach in

terms of examining its reliability and accuracy or even suggesting modifications

for improvements. Moreover it would be beneficial to employ the microwave oven

with the formula (indirect) instead of the vacuum oven because of the reduction

in assay time.

In the case of soluble solids, there are no known direct procedures that

isolate the soluble from insoluble solids and measure them directly. The AOAC

measures soluble solids by refractive index and converts the Rl readings to

percent sucrose with a conversion table, and finally derives the soluble solids

5

concentration. However, since there are many new tomato varieties, these tables

would have to be re-examined and developed based on these new varieties.

Without these new conversion tables, this method could have some serious

limitations.

When measuring tomato paste solids, especially the soluble solids, filtration

is a critical step and may affect the determination. The reason for filtering the

sample is to separate the insoluble components from the soluble components;

the boundary between soluble and insoluble solids is not clear and clean

separation of the two is not always possible.

During the preparation of the soluble solid fraction, some colloidal particles

remains suspended in the supernatant after centrifugation and did not

precipitated with the insoluble solids. This suspended material appear to have

unique properties because they behave like soluble solids but are composed of

large molecular weight macromolecules and lipid material. Disrupting the

complex by enzymatic hydrolysis or by dialysis results in loss in solubility.

Determining the true nature of this suspended material may provide information

about the behavior of the solid fractions in paste and may lead to greater

understanding of tomato product properties or better definition of rheological

property of tomato paste. As well, the ability of this complex to suspend insoluble

components may have applications in other systems if the mechanism is

revealed.

The objectives of this research can be summarized as follows: revealed

6

• Examine the repeatability of total solids, water insoluble solids and soluble

solids measurements as determined by vacuum oven.

• Examine the repeatability of total solids and solids in soluble fraction

measurements as determined by microwave oven.

• Compare the total solids values determined by vacuum oven and microwave

oven methods.

• Compare the insoluble solids values determined by vacuum oven versus

insoluble solids values derived from the model using microwave oven data.

• Compare the soluble solids determined by vacuum oven versus soluble solids

calculated by difference of insoluble solids and total solids using microwave

oven data.

• Characterize the type of particles suspended in the soluble solid matrix.

7

1.2 Literature Review

1.2.1 Tomato

The tomato (Solanum lycopersicum, syn. Lycopersicon lycopersicum) is a

member of the nightshade family (Solanaceae). Based on its botanical structure,

the tomato is a fruit but from a culinary point of view the tomato is used like a

vegetable, which led to the controversy over the categorization of tomato as a

vegetable or as a fruit. In 1893, the supreme court of the United States

categorized the tomato as a vegetable (Nix v. Hedden 1983). The acclamation

was based on the observation that tomato is served more often as part of a salad

and not in a dessert as is done with other fruits. This uncertainty is shared with

other plants such as eggplant, squash and zucchini.

Tomatoes are low in calories and a good source of vitamin A, vitamin C, and

minerals (Figure1.1). A 230 g tomato can supply about 60% of the recommended

daily allowance of vitamin C in adults and 85 % in children (Sainju and Dris

2006).

8

Figure 1.1 Mean Compositions of Tomato Fruit Compiled From Hermann (1979)

and Davies & Hobson (1981).

1.2.2 Tomato Paste

Tomato paste is a thick dark red paste made from ripe tomatoes after

removing the seeds and skin. A large portion of the tomato crop is processed

into tomato paste. Tomato paste plays a major role in industry as an ingredient in

many popular products such as ketchup and pizza sauce.

The industrial processing of tomato paste employs unit operations that serve

two important purposes: the inactivation of the pectinolytic enzymes and the

removal of skins and seeds and extra water. The resulting pulp contains

approximately 24% (W/W) Natural Tomato Soluble Solids (NTSS) (Hayes and

others 1998). This procedure conforms to the definition of tomato paste as set

9

out by the U.S. Department of Agriculture, "a product containing solids not less

than 24% natural tomato solids" (USDA 2000-01). A specially designed

concentration process produces tomato paste, which is a dispersion of solid

particles in an aqueous serum phase (Yoo and Rao 1994).

1.2.3 Tomato Paste Processing

The flow diagram for the processing of tomato paste is shown in Figure. 1.2.

Prior to process, the tomatoes are thoroughly washed and sorted to remove

defects. The tomatoes are chopped into small pieces and heated to a specific

temperature.

There are two ways to heat process tomatoes in the industry: the hot break

process and the cold break process. In the hot break process the tomatoes are

heated as quickly as possible to a temperature higher than 90°C in order to

inactivate pectinolytic enzymes. It is common to simultaneously heat and chop

tomatoes in the hot break procedure. It is also common practice to pass the

tomatoes through a two or three stage pulper/finisher unit to remove seeds and

skins. The final evaporation step concentrates the paste to the desired moisture

content. It has been reported that the rheological behaviour of tomato paste,

such as consistency, depends on variables such as sieve pore size and break

temperature (Sanchez and others 2002).

In the cold break procedure, scalding prior to chopping loosens the tomato

skin. The chopping process is performed at around 66°C. The chopped tomato is

held static for a certain amount of time to allow the enzymatic breakdown of

10

pectins. It is believed that the cold break process gives the paste better color,

flavor and higher levels of vitamin C (Madhayi and Salunkhe 1998).

• Tomato

• Harvesting

• Transporting

Pooling/flumin

• Washing

Sorting

Crushing/Choping/Breaking/reheating/Scalding

Preheating

Pulping/Finishing

Figure 1.2 Simplified Flow Diagram for the Manufacture of Tomato Paste

(Heutink 1985)

11

1.2.4 Total Solids

The total solids content of concentrated tomato products is an important

property that purchasers and producers need to know. This key property can aid

in determining the final product's composition, stability and quality when paste is

used as the main ingredient. For these reasons, considerable attention has been

given to regulations and legislations that relate directly to the measurement of

total solids in tomato products.

The regulations concerning total solids are different in Canada and the USA

The Canadian government utilizes the terminology "total solids" (TS) where the

measurement is determined by oven drying and the salt is deducted, while the

U.S utilizes the terminology 'Natural Tomato Soluble Solid' (NTSS) where the

measurement is determined by refractometry. NTSS is an estimation of total

solids but is much easier to perform than oven drying. Another distinction

between these two definitions is that the natural salt originating from the tomato

is included in NTSS reading but the "salt-free solid" definition used by the

Canadian government excludes both natural and added salt.

Similarly the definition of total solid (TS) put forward by the Canadian

government is different from the total solids determined by the Official Methods of

Analysis (AOAC). The Canadian law defines total solids as the solids determined

by oven drying minus natural and added salt. In the AOAC method, the natural

salt is part of the total solids value.

12

1.2.4.1 Total Solids: AOAC Method

1.2.4.1.a Vacuum Oven Method of AOAC

The most recent investigation of total solids, based on the Official Methods,

was by Frank C. Lamb who was working for the National Canners Association at

the time (Lamb, 1964). Realizing that the determination of the true solids content

was not possible even with the official methods, Lamb (1964) made modifications

to the official method (9th edition 1960) with the intention of shorten the analysis

time and implement conditions that would make the procedure more flexible and

improve its reproducibility. The modified method was compared to the original

official method in a collaborative study that involved 17 analysts in 9 laboratories.

The modification that were made included optimizing sample size, adjusting pre-

drying conditions, employing diatomaceous earth, selecting vacuum oven

pressure, and determining the best drying time.

The sample size used in the analysis was increased from 9-12 mg/sq cm

residue to 9-30 mg/sq cm to increase the flexibility in selecting the proper

sample size. Diatomaceous earth was substituted for pumice to improve the

drying conditions. When pre-drying with diatomaceous earth in a boiling water

bath, in a forced draft oven at 70°C or in a vacuum oven at 70°C with released

cocked left partly open, a moderate amount of over drying or under drying did not

affect the final results. In case of pressure, it was considered advisable that the

pressure should not exceed 50 mm mercury during vacuum drying. One of the

major objectives of their study was to shorten the 4h analysis time specified in

the official method. In the presence of diatomaceous earth it was possible to dry

13

samples in 1h at 70°C but to avoid problems and have an adequate safety

margin, a drying time of 2h at 70°C was recommended.

The suggested recommendations were employed by AOAC as first action in

1964 and final action in 1965 (AOAC 1965) and its status is still in action (AOAC

2000).

In summary the method is carried out by adding diatomaceous earth dispersed

at ca 15 mg /sq cm in a metal dish with a tight fitting cover and dried for 30min at

110°C. The dish is cooled in a desiccator and weighed (W1). The amount of

added sample to the dish should be so that the dry residue ranges from 9-30

mg/sq cm. The weight of sample (W2) should be recorded soon enough to avoid

moisture loss. If necessary, sample can be diluted with H20 and spread

uniformly in the dish.

Pre-drying would be conducted by one of the three specified procedures until

apparent dryness is reached. Apparent dryness was defined as "the point at

which the remaining content is equal to not more than 50% of the weight of the

dried solids" (Lamb 1964). The partially dried samples is transferred to the

vacuum oven with a reduced internal pressure equal to or less than 50 mm of

mercury. Samples should be dried for 2 hours at 69-71 °C and removed from

oven and cool to room temperature in a desiccator. The dish should be covered

and weighed (W3).

The difference between dried weight of sample and initial weight would

determine total solid of sample and should be calculated as a percent.

W 3 - W 1 % Total Solids = „ XAT x100 Equationl

W 2 —Wl

14

Where:

W1 : Weight of dish

W2: Weight of the sample and dish

W3: Weight of the dried sample and dish

1.2.4.1.b Microwave Oven Method ofAOAC

Green and Park (1980) employed the microwave oven to determine solids in

foods and other non-foods items. The advantages of microwave drying are

shorter drying times and reduced sample handling. In microwave drying, the

water molecules absorb electromagnetic radiation directly and heat the sample

from the inside, which reduces the heating time considerably (May and others

2003). These advantages led Chin and others (1985) to standardize and validate

a microwave method for tomato products. The method involves weighting the

sample on a glass fibre pad and drying the pad in a CEM microwave oven Model

AVC-MP for 4 minutes. The sample is automatically weighed before and after the

heat treatment and the loss in weight is used to calculate the moisture content of

the sample.

The validation was done in a collaborative study with 14 laboratories

analyzing 7 samples with solids content ranging from 6.5 to 40.2%. The

repeatability (std dev) ranged from 0.02 to 0.22 and the reproducibility (std dev)

ranged from 0.08 to 0.37 over the concentration range of the samples (Chin and

others 1985).

A comparison of the vacuum oven method (13th edition ofAOAC, 1980) with

the microwave drying method showed no difference at the 95% confidence level

15

indicated that these two methods were in excellent agreement (Chin and others

1985). Based on these results, Chin and others (1985) recommended that the

microwave oven drying method be considered as an alternative to the official

vacuum oven method.

In another study conducted in Canada, the reproducibility and accuracy of the

total solid measurement by microwave were examined on tomato samples

(Wang 1987). However in Wang's experiment, the CEM Model AVC-80

microwave was employed rather than the microwave employed by Chin (AVC-

MP). The results of an eight lab collaborative study indicated that the microwave

oven method produces higher value than the vacuum oven method. Moreover,

their result demonstrated that the AVC-80 model produced higher result than the

AVC-MP model (Wang 1987).

1.2.4.2 Total Solids: Canadian Method

The Canadian regulatory agency has adopted the vacuum oven methods for

the determination of total solids in tomato paste (Health Protection Branch

Ottawa 1981). The regulation states a 'salt-free' total solid in its methodology.

Health protection branch Ottawa method FO-19 states "The method shall be

used for determination of the percent tomato solids in tomato paste under section

B. 11.009 of the Food and Drug Regulation and in concentrated tomato paste

under section B. 11.010 of the Food and Drug Regulation.

Section B. 11.009 states that "Tomato paste shall be the product made by

evaporating a portion of the water from tomato or sound tomato trimmings, may

contain salt and class II preservatives and shall contain not less than 20 percent

16

tomato solids as determined by Official Method FO-19, Determination of Tomato

Solids". Section B.11.010 also states "Concentrated tomato paste shall be

tomato paste containing not less than 30 percent tomato solids as defied by

Official Method FO-19".

The procedure is similar to method Solids (Total) AOAC (2000) with the

exception that the inherent salt is deducted from total solids determined by drying

in a vacuum oven. The official methods FO-1 determines sodium chloride by

titration with 0.1N NH4SCN in the presence of concentrated HN03 and ferric

indicator (Health Protection Branch Ottawa 1981).

1.2.4.3 Total Solids by NTSS: United State Method

The US Food and Drug Administration's standards of identity for tomato pulp

(puree) and paste employs a different term when describing tomato solids.

They use the term 'Natural Tomato Soluble Solids' (NTSS). In the NTSS method,

a refractometer reading is taken at 20°C on the clear serum fraction of a tomato

product containing no added salt. Solid content is expressed as percent sucrose.

Since the official method for total solids determination is labor intensive and time

consuming, many processors have tried faster procedures based on specific

gravity or refractive index. In an attempt to relate the values obtained by

refractometry, vacuum oven and specific gravity, the National Canners

Association (NCA) derived an average factor that links these three

measurements to total solids (Bigelow and Fitzgerald 1915).

17

This attempt resulted in a table providing numerical factors for refractive index,

vacuum oven drying and specific gravity (Bigelow and Stevenson 1923).

However this first table was based on data from sample with concentrations

below 20% solids. A second edition followed extending the values to samples

with solids content up to 35%.

A similar approach was taken by Saywell and Cruess (1932), where a factor

relating refractive index with total solids as established on California tomatoes.

However the proposed factor was very different from the one in the National

Canners Association publication. This poor agreement has appeared in many

other investigations over the years. The main reason for this difference was

attributed to variations in the ratio of soluble solids / insoluble solids in different

pastes.

Due to this problem, the quest to find a valid factor to relate total solids,

refractive index and specific gravity was delayed until the adoption of a new

official method for total solids by vacuum oven (10th edition, AOAC 1965) and the

adoption of the refractive index method employing pectic enzymes. The use of

enzymes accelerates paste filtration time and reduces evaporation during the

test.

Taking advantage of the modified procedure for total solid determination by

vacuum oven in the 10th edition (AOAC 1965) and employing enzymes to

improve the filtration step in the refractive index determination, Lamb (1967) not

only compared the old official method to his modified method but also attempted

to establish a relationship between refractive Index, specific gravity, and total

18

solids in tomato juice, puree and paste. Based on these studies the National

Canners Association developed a table to convert the NTSS value obtained by

refractive index to percent total solids (corrected for added salt).

1.2.5 Water Soluble Solids

The soluble solid is the main solids fraction in tomato and tomato products.

Sugars accounts for almost 50% of the solids in concentrated samples and the

major aroma compounds are also found in this fraction.

1.2.5.1 Soluble Solids: AOAC Method

In 1951, Cheftel made comments on the validity of solids determination by

drying. He questioned the ability of oven drying to distinguish between free and

bound water in paste samples. He recommended that the "refractive index

method" be used to specify the solids content of tomato products. The

recommendation was based on his observation that the refractive index method

was more controllable, had a higher degree of precision and was easy to

perform.

Lamb (1969) initiated a collaborative study to evaluate the methodology for

soluble solids determination in tomato products by refractive index. In that

collaborative study the various laboratories evaluated three paste concentrations,

two filtration methods and the use of pectic enzymes. In that same trial, an

ultracentrifuge (150,000 x g) was used to prepare clear serum samples without

the need for pectic enzyme treatment or filtration. The standard deviation for the

soluble solids measurements varied from 0.15% for samples containing 24%

soluble solids (based on sucrose) to 0.40% for sample with 44% soluble solids.

19

This range of 0.15%-0.40% in standard deviation was comparable with results

obtained with the official AOAC method (vacuum drying method). Although the

ultracentrifugation results showed good agreement with the filtration method,

conclusions were not made because the ultracentrifugation results were from

only one lab (Lamb 1969). Based on the collaborative study and Lamb's

recommendations, the refractive index method employing filtration was adopted

as official method of AOAC for first action in 1970.

The 17th edition (AOAC 2000) describes the refractive index procedure for the

measurement of soluble solids. The method involves measuring the refractive

index (± 0.0001 Rl units) of the clear soluble solids solution from a tomato

product. To isolate the soluble solids, the sample is treated with a pectic enzyme

and depending on the filtration behavior of the sample it may or may not need

dilution. Centrifugation is another option if an ultracentrifuge is available.

Measuring the refractive index and correcting for added enzyme and insoluble

solids determine the solids content. Correction for added enzyme in case of

filtration without dilution is achieved by subtracting the term 1.15*BxC from the

refractive index reading where 1.15 is the correction for insoluble solids, B

accounts for % enzyme preparation and C is the reading as sucrose obtained on

a 1% solution. In case of dilution, the correction term becomes 0.55xD*C where

0.55 is the correction for insoluble solids, D is the % added enzyme, C is the

reading as sucrose obtained on a 1% solution.

In the case of added salt, the refractometer value expressed as % sucrose

should be corrected for salt by the following equation:

20

Equation 2

NTSS= (Refractometer sugar scale reading at 20 °C - % Total salt) x 1.016

1.2.5.2 Soluble Solids: Formula Method

Another way to determine soluble solids is to measure the total solids and

subtract the insoluble solids (Bohart 1940). The principle behind this procedure

assumes that a negligible amount of soluble solids is adsorbed by the insoluble

fraction and with appropriate centrifugation of the sample a clear supernatant

containing all the soluble solids can be made. In this method, both total solids in

the paste and the solids content of the supernatant fraction are measured. It

should be noted that the % solids in the supernatant fraction (%SSF) is not the

same as the % soluble solids (%SS) in the paste. Equation 3 is used to calculate

the % water insoluble solids (%WIS).

%WIS = 100(%TS-%SSF) Equation 3 100-%SSF

Where:

%WIS = % Water Insoluble Solids in paste

%TS = % Total Solid in Paste

%SSF = % Solid in Supernatant fraction after one centrifugation

The soluble solids (%SS) in the paste can then calculated with Equation 4.

%SS =%TS-%WIS Equation 4

Where:

%SS = % Soluble Solids in paste

%TS= % Total Solid in paste

%WIS=% Water Insoluble Solids in paste

21

1.2.6 Water Insoluble Solids

There is wide agreement among researchers that the amount of insoluble

solids (WIS) has the greatest influence on gross juice viscosity, but titrable

acidity, serum viscosity and the nature of the suspended particles may also

contribute to the gross viscosity (Kertesz and Loconti 1944, York and others

1967, Bartolome 1972).

1.2.6.1 Water Insoluble Solids: AOAC Method

The AOAC Methods (AOAC 2000) determines water insoluble solids by

adding a certain amount of tomato products to boiling water and separating out

the soluble fraction by centrifugation. Multiple washings and centrifugations

achieve complete removal of the soluble fraction. The weight of the dried residue

represents the WIS fraction.

In brief, the official AOAC Method subjects 20 g of paste to 4 or 5 washings

with hot water. Each washing step is centrifuged to produce a clear supernatant.

The supernatant is filtered through tared filter paper in a Buchner funnel. The

pallet is collected on the same filter paper and the residue dried in an uncovered

dish for 2 hours at 100 °C, cooled in a desiccator and then weighed.

1.2.6.2 Water Insoluble Solids: Formula Method

To determine insoluble solids by the formula method, the procedure outlined

in section (1.2.5.2 soluble solids) is followed. The principle is based on the

measurement of total solid and the % solids in the supernatant fraction (%SSF)

after one centrifugation and utilizing Equation 3.

22

One of the most scholarly sources of information on tomato and tomato

product testing is the National Food Processor Association Bulletin 27-L. In the

7th edition Equation 3 is given high marks. The bulletin describes the procedure

for determining %TS and %SSF. An unfiltered paste sample is vacuum dried to

determine %TS. For %SSF determination, the paste sample is diluted with water

and filtered and the clear filtrate is vacuum dried. To overcome the possibility of

evaporation during filtration, the method recommends using centrifugation of the

diluted sample (approximate 12% solid) for 10 minutes at 2,000 RPM. The

supernatant is easier to filter and the possibility of evaporation is lower. The

results (%TS and %SSF) are multiplied by the dilution factor and used in

Equation 3 to calculate %WIS. However, this publication acknowledges the

possibility of error if appreciable amount of water was absorbed by the insoluble

solids or by the filter paper.

Although it appears to be a simple task, the determination of solids in tomato

products and especially concentrated tomato pastes has been a challenge over

the years. The lack of a recognized definition for the different solids fractions has

led to controversy not only in international commerce but also among industrial

processors and the scientific community. However, all the proposed methods that

were developed for scientific purposes or for commerce, were intended to be

rapid and with sufficient reproducibility to serve their intended purpose.

Because different definitions and approaches have been employed to

measure solids, we will use the following definitions of solids in our study. The

following are the solids definitions:

23

A. Total Solids: The residue that remains after all the moisture has been

removed from the paste by the conditions specified by the vacuum or microwave

oven methods and without subtracting inherent salt.

B. Insoluble Solids: The water insoluble fraction free of all soluble compounds.

C. Soluble Solids: The fraction containing the compounds that dissolves in water.

D. Solids in Soluble Fraction: The supernatant from first centrifugation of diluted

paste containing soluble solids and colloidal particles.

Although the principles behind the procedures used in this study are similar to

the AOAC official method, some modifications were made to facilitate the

removal of water and to reduce some of the sources of errors. The repeatability

of the measurements was used to assess the effects of these modifications on

the robustness of the procedures. In addition it was considered important to

compare the results obtained by the vacuum oven method with the results

obtained by the microwave method and applying Equation 3 and 4.

It is known that one of the drawbacks in the soluble solid measurement in the

official method is the filtration step to remove some suspended particles, which

appear to be soluble. The nature of these particles is not known and

consequently can not be categorized as soluble or insoluble but for now is

considered to be soluble as long as their composition is unknown.

24

1.3 Experimental

The Figure 1.3 depicts the analysis of the diluted paste sample for total solids,

insoluble solids and soluble solids. Two methods are used to analyze the same

paste sample. The vacuum oven is a direct method and is recommended by the

AOAC. In the direct method, all three solids fractions are individually separated

and weighed. The microwave oven is an indirect method and is recommended by

the National Canners Association. For the indirect method the total solids is

determined directly by microwave drying but the soluble and insoluble solids are

determined by equation using microwave.

I Dilluted Sample

Figure 1.3 Flow Chart for Solids Analysis by the Vacuum and Microwave

Methods.

1.3.1 Material and Equipment

Analytical balance model Mettler AE 240 (Mississauga, ON. Canada),

centrifuge model J2-21 and rotor Ja-20 capable of producing approximately

20000 rpm, 31,360xg force (Beckman, Mississauga, ON, Canada), plastic

centrifuge tube 50 ml_ (Fisher Scientific, Mississauga, ON, Canada), aluminum

25

70mm x 32mm pans with covers (Dual Manufacturing Co. Inc. Chicago, IL, USA),

water bath equipped with digital thermostat model HAKKE W26 (Thermo Fisher

Scientific, Mississauga, ON, Canada), sintered glass filter: 50 mil Pyrex® coarse

40-60 ASTM (Fisher Scientific, Pittsburgh, PA, USA), jumbo bulb 10 cm pipette

(Curtin Matheson Scientific, Wood Dale, IL, USA), celite acid wash (Sigma

Aldrich, St. Louis, Mo, USA), vacuum oven model 281 capable of maintaining

temperature at 70°C ±1° with no more than 2°C variation between shelves

(Fisher ISoTemp® Co., Pittsburgh, PA, USA), vacuum pump operating pressure

-30 inches Hg (-100 Kpa) (Duoseal 1380 Welsh Vacuum .Thomas Industries

Inc., IL,USA), microwave oven solid analyzer (CEM model AVC-80), glass fibers

10x10cm sample pads suitable to be used in CEM microwave oven model AVC-

80, convention drying oven (Memmert), stomacher (400 lab blender), laboratory

hot plate, desiccators with silica gel absorbent, commercially available tomato

paste samples in range 25-30 % total solids.

1.3.2 Methods

1.3.2.1 Vacuum Oven Method

1.3.2.1.a Total Solids (Vacuum Oven)

The total solids were determined by drying the sample in a vacuum oven.

1.3.2.1.a.b Sample Preparation

Due to the high solids content of the samples, a dilution step was required.

50g of paste was weighed into a stomacher bag and diluted with 100g of distilled

water. The level of dilution was determined in preliminary trials. The diluted

sample was thoroughly mixed in the stomacher until no paste clumps were

26

visible. By turning the bag over at intervals in the stomacher this procedure

quickly dispersed the paste into a homogenous mass. Three replicate samples

were prepared with this procedure.

1.3.2.1.a.c Procedure

Approximately 15 mg/sq cm of diatomaceous earth was added to drying pans

and heated in an oven set at 110°C for 30 minutes to dry the pan and

diatomaceous earth. The dried pans were transferred to a desiccator and cooled

for 30 minutes. The dried pans were weighed on an analytical balance and the

initial weight recorded.

Approximately 7g of diluted sample were weighed into the pan in triplicate.

The initial weight was adjusted to give a final dry residue weight in the pan of 9-

30 mg per square centimetre. To reduce problems due to evaporation during the

weighing process, this step has to be performed quickly or alternatively the pan

has to be covered during the weighing procedure. After weighing the sample, a

small amount of water was added to the mixture of diatomaceous earth and

sample to evenly distribute the sample in the pan.

To facilitate the drying process, the samples were pre-dried in a boiling water

bath prior to transferring them to the vacuum oven. The vacuum oven was set at

70°C and the temperatures of the shelves were measured with a thermometer in

direct contact with the shelves. Following the procedure of Lewis and Kimbal

(1961), two 250 ml_ bottles were connected in series with the petcock release

valve on the vacuum oven. The nearest bottle to the petcock was filled with

glass wool and the farthest was filled with 90 ml_ of concentrated sulfuric acid.

27

This design worked as a trap to remove moisture and sulfuric acid from the air

entering the chamber. When the volume of sulfuric acid increased by more than

5 mm due to absorbed moisture, the acid was replaced with fresh concentrated

sulfuric acid. Replicates of each sample were uncovered and placed in rows from

back to front as it seemed that the back of the chamber had a slightly higher

temperature. The applied pressure of <50 mm Hg was applied to the chamber.

Dry air (pass through concentrated sulfuric acid) was allowed to purge the

chamber at a rate of 3 bubbles/second. The temperature of the vacuum oven

chamber dropped initially, but reached the set temperature of 70 °C±rC after 15

minutes. Samples were kept in the vacuum oven for exactly 2 hours. The

vacuum was turned off after 2 hours and the rate of air entering the chamber

increased to 6-8 bubble/second. When the vacuum was completely released the

oven was opened and lids were placed on the pans and transferred to a

desiccators to cool. The cooled samples were weighed on an analytical balance.

The total solids content of each replication was calculated based on weight

loss. Because the samples were diluted 1:3, a dilution factor of 3 was applied to

calculate the true total solids.

Equation 5

% Total solids = (Weight of dry residue + dish) - (Weight of dish )x100 * 3 (Weight of sample +dish)- (Weight of dish)

1.3.2.1.b Water Insoluble Solids (Vacuum Oven)

The water insoluble solids were separated from the soluble solids by

successive washing of the paste with water. To avoid solubilizing the cell wall

28

materials, hot water was not used. However to compensate for the reduced

efficiency, the total number of washing steps was increased.

1.3.2.1.b.a Sample Preparation

The same procedure detailed in section 1.3.2.1.a.b was used to prepare the

diluted sample.

1.3.2.1.b.b Procedure

23-25 g of diluted sample were weighed into three 50 ml_ centrifuge tubes. To

aid with the subsequent filtration step, 10 to 15 mL of water (adequate to balance

the tubes for centrifugation) was added to each tube. It was shown in preliminary

trials that the addition of water at this stage was better than diluting the original

sample down to this solids level. By vortexing the centrifuge tube, the added

water was mixed thoroughly with the diluted sample.

The three replications were centrifuged for 18 minutes at 26000*g to

separate the soluble solids from the insoluble solids. Glass beads (3 mm) were

added to a sintered glass filter to act as a filter aid during the filtration of the

supernatant after each washing/centrifugation step. The filter and filter aid was

dried in the oven set at 110 °C for 2 h prior to their use.

Aluminum pans were prepared by adding approximately 15 mg/sq cm of

diatomaceous earth and drying them in an oven set at 110°C for 30 minutes.

The weight of the pan and diatomaceous earth was recorded after cooling in a

desiccator for 30 minutes to 1 hour. The supernatant from the centrifugation was

filtered through the sintered glass filter and collected in the weighed pan. This

material represents the soluble solids and will be discussed in the next section.

29

The recommended procedure is to repeat the washing step until no soluble

solids is detected in the supernatant. The best indication of this point is the

refractive index measurement of the supernatant. A Brix value of zero would

indicate zero soluble solids in the supernatant. After a few trials, it was

determined experimentally that by adding 10ml_ to 15ml_ water (adequate to

balance the tubes for centrifugation) to each centrifuge tube and washing the

sample 5 times, a Brix value of zero can be obtained. However, to have a little

safety margin, a total of 6 washing steps were used in this study.

The pellet resulting from 6 washed steps was transferred to a pre-dried and

weighed aluminum pan. For each centrifuge tube there was a designated pan.

By adding small amount of water and using a spatula, the insoluble solids were

spread uniformly over the bottom of the pan. Because this sample is free of

sugars and is less prone to caramelize, it was possible to subject it to higher

temperatures and longer drying times. The experimental practice showed that

subjecting this sample to a temperature of 100°C for 8h or over night resulted in

the efficient evaporation of water. The dried sample was covered and placed into

a desiccator and weighed when they reached room temperature.

As the number of washing steps increased, it became more difficult to form a

firm pellet during centrifugation. These loose pellets would release insoluble

material into the supernatant liquid during decanting and lead to a loss in

insoluble solids. To prevent this error, the supernatant is decanted into a

sintered glass filter to collect the released insoluble material. This amount of

trapped insoluble solids on filter is dried and added to the dried insoluble pellet.

30

The water insoluble solids (WIS) of each replication was calculated as the dry

weight of the residue after 6 washings divided by the initial weight of the paste

while the dried weight of the residue itself is the sum of dried weight in the pan

and on the sinter glass filter.

Equation 6

Dried weight of residue in pan = (Weight of dry residue + pan) - (Weight of pan)

Equation 7

Dried weight of residue in filter=(Weight of dry residue + filter) - (Weight of filter)

Because the samples were diluted 1:3, a dilution factor of 3 was applied to

calculate the true WIS.

Equation 8

% Insoluble Solids = ( Equation 6 + Equation7 ) x 3 * 100

Weight of the initial sample in the centrifuge tube

1.3.2.1.c Water Soluble Solids (Vacuum Oven)

None of the recognized methods measure soluble solids directly by isolating

soluble solids from insoluble solids. The method developed in the present study

is the first reported procedure that measures soluble solids directly.

1.3.2.1.c.a Sample Preparation

The same dilution procedure that was used for total solids measurement

(1.3.2.1 .a.b) was used for soluble solids.

1.3.2.1.c.b Procedure

The same procedure used for the isolation of WIS (section 1.3.2.1.b.b) was

used for the isolation of soluble solids. In this experiment, the supernatant is

collected rather than the pellet. The supernatants from six successive washing

31

steps were passed through a sintered glass filter with glass beads and collected

in a dried and pre-weighed pan.

The filtered soluble solids solution contains a large amount of water and was

therefore pre-dried in a water bath set at 70°C to prevent the caramelization of

the sugars. The pre-weighed pans containing diatomaceous earth and the

soluble solids solution were placed in the water bath. Approximately 24 h was

needed to partially dry these samples. The pre-dried samples were transferred to

a vacuum oven set at 70°C and the applied pressure of <50 mm Hg. After exact

2 h the vacuum was released as describe previously. The sample were covered

with a lid and transferred to a desiccator and weighed after cooling to room

temperature.

The water soluble solid content (%SS) of each replication was calculated as

the difference in the dry weight of the supernatant from 6 centrifugations divided

by the weight of the initial paste. Because the samples were diluted 1:3, a dilution

factor of 3 was applied to calculate the true soluble solids content.

Equation 9

Soluble Solids (%) = (Weight of dry residue + dish) - (Weight of dish)x100 *3

Wight of the initial sample in the centrifuge tube

It should be noted that in our experiment we collected the soluble solids from

the same centrifuged tube that the insoluble solids was collected, but it is not

necessary to collect the insoluble solids if only the soluble fraction is required.

1.3.2.2 Microwave Oven Method

1.3.2.2.a Total Solids (Microwave Oven)

The following procedure is based on the official method of AOAC sec 42.1.09

32

(AOAC 2000) for the total solids content of paste determined by the microwave

oven method.

1.3.2.2.a.b Sample Preparation

The same procedure detailed in section 1.3.2.1.a.b was used to prepare the

diluted sample.

1.3.2.2.a.c Procedure

The microwave oven was set for power level 100% and time 4 minutes. Two

glass fibre pads were placed on the balance ring in the microwave and the

complete cycle of 4 minutes was run. By performing this cycle any moisture in

the pads was removed. The pre-dried pads were placed on the scale in the oven

and tared. The balance displays 0.0000 with a deviation of ±0.0002.

Approximately 2 g of the diluted paste was removed with a jumbo bulb pipette

and deposited on the first pad and then covered with the second pad. The

diluted sample is composed of large paste particles suspended in water that

could clog the pipette and not deliver a homogeneous sample. To remedy this

problem, the pipette tip was cut off to increase the outlet pore size and allow the

free flow of the sample. The deposition of the sample on the pad was performed

quickly to reduce absorption of air moisture and/or evaporation of samples

moisture. After placing of the sample on the balance, the microwave door was

closed and the microprocessor displays the weight of the sample in less than 5

second. When the weight starts to decrease the run button was pressed and the

drying cycle starts. The percent solids content of samples was automatically

displayed at the end of the 4 minutes cycle.

33

Three determinations were performed on each sample and if the variation

between readings was greater than 0.02, the reading was not accepted and more

replications were performed.

1.3.2.2.b Water Insoluble Solids (Microwave Oven)

The insoluble solids were determined by calculation using values for total

solids (%TS) and % solids in the supernatant fraction (%SSF) in Equation 3

(see 1.2.5.2). %TS was determined by microwave oven (see 1.3.2.2).

%WIS = 100(%TS-%SSR 100-%SSF

Where:

%WIS = % Water Insoluble Solids in paste

%TS = % Total Solid in paste (see 1.3.2.2.a)

%SSF = % solids in the supernatant fraction from one centrifugation (see

1.3.2.2.C)

1.3.2.2.C Solids in the Supernatant Fraction (Microwave Oven)

The % solid in the supernatant fraction (%SSF) is the concentration of

dissolved solids in the supernatant of a paste sample that was centrifuged once

to separate the water insoluble solids from the aqueous phase (supernatant

fraction). The (%SSF) should not be confused with the % soluble solids (%SS)

term which represents the total soluble solids in the paste.

1.3.2.2.c.a Sample Preparation

Three replications of each sample were prepared by pipetting 25 g of diluted

paste into a 50 ml_ centrifuge tubes (details given at 1.3.2.1.b).

34

1.3.2.2.c.b Procedure

Replicate samples were centrifuged for 18 minutes at 26672xg. The clear

supernatant was collected and used directly without filtration to avoid absorption

of serum on the filter medium and moisture evaporation during filtration. The

solids in the supernatant was determined by evaporating the moisture in a CEM

microwave set at 100% power level and run time of 5 min. Before starting, the

empty microwave oven was pre-conditioned by running the cycle once. Two

glass fiber pads were dried by placing them on the scale in the microwave oven

and running one cycle. The dried pads were tared on the scale. To one pad, 2.5-

3.5 g of sample were spread out evenly and then covered with the second pad.

The sample containing pads were placed on the microwave internal balance and

automatically weighed by pressing the run button. After completing the heating

cycle, the microwave reports the soluble solid in the supernatant as a percentage

of the starting weight.

1.3.2.2.d Water Soluble Solid (Microwave Oven)

The percent water soluble solids (%SS) is the final component that was

determined by the microwave method. The concept of soluble solids defines it as

the portion of solids in the paste that is soluble. The water soluble solids were

determined indirectly by calculation. The difference between total solids and

insoluble solid is equal to the soluble solids in the paste. The total solid was

determined by microwave method (section 1.3.2.2.a) and the water insoluble

solids were determined by calculation (section 1.3.2.2.b). Soluble solid was

calculated with the following equation:

35

Equation 10

%Water Soluble Solids = %TS - %WIS

%TS = % Total Solid in paste (see 1.3.2.2.a)

%WIS=% Water Insoluble Solids (see 1.3.2.2.b)

As the samples were diluted prior to analysis, all values have to be multiplied by

a factor of 3 to obtain the final concentrations.

36

1.4 Results

The results from these experiments were used to examine the repeatability of

each method and to compare the two methods (the microwave method and the

vacuum oven method).

1.4.1 Vacuum Oven Method

1.4.1.1 Total Solids (Vacuum Oven)

The % total solids were determined in triplicate following the procedure

outlined in section 1.3.2.1.a. The mean of three determinations and the standard

deviation for 20 different paste samples are given in Table 1.1.

Table 1.1 Percent Total Solids (%TS) in Tomato Pastes by Vacuum Oven.

Mean of Three Determinations (%) ± Standard Deviation.

ID

1

2

3

4

5

TS (%)

28.51±0.05

28.03±0.07

27.45±0.12

28.44±0.09

27.68±0.02

ID

6

7

8

9

10

TS (%)

27.07±0.05

24.85±0.05

26.58±0.09

27.83±0.10

26.72±0.08

ID

11

12

13

14

15

TS (%)

25.53±0.06

24.63±0.07

28.37±0.06

28.21±0.06

28.19±0.06

ID

16

17

18

19

20

TS (%)

26.63±0.04

29.69±0.07

27.88±0.09

25.15±0.00

25.02±0.02

1.4.1.2 Water Insoluble Solids (Vacuum Oven)

Similar to total solids, the water insoluble solids were determined in triplicate

on 20 different paste samples following the procedure in section 1.3.2.1.b. The

mean of three determinations and the standard deviation for 20 different paste

samples are given in Table 1.2.

37

Table 1.2 Percent Water Insoluble Solids (%WIS) in Tomato Pastes by

Vacuum oven. Mean of Three Determinations (%) ± Standard Deviation.

ID

1

2

3

4

5

WIS (%)

5.67±0.08

5.52±0.08

5.93±0.05

6.31±0.08

5.20±0.02

ID

6

7

8

9

10

WIS (%)

5.54±0.04

5.28±0.49

6.43±0.23

6.10±0.09

5.68±0.22

ID

11

12

13

14

15

WIS (%)

5.56±0.07

5.23±0.03

5.98±0.06

5.58±0.06

6.66±0.01

ID

16

17

18

19

20

WIS (%)

5.01 ±0.08

5.53±0.8

6.1U0.04

5.42±0.02

5.47±0.10

1.4.1.3 Water Soluble Solids (Vacuum Oven)

Following the procedure detailed in section 1.3.2.1.C, the soluble solid fraction

of tomato paste was determined in triplicate. The mean of three determinations

and the standard deviation for 20 paste samples are given in Table 1.3.

Table 1.3 Percent Water Soluble Solids (%SS) in Tomato Pastes by Vacuum

Oven. Mean of Three Determinations (%) ± Standard Deviation.

ID

1

2

3

4

5

SS (%)

22.77±0.07

22.19±0.07

21.55±0.18

22.21±0.09

22.94±0.03

ID

6

7

8

9

10

SS (%)

22.05±0.05

19.61±0.07

21.47±0.54

22.14±0.08

21.15±0.08

ID

11

12

13

14

15

SS (%)

20.18+0.10

19.41±0.11

22.34±0.12

22.93±0.17

21.92±0.06

ID

16

17

18

19

20

SS (%)

22.10±0.07

23.54±0.25

22.05±0.12

19.97±0.01

19.98+0.05

1.4.2 Microwave Oven Method

The total solids content of tomato paste were determined directly by

microwave oven drying. However the soluble solids and water insoluble solids

were calculated with a formula that requires values for the % total solids (%TS)

and % solids in the supernatant (%SSF).

38

1.4.2.1 Total Solids (Microwave Oven)

The % total solids were determined by microwave as described in section

1.3.2.2.a. The mean of three determinations and the standard deviation for 20

different paste samples are given in Table 1.4.

Table 1.4 Percent Total Solids (%TS) in Tomato Pastes by Microwave. Mean of

Three Determinations (%) ±Standard Deviation

ID

1 2 3 4 5

TS (%)

28.28+0.09 28.20±0.06 27.77±0.06 28.66±0.05 27.50±0.02

ID

6 7 8 9 10

TS (%)

27.48±0.00 25.32±0.04 28.13±0.06 27.92±0.06 26.91+0.00

ID

11 12 13 14 15

TS (%)

25.52±0.06 24.89±0.02 28.47±0.04 27.99±0.04 28.29+0.04

ID

16 17 18 19 20

TS (%)

26.88±0.04 29.99±0.02 28.26±0.09 25.28±0.02 25.40±0.06

1.4.2.2 Solids in the Supernatant Fraction (Microwave Oven)

The % solids in the supernatant fraction (%SSF) were determined using the

procedure described in section 1.3.2.2.C. Three replicate samples were

centrifuged and the supernatant dried in the microwave oven. The readings were

repeated at least three times and in some cases where high variations were

detected, it was repeated more than 3 times. The results are shown in Table 1.5.

Table 1.5 Percent Solids in the Supernatant Fraction (%SSF) in Tomato

Pastes by Microwave. Mean of Three Determinations (%) ± Standard Deviation.

ID

1 2 3 4 5

SSF(%)

23.25±0.06 22.78±0.11 22.12±0.09 23.01±0.00 22.40+0.02

ID

6 7 8 9 10

SSF (%)

22.22±0.02 20.12±0.02 21.84±0.00 22.23±0.08 21.38±0.06

ID

11 12 13 14 15

SSF (%)

20.34±0.04 19.64±0.02 22.64±0.04 22.845±0.02 22.28±0.02

ID

16 17 18 19 20

SSF (%)

22.28±0.06 23.90±0.11 22.58±0.02 20.22±0.04 20.1 ±0.00

39

1.4.2.3 Water Insoluble Solids (Microwave Oven)

The % water insoluble solids (%WIS) were determined by calculation using

the equation in section 1.3.2.2.b. The means values for (% TS) and (%SSF)

were used in the equation. The standard deviation was not included in Table 1.6

as these values were calculated with the means of the (%TS) and (%SSF).

Table 1.6 % Water Insoluble Solids (WIS) in Tomato Pastes by Equation 3

(Microwave Oven)

ID

1 2 3 4 5

WIS (%)

5.45 5.87 6.10 6.12 5.51

ID

6 7 8 9 10

WIS(%)

5.69 5.58 6.78 6.14 5.96

ID

11 12 13 14 15

WIS(%)

5.55 5.62 6.31 5.57 6.50

ID

16 17 18 19 20

WIS(%)

4.97 6.12 6.12 5.50 5.65

1.4.2.4 Soluble Solids (Microwave Oven)

The water soluble solids were calculated with Equation 10 in section

1.3.2.2.d by the difference between percent total solids and percent insoluble

solids. The standard deviation was not included in Table 1.7 as these values

were calculated with the means of the total solids and the insoluble solids.

Table 1.7 Percent Soluble Solids (%SS) by Difference Between Total and

Insoluble Solids (Microwave Oven)

ID 1

2

3

4

5

SS (%) 22.83

22.33

21.67

22.54

21.98

ID 6

7

8

9

10

SS (%) 21.79

19.74

21.35

21.78

20.95

ID 11

12

13 14

15

SS (%) 19.97

19.27

22.16

22.42

21.79

ID 16

17

18

19

20

SS (%) 21.91

23.87

22.14

19.85

19.72

40

1.4.3 Comparison of Methods (Microwave vs. Vacuum oven)

The mean values for percent total, insoluble and soluble solids determined by

the microwave oven method and the vacuum oven method are shown in Table

1.8. The means for each method were compared.

Table 1.8 Comparison of Mean Values of % Total, % Water Insoluble and

% Water Soluble Solids Measured by Vacuum and Microwave Methods.

ID

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20

Ave.

Total

Solids%

Vacuum 28.51 28.03 27.45 28.44 27.68 27.07 24.85 27.58 27.83 26.72 25.53 24.63 28.37 28.21 28.19 26.63 29.69 27.88 25.15 25.02 27.17

Microwave 28.29

28.2 27.78 28.65 27.51 27.48 25.32 28.14 27.93 26.91 25.52 24.89 28.47 27.99 28.29 26.88 29.99 28.26 25.35 25.37 27.36

Water Insoluble

Solids%

Vacuum 5.67 5.52 5.93 6.3JL 5.20 5.54 5.28 6.43 6.10 5.68 5.56 5.23 5.98 5.58 6.66 5.01 5.53 6.11 5.42 5.47 5.71

Microwave 5.45 5.87 6.10 6.12 5.51 5.69 5.58 6.78 6.14 5.96 5.55 5.62 6.31 5.57 6.50 4.97 6.12 6.12 5.50 5.65 5.86

Water Soluble

Solids %

Vacuum 22.70 22.32 21.65 22.29 22.88 21.99 19.64 21.28 22.09 21.19 20.15 19.54 22.47 22.82 21.97 22.06 23.67 22.14 19.98 19.92 21.64

Microwave 22.83 22.33 21.67 22.54 21.98 21.79 19.74 21.35 21.78 20.95 19.97 19.27 22.16 22.42 21.79 21.91 23.87 22.14 19.85 19.72 21.50

41

1.5 Statistical Analysis

Data analysis was done by S-PLUS, Copyright (c) 1988, 2007 Insightful Corp.

S: Copyright Insightful Corp. Enterprise Developer Version 8.0.4 for Microsoft

Windows: 2007. All data used in the statistical analysis were from the data set

where the total solids in the 20 paste samples ranged from 24 to 29%.

1.5.1 Repeatability (Vacuum and Microwave Oven Methods)

1.5.1.1 Total Solids (Vacuum Oven)

To compare the repeatability of the microwave and vacuum oven methods,

the Average Standard Deviation (ASD) was calculated. ASD is defined as the

square root of the arithmetic mean of the square of the deviations from the

average value for a set of observations.

Equation 9

MD= |Z?°ZJ(w-W (3 - 1) X (20)

Where:

yij: Observation for one replication of a sample

yi: Mean of replications for a sample

ASD can be considered as a measure of statistical dispersion, measuring how

widely spread the values are in a data set. If many data points are close to the

mean, then the ASD is small; if many data points are far from the mean, and then

the ASD is large. If all data values are equal, then the ASD is zero. The source of

the errors is both systematic errors (calibration of instruments, changes in the

environment, imperfect observational measurements) and random errors

42

(inherent limitations of the instrument or the experimenter's inability to precisely

make measurements or take measurements). In this project, ASD was calculated

for the 20 samples with 3 replications on each sample. For Total Solids, the

vacuum method produced an ASD of 0.067%.

1.5.1.2 Water Insoluble Solids (Vacuum Oven)

The ASD of 0.23% for insoluble solids was determined by the vacuum oven

method.

1.5.1.3 Water Soluble Solids (Vacuum Oven)

The ASD for soluble solids determined by the vacuum oven method was 0.21%.

1.5.1.4 Total solids (Microwave Oven)

The microwave method for total solids was evaluated for repeatability in the

same manner as was done with the vacuum oven method. The ASD of

measurements determined by microwave was 0.045%.

1.5.1.5 Solids in Supernatant Fraction (Microwave Oven)

The average standard deviation for solids in the soluble solids fraction was

0.050%.

1.5.2 Comparison of Methods (Microwave vs. Vacuum oven)

1.5.2.1 Total Solids (Microwave vs. Vacuum oven)

1.5.2.1.a Equality of the Methods (Total Solids)

A paired t-test was performed at the 5% significant level to test equality of

means between the two methods. A mean difference of -0.187 (27.17- 27.36)

was calculated for total solids (Table 1.8, section 1.4.3) The p-value was 0.001

43

which indicates that the mean of the total solids by the vacuum oven method is

significantly smaller than the mean of the microwave oven method.

Table 1.9 Total Solids, t-Test: Paired Means Variablel: total solid contents determined by vacuum oven Variable2: total solids contents determined by microwave oven

Variable 1 Variable 2 Mean 27.17350107 27.36 Variance 2.068291792 1.932631579 Observations 20 20 Pearson Correlation 0.98873264 Hypothesized Mean Difference 0 df 19 tStat -3.832751402 P(T<=t) two-tail 0.001122624 t Critical two-tail 2.09302405

1.5.2.1.b Regression Equation of the Methods (Total Solids)

The means of the 20 microwave samples were regressed with the means of

the 20 vacuum oven samples using a simple linear model. This resulted in a R2

value of 0.9776 in the model:

{Total solids (microwave oven) = 0.956(vacuum oven) +1.389}

1.5.2.1.c Regression of Exact Equality (Total Solids)

An unpaired t-test was performed to test whether the slope of this regression

line was significantly different from 1. This value represents the slope of the

perfect line, in which the total solids determined by vacuum oven is exactly equal

to the value determined by microwave oven. A p-value of 0.213 indicates that

the slope is not significantly different from the value 1 at the 5% significance

level.

44

Total Solids

y K °

25 26 27 28 29

Total Solids Using Vacuum Method

Figure 1.4 Regression of Exact Equality Between Vacuum and Microwave

Oven Methods in Total Solid Determination.

Dashed line= Perfect line, Solid line=Fitted Line, O = Experimental Points

1.5.2.1.d Average Error Between Methods (Total Solids)

The average difference between two methods was calculated via the root

mean square error (RMSE).

^fi&i *02

20 RMSE=AJ

Where:

yi : Total solids by vacuum oven

xi : Total solids by microwave

The RMSE for total solids was 0.283%.

45

1.5.2.2 Water Insoluble Solids (Microwave vs. Vacuum oven)

1.5.2.2.a Equality of the Methods (Water Insoluble Solids)


means between the two methods. The mean difference of -0.145 (5.71 - 5.86)

was calculated for insoluble solids from Table 1.8, section 1.4.3. The p-value

was 0.0078 showed that the water insoluble solids determined by vacuum oven

were significantly smaller than microwave method.

Table 1.10 Water Insoluble Solids, t-Test: Paired Means Variablel: total solid contents determined by vacuum oven Variable2: total solids contents determined by microwave oven

Variable 1 Variable 2 Mean 5.710620376 5.855253206 Variance 0.192490357 0.178781612 Observations 20 20 Pearson Correlation 0.872924537 Hypothesized Mean Difference 0 df 19 t Stat -2.97091443 P(T<=t) two-tail 0.007850883 t Critical two-tail 2.09302405

1.5.2.2.b Regression Equation of the Methods (Water Insoluble Solids)

The simple regression of the means of insoluble solids from microwave and

vacuum oven gave a R2 value of 0.762. The regression model is a follow:

{Insoluble solids (microwave oven) = 0.841(vacuum oven) +1.051}

1.5.2.2.C Regression of Exact Equality (Water Insoluble Solids)

Unpaired t-test tested the difference of the regression slope from one. The p-

value of 0.17 showed that the slope was not different from one and so the two

slopes are identical. Moreover the intersection of perfect line and the

46

experimental line at the higher levels of insoluble solids indicates the two

methods will have better agreement at higher level of insoluble solids.

Water I nso lub le So l i ds

Water insoluble Softds Using Vacuum Method


Oven Method in Water Insoluble Solid Determination.

Dashed line= Perfect line, Solid line= Fitted Line, O =Experimental Points

1.5.2.2.d Average Error Between Methods (Water Insoluble Solids)

The root mean square error (RMSE) of 0.257% was calculated for water

insoluble solids measurements.

1.5.2.3 Water Soluble Solids (Microwave vs. Vacuum oven)

1.5.2.3.a Equality of the Methods (Water Soluble Solids)


means determined by the two methods. A mean difference of 0.13 (21.63 -

21.50) was calculated for soluble solids from Table 1.8, section 1.4.3. The p-

47

value was 0.015 which indicates that the vacuum oven mean was significantly

greater than the microwave mean.

Table 1.11 Water Insoluble Solids,t-Test:Paired Means. Variablel: total solid contents determined by vacuum oven Variable2: total solids contents determined by microwave oven

Variable 1 Variable 2 Mean 21.63712272 21.50199679 Variance 1.438125639 1.483295167 Observations 20 20 Pearson Correlation 0.977603697 Hypothesized Mean Difference 0 df 19 t Stat 2.356342375 P(T<=t) one-tail 0.014672328 t Critical two-tail 2.09302405

1.5.2.3.b Regression Equation of the Methods (Water Soluble Solids)

The means of soluble solids determined by vacuum oven were regressed with

the means of the soluble solids determined by the microwave oven. The simple

linear model has the following relationship:

{Soluble solids (microwave oven) = 0.993(vacuum oven) +0.020}

A R2 value of 0.956 demonstrates that 95.6% of the variance is shared between

the microwave and vacuum oven methods.

1.5.2.3.C Regression of Exact Equality (Water Soluble Solids)

An unpaired t-test determined how close the slope of the regression line was

to one. The p-value of 0.89 indicated that the slope was not significantly different

from one.

48

Water So lub le So l ids

o to

22 23

Water Soluble Solids Using Vacuum Method

Figure 1.6 Regression of Exact Equality Between Vacuum and Microwave

Oven Method in Water Soluble Solids Determination

Dashed line= Perfect line, Solid line= Fitted Line, 0 =Experimental Points

1.5.2.3.d Average Error Between Methods (Water Soluble Solids)

The average difference between two methods was calculated via the root

mean square error (RMSE). The RMSE of 0.284% was determined for water

soluble solids.

49

1.6 Discussion

The comparison of solids measurements by vacuum oven and the

microwave/formula methods on tomato paste samples with total solids in the

range of 24-29% revealed that higher values were produced by the microwave

method when determining total solids and insoluble solids. For total solids the

same diluted paste sample was used for both methods so the most likely source

of error would be the drying step. For the vacuum oven the decomposition of

sugars and other labile compounds or loss of volatiles other than water could

contribute to lower values. However, care was taken to maintain the temperature

of the vacuum oven at 70°C to reduce these decomposition reactions. For the

microwave oven it should be noted that due to the rapid evaporation of water in

the microwave the exposure of the sample to heat is much shorter than the

vacuum oven. This would limit the weight loss due to decomposition reactions

which is important in product containing high amount of sugar such as paste and

especially soluble solids. The microwave oven could give higher values if the

sample was not completely dry after the 4 minute drying cycle. Additionally it has

been reported that the microwave oven model AVC-MP which is used in method

42.01.09 (AOAC, 2000), could be substituted with the AVC-80 model (Chin and

others ,1985). However, in another study by Wang (1987), the AVC-80 model

produced results higher than the AVC-MP model. This study employed the AVC-

80 model which might be the source of higher results for total solids.

In the case of the water insoluble solids (% WIS) the determination is

calculated with an equation that requires data obtained by extracting the diluted

50

paste sample with water. The assumption here is that all the soluble solids are

extracted by the water and is in the supernatant fraction (%SSF) after

centrifugation. Higher values for %WIS would be calculated if the %SSF value

was lower than the true value because not all the soluble solids were extracted.

On the other hand, the results produced by microwave/formula for soluble

solids (%SS) were slightly lower than the vacuum oven results. This difference

could be due to way it was calculated. Using the equation, %SS = %TS - %WIS,

the value for %SS will decrease if %WIS increases. Because the %SS is

determined by the difference between %TS and %WIS, any errors in these two

values will be reflected in an error in %SS. However, in the absence of clear-cut

definition for moisture content (free or bound) it is unclear which of the methods

would produce the true value. More over the good linear correlation of the

methods and the high repeatability of the microwave oven method in addition to

its ease of performance would suggest that the microwave/formula method is a

good alternative method for the determination of solids in tomato paste.

51

Chapter 2

THE COMPOSITION AND PROPERTIES OF THE

SOLUBLE SOLIDS FRACTION

2.1 Introduction

From a simplified point of vie,w, tomato juice and tomato paste are

considered to be composed of suspended particles (pulp) dispersed in a liquid

medium (serum), which can be separated by high speed centrifugation. These

tomato products can be viewed as a special type of dispersion in which the pulp

is suspended in a colloidal medium called serum. Depending on the processing

conditions, the serum is believed to contain soluble pectic substances, sugars,

salts and organic acids.

The soluble solid or serum solid phase has been extensively investigated for

its contribution to the consistency of tomato products, but much less attention

has been paid to its chemical interactions or nutritional value. Investigations that

have been conducted so far can be placed into the following categories:

measurements of soluble constituents, changes during harvesting or process and

measurement of its viscosity or consistency behavior.

This study investigated the chemical interactions of different constituents

present in the soluble solid fraction of tomato paste. The bright red appearance

of soluble solids separated by centrifugation denotes the presence of lycopene in

this fraction. Since lycopene is a hydrophobic compound it was not expected to

be in the water soluble fraction. It was hypothesized that lycopene interacts and

52

associates with hydrophilic compounds during the processing of tomato paste

and form a complexes that suspend lycopene in the aqueous environment.

2.2 Literature Review

2.2.1 Tomato Composition

To better understand the properties of the soluble solids fraction in tomato

paste, it is important to investigate the composition of the tomato.

2.2.1.1 Solids in Tomato

Water is the predominant constituent in tomatoes and represents about 90 to

95% of the total weight. The remaining constituents are the tomato solids. A

project in California on commercially grown tomatoes reported a variation of 4.56

to 9.55% in the total solids content of their tomatoes. However, the insoluble

solids were reported to be about 1 to 2%, leaving the soluble solids as the main

solids in tomato (Saywell and Cruess 1932).

The solids in tomato can be placed into 5 groups: carbohydrates, proteins/amino

acids, organic acids, minerals and lipids.

Carbohydrates: The predominant carbohydrate components in tomato are

reducing sugars (El Miladi and others 1969) while sucrose accounts for less than

0.1% (Goose and Binsted 1964). Glucose and fructose are the predominant

reducing sugars with relatively more fructose than glucose (Davies 1964). Among

the polysaccharides, pectins and arabinogalactans account for 50%; cellulose

about 25%; and the xylans and arabinoxylans constitute the rest of the fraction

(El Miladi and others 1969).

53

Organic Acids: In processed tomatoes such as juice and paste citric acid is the

most abundant organic acid followed by malic acid (El Miladi and others 1969).

Acetic acid levels can increase during processing as the oxidation of aldehydes

and alcohols may occur.

Table 2.1 Organic Acids in Fresh and Processed Tomato (Gould 1992)

Acid Acetic Lactic

Succinic Alpha-ketoglutaric

Pyrolidone-Carboxylic Unknown

Malic Citric

m Fresh 1.06 1.37 0.60 1.10 0.81 0.17 3.72

60.92

Eq/liter Processed

1.56 1.46 0.49 0.53 8.10 0.28 5.39

66.92

Minerals: The average mineral content of tomatoes ranges from 0.3-0.6%.

Proteins and Amino Acids: Among the 19 known soluble amino acids present

in tomatoes, glutamic acid accounts for 45 to 48 % of the total followed by

aspartic acid. Proline is found in the lowest amounts (El Miladi and others 1969).

Heat processing is believed to increase free amino acid levels by denaturing and

hydrolyzing native proteins and by the deamination of glutamine and asparagine.

Tomato cells are filled with organelles called chromoplasts (carotenoid-

containing plastids). The plastids are the site of protein, lipid, carotenoid, and

sugar biosynthesis (Galili 1995) and are a rich source of essential and

nonessential amino acids (Table 2.2) (Hansen and Chiu 2005).

Lipids and Fatty Acids: Tomato seeds are recognized as the main source of

fatty acids in tomato and tomato products. In an investigation that determined the

54

fatty acid composition of tomato seed oil from processing wastes, palmitic acid

was identified as the major saturated fatty acid (23.4%), while linoleic (42.8%)

and oleic (18.3%) was the major unsaturated fatty acids (Cantarelli 1993). The

analysis of tomato plastids showed 47% linoleic and 37% palmitic as the major

fatty acids (Table 2.3).

An important and much studied carotenoid in tomatoes is lycopene. There

are increasing numbers of clinical studies that support the role of lycopene in the

protection against prostate cancer, lung cancer and a broad range of epithelial

cancers. This important micronutrient is synthesized in the chromoplasts of

tomatoes during its maturation. The investigation of lycopene's behavior and its

interaction with other compounds requires a clear description of the tomato

chromoplast and its major constituents.

55

Table 2.2 Free Amino Acids in Pastes Made from Red Tomatoes (Liu and Luh

1979) and Amino Acid Composition of Water-Soluble Proteins in Tomato Juice

(Stein and Mohr 1949) and Tomato Plastids (Hansen and Chiu 2005).

Amino acid

Aspartic acid

Threonine

Serine

Asparagine

Glutamic acid

Glutamine

Proline

Glycine

Alanine

Valine

Cystine

Methionine

Isoleucine

Leucine

Tyrosine

Phenylalanine

y-amino-butyric acid

Lysine

Histidine

Arginine

Tryptophan

mg/100g paste

112.91

15.61

24.43

84.79

320.70

4.00

Trace

2.89

14.17

2.54

0.58

1.08

6.32

4.24

3.93

15.92

240.08

8.81

10.84

6.72

—

Molar ratio of amino acids in tomato

water soluble protein 12.71

5.70

4.02

—

11.73

—

6.22

9.02

7.17

6.53

0.00

1.22

5.68

10.21

3.29

4.08

—

6.67

2.00

3.75

—

mg/g of plastid protein

105.4

49.7

56.8

—

161.5

—

46.6

48

58.4

47.6

6.2

17.9

52.4

85.5

40.9

93.2

—

71.6

31.4

63.5

28.4

56

Table 2.3 Fatty Acid Composition of Tomato Seed Oil (%) from the Hot Break

Process (Cantarelli and others 1993) and Fatty Acid Composition of Tomato

Plastids (Hansen 2005).

FATTY ACID UNSATURATED

Myristoleic C14:1 Palmitoleic C16:1 Oleic C18:1 Linoleic C18:2 Linolenic C18:3

Arachiadoinc C20:4

TOTAL FATTY ACID SATURATED

Laurie C12:0 Myristic C14:0 Palmitic C16:0 Stearic C18:0 Arachidic C20:0 Monosaturated oleic C18:1 TOTAL

% in Seeds Hot Break

Trace 6.8 18.3 42.8 0.7

*

68.6 % in Seeds Hot Break

0.3 2.3

23.4 4.0 1.3 *

31.3

% in Tomato Plastids

*

* *

47.0 2.1

0.37

49.6 % in Tomato

Plastids *

*

37.0 3.0 8.5 1.0

49.6

2.2.1.2 Tomato Chromoplasts

Chromoplasts or plastids are organelles present in the tomato cell and are a

rich source of macro and micronutrients such as proteins, lipids, sugars and

lycopene. Plastids can be described as a membrane encapsulated vesicle

containing these concentrated nutrients. Plastids can be isolated from whole

tomatoes. The tomatoes are cut into pieces, homogenized in a Waring blender

and the seeds, skins, membranes and cell wall material are removed by filtration.

The filtrate is centrifuged to separate the deep red intact plastids from the clear

amber-colored supernatant liquid. The supernatant contains a high level of

57

ascorbic acid, sugars and other water-soluble compounds (Hansen and Chiu

2005). The precipitated plastids are rich in proteins, lipids, carbohydrates and

dietary fibre.

Amino Acids in Plastids: The nonessential amino acids, glutamic, aspartic and

phenylalanine, are found in the highest amounts in plastids. However, plastids

have a good balance of essential amino acids and its high lysine levels can be

used to supplement low levels found in white rice flour and white wheat flour

making these flours a more complete protein source. Also, the low content of

methionine in plastids can be an advantage when used as a supplement by

reducing the potential buildup of homocysteine. Homocysteine is formed when

methionine loses a methyl group by the action of methyl transferase (Hensen and

Chiu 2005). Amino acid profiles of plastids are shown in Table 2.2.

Dietary Fiber in Plastids: The dietary fiber content of the total, soluble and

insoluble solids in plastids are 21.1%, 17.7% and 3.4 %, respectively. Pectin is

the main soluble dietary fiber in plastids. Human digestive enzymes do not

hydrolyze pectin. The colonic bacteria however are capable of hydrolyzing the a-

glycosidic linkage in galacturonic acid polymers and produce butyric acid which is

known to induce the apoptosis process and cause cancerous cells to die (Hague

and others 1993, Smith and others 1998). Also insoluble fibers are believed to

prevent digestive tract cancers by complexing with food material and accelerating

the intestinal transit time of fecal matter.

Fatty acids in Plastids: Tomato plastids have a very high level of the essential

fatty acid linoleic acid (47%) which is a cholesterol-lowering fatty acid followed by

58

linolenic acid (2.1%). Meanwhile palmitic acid (37%) followed by arachidic acid

(8.5%) is present as saturated fatty acids in plastids (Hansen and Chiu 2005).

The fatty acid profile of plastids is shown in Table 2.3.

2.2.2 Lycopene

2.2.2.1 Lycopene in Tomato

In their investigation of plastids, Hansen and Chiu (2005) reported on the high

concentration of ascorbic acid, sugars and others soluble solids but they didn't

comment on the source of the amber colour in the supernatant while isolating

tomato plastids. A large amount of lycopene is precipitated with the plastids but

a significant amount remains in the supernatant. Because lycopene is a lipophilic

compound, it should not be in the water soluble fraction. This solubilization or

suspension of lycopene requires a special type of complex formation or an

association between lycopene and a more hydrophilic component(s) present in

tomato.

Lycopene is an important carotenoid that exists as a microcrystal, and

imparts the familiar red color to tomato, watermelon and a few other fruits.

However, tomato and tomato products are one of the best sources of lycopene.

The outer pericarp of the tomato has the highest concentration of lycopene

(McCollum 1955). Although there are different reports on the concentration of

lycopene in different parts of the tomato, the skin accounts for 3 or 5 times more

than the pulp, which indicates that lycopene is associated mainly with the

insoluble fiber components.

59

The content of lycopene in tomatoes depends on many factors such as the

variety [Hart and Scott,(1995) reported the highest in red and lowest in yellow

variety], maturity [Ellis and Hammer, (1943) reported that lycopene increases

during maturity], harvesting season [Heinonen and others (1989) reported higher

levels in summer than winter], and temperature [Lurie and others (1996) showed

an increase in lycopene content under moderate conditions ].

2.2.2.2 Lycopene Structure

Lycopene is an apolar, acyclic carotenoid (C40H56 poly-isoprenoid). It is

assembled from 8 isoprene units and has 13 double bond, 11 of which are

conjugated. There are theoretically 2048 possible geometrical configurations but

due to steric hindrance only 72 isomers exist in nature (Zechmeister 1962).

Figure 2.1 The Basic Structure of Lycopene.

2.2.2.3 Lycopene Isomers in Tomato

All- trans lycopene is the predominant geometrical isomer in tomatoes and is

the most thermodynamically stable form, however the 5-c/s, 9-c/s, and 15-c/s

isomers of lycopene have also been identified in tomato-based foods. The cis-

isomers of lycopene are more polar and more soluble in oil and hydrocarbon

solvents (Nguyen and Schartz 1999) and represent more than 50% of the total

lycopene in human serum (Krinsky and others 1990). The c/'s-isomers have less

color intensity and lower melting points than their trans counterparts.

60

The conversion from trans to the cis form (unstable, energy-rich) is a reaction

that has been shown to take place during tomato processing. The formation of di

-cis isomers has been detected during heat processing while heat, light, acids

and some chemicals can be used to promote isomerization of lycopene in

laboratory experiments.

2.2.2.4 Lycopene and Health Benefits

Lycopene's configuration enables it to trap peroxyl radicals (ROO ) and act

as a very efficient quencher of singlet oxygen (Di Mascio and others 1991). The

ability of lycopene to quench singlet-oxygen is reported to be twice that of (3-

carotene and 10 times greater than a-tocopherol (Weisburger 2002).

Various epidemiological studies have suggested that lycopene can lower the

risk of certain types of cancers. The treatment of diseases such as skin cancer

and prostate cancer by lycopene has been reported in the literature (Ribayo-

Mercado and others 1995). Dietary intake of tomato and tomato products

containing lycopene has been shown to be associated with a decrease in the risk

of chronic diseases such as cardiovascular disease (Clinton 1998). Also the

consumption of tomato-based foods can reduce the susceptibility of lymphocyte

DNA to oxidative damage (Riso and Porari 1997).

2.2.2.5 Lycopene Extraction by Enzyme

The increasing evidence of the health-promoting benefits of consuming

lycopene has induced many researchers to investigate novel approaches to

isolate and purify lycopene from tomatoes. Studies conducted by Sharma and

Le Maguer (1996) reporting high amounts of lycopene in tomato skin, led

61

Choudhari and Ananthanarayan (2007) to employ enzymes to extract lycopene

from tomato tissues. Their results showed the effectiveness of using pectinase

and cellulase for lycopene extraction. Optimum conditions for pectinase activity

were 2% w/w enzyme at pH 5, 60°C for 20 minutes. Cellulase required 3% w/w

enzyme at pH 4.5, 55 °C for 15 minutes.

2.2.2.6 Lycopene and Tomato Processing

The lycopene content in concentrated tomato products is generally lower than

expected because of losses during tomato processing (Tavares and Rodriguez-

Amaya 1994). Thermal and mechanical treatments are involved in tomato

processing which can cause lycopene degradation. Isomerization and oxidation

are the main reasons for lycopene degradation during tomato processing. High

temperatures, light intensity and oxygen levels accelerate these degradation

reactions.

2.2.2.7 Lycopene and Temperature

Heat treatment promotes the isomerization of lycopene from trans to cis.

The degree of isomerization is directly correlated with the intensity and duration

of the heat processing conditions (Schierle and others 1996, Shi and others

1999). To determine the effect of heat treatment on lycopene, an experiment

was conducted on lycopene dissolved in hexane and heated at 50°C, 100 °C and

150 °C for different times. The HPLC analysis of the lycopene solutions following

treatment demonstrated that at 50°C the all-frans-lycopene showed no significant

change for the first 12 hours, but it began to degrade afterwards. In that study an

increase in di-c/'s isomers was observed, indicating that the mono-c/'s-lycopene

62

was being converted into the di-c/'s. The decrease in mono-cis-lycopene when

the heating time was increased, could also suggest that its degradation rate was

greater than its formation rate. They concluded that the isomerization reaction

was dominant during the early stages of the heat treatment and degradation was

the dominant reaction at the latter stages (Lee and Chen 2001).

2.2.2.8 Lycopene and Storage

To study lycopene storage stability, Sharma and Le Maguer (1996) placed

fiber-rich tomato pulp samples under 3 different storage conditions (vacuum/dark,

air/light, air/dark) at -20, 5 and 25 °C for 60 days. The degradation of lycopene

followed pseudo first order kinetics and the maximum losses occurred in the

presence of air and light at 25°C. The small amount of lycopene loss in the

samples stored at -20 °C under vacuum/dark shows the possibility of an

autocatalytic reaction.

2.2.2.9 Lycopene and Illumination

Lee and Chen (2002) placed lycopene in an incubator at 25°C for 6 days

under four fluorescent 20 W tubes. The result showed a decrease in the a\\-trans-

lycopene content with increasing incubation time, but the mono-c/s isomers

showed inconsistent changes (an increase at the beginning and a decrease after

2 h) suggesting that isomerization and degradation of lycopene was proceeding

simultaneously. In another study, the effects of light exposure on tomato powder

under different temperature conditions were investigated. An increase in cis-

isomer that ranged from 14-18% was reported (Anguelova and Warthesen 2000).

63

2.2.2.10 Lycopene in Different Food Systems

A study conducted by Ribeiro and Schubert (2003) showed the influence of

food systems on the stability of lycopene. In their study an emulsion of lycopene

was diluted in three food systems, (skimmed milk, orange juice and water). The

lycopene emulsion diluted in orange juice had the greatest lycopene stability.

This result showed the stability of lycopene in food systems was also influenced

by the presence of antioxidants. The presence of a-tocopherol in orange juice

enhanced the stability of lycopene.

2.2.2.11 Lycopene and its Bioavailability in Processed Tomato

It has been suggested by scientists that lycopene in processed tomatoes is

more bioavailable than in fresh tomatoes (Hadley and others 2003). The

bioavailability of lycopene has been strongly associated with the trans and cis

isomer content of the tomato product. The cis form is thought to be more

bioavailable than the trans form as serum contains more cis- lycopene than the

trans, which is the naturally occurring form of lycopene. This trend is based on

the cis isomer's greater solubility in bile acid micelles and its preferential

incorporation into chylomicrons (Boileau and others 1999). The chylomicrons

transport carotenoids from the intestinal mucosa to the blood via the lymphatic

system (Parker, 1996). In plasma, the carotenoids are carried by lipoproteins,

which surround the lipophilic carotenoids and increase their solubility in the

aqueous plasma.

In a study by Gartner (1997) focusing on the concentration of lycopene in

chylomicrons, he demonstrated that the lycopene that originated from tomato

64

paste was more bioavailable than the lycopene from fresh tomatoes. His study

showed that 65% of lycopene in the chylomicrons was in the trans form

(predominant form in the food) but only 45% were present in the trans form in

serum. The isomerization from trans to cis appears to be more prevalent in vivo

through biochemical or physiologic mechanisms than the pre-formed cis

lycopene found in paste forms during processing of fresh tomatoes.

Additional factors that can explain the greater bioavailability of lycopene in

processed tomato are food matrix effects and the disruption of the chromoplast,

which makes lycopene more accessible due to the breakage of cell wall

structures and membrane. In addition, the bioavailability of carotenoids can be

promoted by heat treatment of the food matrix, which dissociates the protein-

carotenoid complexes (Erdman, 1993).

It has also been shown that an oil medium improves the extraction of

lycopene into a lipophilic phase (Stahl and Sies 1992&1996). The complexes of

lipid-carotenoid that enters the duodenum (following the action by pancreatic

lipases and bile salts) are in the form of multi-lamellar lipid vesicle (Parker 1996).

In another study, it was suggested that there are interactions between

carotenoids such that lycopene ingested with p-carotene will be absorbed better

than lycopene ingested alone (Jackson 1997).

Dietary fiber can reduce the bioavailability of carotenoids due to matrix

interactions. Pectin for example, can produce high viscosity conditions that can

delay gastric emptying and interfere with micelle formation, which is needed for

65

carotenoid absorption (Rock and Swendseid 1992 and Di Lorenzo and others

1988).

2.2.2.12 Lycopene Rich Granules in Tomato Juice

During the investigation of the influence of insoluble solids on the viscosity of

tomato juice, Whittenberger and Nutting (1958) encountered an unexpected

phenomenon. When the serum (soluble solids fraction) was removed and

substituted with water, they noticed an increase in viscosity accompanied by a

decrease in the conductivity of the sample. In their microscopic examination,

they described tomato cell walls with visible lines outlining the cells along with

numerous small granules. These granules occurred commonly in clusters within

the cells and singly outside the cells and could be washed out along with the

soluble solids. The presence of these proteinaceous granules accompanied by

carotenoids and cellulosic cell wall material in the insoluble solid fraction was

previously reported by Kimball and others (1952). However, neither of these

researchers reported on the nature of these so called proteinaceous granules.

Linder and others (1984) assumed that lycopene was located in the chloroplast

and its distribution could be used as a marker for the distribution of coagulated

cytoplasmic material. Using this assumption he estimated that the cytoplasmic

material accounts for 34% of the total insoluble solids in tomato juice and that

25-30% of the total calcium is associated with this insoluble fraction (Linder,

Shomer and Vasilver 1984).

66

2.2.2.13 Lycopene, Protein and Different Elements (Ca, Mg, P and N) in

Various Fractions of Tomato Juice

Processed tomato products (tomato juice) have been analyzed for some of

the common compounds in their various fractions. The common criteria used to

evaluate tomato and tomato products have been based on the total contents of

their constituents or their relative ratio. However, to evaluate the nutritional value

or other attributes of the tomato products, it is important to determine the

distribution of the different components in the various fractions because their

localization and compartmentalization may explain some of the reactions that

take place during processing.

The first report on the distribution of certain elements and other constituents

in the various fractions of tomato juice was given by Lindner, Shomer and

Vasiliver (1984). The elements such as Ca, Mg, P and N along with protein and

lycopene were analyzed in juice and its 4 fractions; serum, cell walls,

extracellular granules, and the alcohol insoluble solids (AIS). The amount of each

element in each fraction is reported in Table 2.4.

Serum: Serum carries most of the nitrogen in the juice and most of this N doesn't

precipitate when treated with trichloroacetic acid (15%) which suggests that the N

content in the soluble fraction came from amino acids or small peptides rather

than large molecular weight proteins. Moreover just small amounts of the N

content (3%) precipitated with alcohol.

Insoluble solids and cell walls: The N content of insoluble solids is considered

to originate from proteins. Insoluble solids carry about 25-30% of the Ca, which

67

are primarily bound to the pectic material. It is believed that processing can

destroy the intracellular compartmentalization of Ca through mechanical rupture

or heat coagulation. This facilitates Ca binding to cytoplasmic materials such as

phospholipids and proteins and results in the redistribution of Ca between the

cytosol and cell wall material.

Extracellular granules: To isolate this fraction Lindner, Shomer and Vasiliver

(1984) diluted the juice and passed it through a series of sieve (the finest one

was 0.074 mm in size). The material that passed through the sieve was collected

by centrifugation at 27000*g for 25 min. Although this fraction only accounted for

24% of the insoluble solids, it contained 65% of the juice's protein and lycopene.

Alcohol Insoluble Solids: Less than 3% of the N in the soluble solids strongly

interacted with pectin and precipitated with alcohol. In contrast, large portion of

calcium in the serum (60-80%) was alcohol insoluble.

Table 2.4 Distribution of Protein, Lycopene, and Ca, Mg, P and N among Tomato

Juice Fractions3 (Lindner, Shomer and Vasiliver 1984).

a: mg/100g juice b insoluble N*6.25 c :Mean

Insoluble solids Ca Mg P N Protein" Lycopene

Juice

650-900

8.7-13.5 9.6-11.6 12.5-17.5 117-157 97-153 5.8-9.0

Serum Total

—

4.5-6.5 9.2-11.1 10.4-15.1 96-130

—

—

AIS

—

3.2-4.4 0.8-1.2

<2 <3

<18 —

Insoluble Solids Total

650-900

3.6-8.1 <0.6 1.3-2.7 15.5-24.5 97-153 5.8-9.0

In extracellular granule mg/g juice

154-195

1.2-1.9 <0.2 0.9-1.2 10.4-14.0 65-87 4.6-5.8

% c

24%

31%

56% 66% 66% 67%

68

In this experiment lycopene was detected in the serum fraction of

homogenized juice when the serum was obtained through centrifugation at

27,000*g for 30 min. Disintegration of some granules into submicron size

particles due to homogenization can account for this observation. Apparently the

conditions introduced by centrifugation (27,00Og) could not precipitate these

submicron particles.

These results reflect the variation in the distribution of tomato constituents in

the different fractions of tomato juice caused by processing.

2.2.2.14 Stabilization of Lycopene

The high sensitivity of lycopene to oxidation and other degradation reactions

have lead to various protective treatments such as the coating of this lipophilic

nanoparticle with a hydrophilic matrix. This treatment not only protects the

lycopene from oxidation but may also serve as a vehicle to disperse lycopene in

an aqueous medium.

2.2.2.14.a Encapsulation of All-Trans- Lycopene by Cyclodextrins

The nutritional importance of lycopene and its susceptibility to oxidative

degradation lead many researchers to investigate different methods of stabilizing

this compound. Blanch and others (2007) used cyclodextrins (CD) to

encapsulate all trans lycopene. They compared a supercritical fluid-C02 (SF-

C02) method to a conventional method in performing the encapsulation

procedure. The macrocycles of (3-CD in the torus-shaped structure with a

hydrophobic cavity hosting the lipophilic guest were favoured in comparison with

a-CD and y-CD in this study. The conventional method demonstrated a greater

69

encapsulation yield over the SF-C02 method (93.8% vs. 67.5%) but the

supercritical method has some important advantages. The authors

recommended the SF-C02 method due to its shorter time (1/6 of the

conventional method) and the extraction, fractionation and encapsulation could

be done in one step.

2.2.2.14.b Lycopene Coating with Protein

A patent by Garti and others (2003) describes a procedure to prevent

lycopene from migrating into the lipid, oil or fat phase and in so doing prevent the

fading of lycopene's bright red color in the product. The procedure involves

coating lycopene with a non-soluble film containing an amphiphilic protein which

itself can be attached to a colloid (proteinaceous polysaccharides). A preliminary

grinding process is a prerequisite to enable coating of lycopene by protein

(Patent Number WO/2003/045167 carotenoid formulation). The lycopene source

was either synthetic or an extract from tomato. However, crystalline micronized

lycopene (1-10 \xm) from tomato pulp was preferred in this patent.

The protein should contain lipophilic amino acids such as leucine, isoleucine,

phenylalanine and valine. However, the three dimensional conformation of the

protein has to also be modified. This transformation was achieved by heating the

protein dispersion in water in a pH range of 9-10 followed by cooling and

lyophilization. The protective colloids can be a polysaccharide such as amidated

pectin, xanthan gum or modified methylcellulose.

2.2.2.13.c Nano-encapsulation of Lycopene by Casein

Some attempts to incorporate hydrophobic ingredients into an aqueous

70

environment or to protect and deliver sensitive nutraceuticals have been based

on sodium caseinates. Caseins can be used to form nanocapsules that can hold

sensitive nutraceuticals and serve as a vesicle for their dispersion in an aqueous

beverages (Livney 2007).

The procedure to encapsulate hydrophobic molecules such as vitamin D or

lycopene is done in stages. The encapsulation is initiated by adding the

hydrophobic molecule to an aqueous solution of casein. In consecutive steps,

citrate phosphate and calcium ions from different sources are added to the

solution. The solution is adjusted to pH 6.5-7 and if needed the formed micelle is

dried. The result would be the non covalent bonding of nutraceuticals to sodium

caseinate. This procedure has been reported in another publication introducing

casein micelle as a natural nano-capsular vehicle for nutraceutical (Semo 2007).

2.2.2.14.d Pectin and lycopene in Tomato and Tomato Products

Pectin has been extensively studied in the tomato industry, as it is believed to

be an important contributor to the textural properties of tomato products. Most

studies have focused on the quantitative measurement of pectin in different

tomato fractions or pectin's gel-forming and aggregation capabilities. However

none of these studies have investigated the influence of pectin on lycopene

especially after processing.

In a study performed by Lee and Chen (2002), the degradation rate constants

for standard lycopene were determined at 150 °C, 100 °C and 50 °C. The

standard lycopene degradation rate constant was 0.0124 (min ~ 1) at 100°C.

However, in a study performed by Sharma and Le Maguer (1996) on tomato pulp

71

lycopene, the degradation rate constant was much lower (0.0023 min - 1). Their

results suggest that some macromolecules in tomato pulp (such as pectin), may

have a protective effect on lycopene during heat processing.

2.2.3 Pectin

Pectins are polysaccharide present mainly in plant cell walls and consists of

more than 100 a (1 _+.4) galacturonic acid units. The galacturonic acid residues

can be partly esterified with methyl groups (Gamier and others 1993).

H H, 'HO

H3COOC ^ ^ ^

Figure 2.2 Structural Formula for Partly Methylated Poly-Galacturonic (Tho and

others 2005).

The degree of methylation is different among pectins. High-methoxy pectins

have more than 50% methoxylation in their backbone while low-methoxy (LM)

pectins have less than 50% methoxylation and can be prepared by acid de-

esterification of the high methoxy pectin. If ammonia is employed in the de-

esterifaction process, the resulting LM pectin would be amidated and would

demonstrate different properties such as the ability to form gel in the presence of

lower calcium levels with thermo reversible properties (Reitsma and others,

1984).

72

One characteristic of LM is its ability to form a gel in presence of divalent ions

such as calcium ions through cross-linking. The cavities formed from junction

zone of side-by-side chains of galacturonic acid known as the egg-box model

would confine the Ca ions (Grant and others 1973). Calcium ions are capable of

linking other galacturonic acid chains together through electrostatic or ionic

bonds to form gels (Powell and others 1982; Morris and others 1982). Figure 2.3

illustrates the egg box model.

Poly-Qaiacturonic acid sequences • of pectin chains

Figure 2.3 Schematic Illustration of the Egg-box Model (Thon and others 2005).

The factors that can affect pectin gel formation are the degree of methoxylation,

molecular weigh of the galacturonic acid chain, charge distribution on the pectin

backbone, pH, ionic strength, temperature and co-solutes (Axelos and Thibault

1991; Clark and Farrer 1996; Lootens and others 2003).

2.2.3.1 Peptide-Pectin Interaction and Gelation Behavior of Plant Cell

Wall Pectin

Hydroxyproline-rich plant glycoproteins (HRGPs) is a class of structural

proteins in plant cell walls. These proteins consist of serine and hydroxyproline

amino acids glycosylated with arabinose. A protein network has been

73

hypothesized that involves covalent crosslinking through tyrosine residues (Qi

1995, Bradly 1996) while an ionic complex between pectin and HRGPs has also

been speculated (Showalter 1993, Sommer_Knudsen 1998, Kieliszewski 1994).

In a study conducted by MacDougall and others (2001), the ionic interaction of

pectin (from unripe tomato pericarp ) and basic peptides (poly-L-lysine, poly-L-

arginine ) and carrot extensin were examined. Additionally the swelling behavior

of pectin-peptide gels was also monitored.

The experiment was performed by acidifying the pectin solution (pH=2) to

suppressing the pectin charge, then allowing the basic amino acids components

perfuse into the pectin network and then readjusting the pH to 5.5-6.0. Under

these conditions the basic peptides formed a gel within the pectin network.

Monitoring the shear modulus (G) of these peptide-pectin gels and pectin-

calcium gels demonstrated that the cross-linking effectiveness was in the order

poly-L-arginine > poly-L-Lysine and finally > calcium ions. These results indicated

that a multiple charged peptide can initiate crosslinking reactions and that

calcium crosslinking will enhance it (MacDougall and others 2001).

2.2.3.2 Pectin-Protein Interaction in Tomato Products

In an investigation concerning the contribution of various tomato components

to the consistency of tomato products, the critical contribution of pectic

substances (McColloch and others 1950) was recognized. Others have reported

that proteolytic enzyme treatment caused only a small loss in tomato juice

consistency but cellulase treatment greatly decreased it (Foda and McCollum

1970). The role of protein was investigated more thoroughly by Takada and

74

Nelson (1983) using a pectin-protein model system. Their research studied the

interaction between bovine serum albumin and pectin from citrus fruit (low

methoxy pectin). The results from the study indicated that the viscosity of the

pectin-protein complex was dependent on pH but the same effect was not

observed when each of components where treated separately. This behaviour

was attributed to pectin-protein interactions. The pH examined in their

experiment ranged from acidic to neutral. The maximum viscosity was achieved

at pH 4.2, which is close to the pH of tomato juice and paste. The effect of pH on

viscosity was reversible, which indicated the formation of a reversible

electrostatic complex between pectin and protein (Figure 2.4). However, an

irreversible pH effect has also been reported by Dougherty and Nelson (1974).

Considering that tomato pectin and bovine serum albumin have a similar

isoelectric point of 4.7-4.9 (Young 1963), the possible difference in pKa of pectin

and protein could explain their electrostatic interaction or repulsion.

c o o C O O C H 3 C O O "

( i.) p H > p l p r o t e i n

( i i ) p H = P I p r o t e i n

( H i ) p K a , p e c t i n < p H < P I p r o t e i n

( i v ) p H < p K o , p e c l i

C O O C M 3 c o o -

C O O C M 3 C O O -

Figure 2.4. Suspected Schematic Model of Pectin-Protein Interactions in

Tomato Products (Takada and Nelson 1983).

75

In spite of all the information available on the components present in tomato

paste products, little is known about their interactions and the complexes formed.

The present study intends to clarify the interactions that are possible among

pectin, protein and lycopene in tomato paste products. Studying these

interactions will provide information on lycopene's ability to form soluble

complexes in an aqueous environment.

2.3 Experimental

2.3.1 Sample Preparation

In order to properly conduct the experiments in this study, four different sample

preparation protocols were employed to produce the samples for all the required

analyses. Some of the assays required a liquid sample while others were better

conducted on dried samples. Also, to gain a better understanding of the behavior

of soluble solids, some samples were dialyzed. Figure 2.6 outlines the sample

preparation used for each assay.

Dried Paste Dr ied

So lub leSo l ids

Dilute Paste

• U i l So

"1 ute lub ieSol ids

_ J

Figure 2.5 Schematic Illustration of Sample Preparation and Related

Measurement.

76

It should be noted that this study intended to investigate the quality

characteristics of the soluble solids that enable them to form soluble complexes

with lycopene and not just its quantitative levels. Therefore the soluble solids

employed in this study was not exhaustively extracted but rather were collected

from a single centrifugation (first centrifugation) as described in detailed in

section 2.3.1.2.

2.3.1.1 Paste (Diluted)

A dilution of 1:2 ( paste:water) was prepared by adding 100 ml_ of water to 50

g of tomato paste. The diluted paste was stomached for 2 minutes to completely

disperse the paste.

2.3.1.2 Soluble Solids (Diluted)

A dilution of 1:2 (paste: water) was prepared following procedure detailed at

2.3.1.1. The soluble solid fraction was prepared by placing 20±3 g of the diluted

paste into a 25 ml_ centrifuge tube and centrifuging at 25,000xg for 18 min

(Beckman JA-20 rotor). The supernatant was decanted into a coarse sintered

glass filter to remove any solid materials. This filtered sample was used for

lycopene determination, transmission electron microscopy (TEM) examination

and ion exchange chromatography.

2.3.1.3 Soluble Solids (Dried)

The soluble solid collected from the first centrifugation of diluted paste was

frozen (VIP series) at - 80 °C then freeze dried (Gardiner, VirTis Co.) for 48 h.

This dried sample was used to determine total nitrogen, pectin content and

protein composition by SDS-PAGE.

77

2.3.1.4 Paste (Dried)

A dilution sample of paste (section 2.3.1.1) was spread in an aluminum pan,

frozen and lyophilized following the same procedure as soluble solids.

2.3.1.5 Soluble Solids (Dilute-Dialysis)

Some assays were conducted on dialyzed samples of dilute soluble solids.

One hundred ml_ of diluted soluble solids (section 2.3.1.2) was filled into dialysis

tubing (Fisher Scientific, Mississauga, ON, Canada) with MW cut off of 3500 Da

and dialyzed in 4 L of Milli-Q water for 48 h with a water change every 4 h. This

sample was used in the determination of minerals.

2.3.1.6 Soluble Solids (Dried- Dialysis)

Dilute- dialyzed soluble solid samples from the previous step (2.3.1.5.) were

frozen at 80 °C and freeze dried for 48 h resulting in dried samples of dialyzed

soluble solids. These samples were employed for determination of nitrogen

content of soluble solid and proteins by SDS-PAGE.

2.3.2 Total Solid and Total Soluble Solids

The total solids were determined according to the procedure in section

(1.3.2.1.a). The total soluble solid was determined on the same samples using

the procedure outlined in section (1.3.2.1.c). The mean value of replicates was

calculated and reported as total solids and total soluble solids.

2.3.3 Soluble Solid Dry Weight (1s t centrifugation)

The solid content of the soluble solids from the first centrifugation (see 2.3.1.3)

was determined by the difference in weight before and after freeze drying.

% Soluble Solids =Weiqht before drying - Weight after drying *100 Weight before drying

78

This measurement was necessary, as some procedures using diluted soluble

solids samples required the results on a dry weight basis.

2.3.4 Pectin Determination

The uronic acid content of the soluble solids and paste was determined by a

colorimetric method developed by Blumenkrantz and Asboe-Hansen (1973) and

modified by Ahmed and Labavitch (1977). The principle of the method requires

the hydrolysis of the pectin to free the galacturonic acid units. This procedure

also releases the side chain sugars from the polygalacturonic acid backbone and

the color produced by these side chain sugars is corrected in the assay

procedure.

2.3.4.1 Material and Equipment

UV-visible spectrophotometer model 260 (Shimatzu, Tokyo, Japan), D-(+1)

Galacturonic acid powder (Fluka, Buchs, Switzerland), glass cuvettes 10 mm

pathlength (Hellma, Concord, ON, Canada), 3-phenylphenol 85% (Sigma-

Aldrich, Oakville, ON, Canada), pyrex centrifuge tube (30ml_), sodium tetraborate

(99.98%), sulfuric acid (Fisher Scientific, Mississauga, ON, Canada).

2.3.4.2 Methods

The galacturonic acid stock solution was prepared by dissolving 100 mg dry

galacturonic acid powder in 100 ml_ of deionized water. The 1 mg/mL stock

solution was diluted to prepare the calibration curve. A 0.0125 M solution of

sodium tetraborate was prepared by dissolving 1.192 g of sodium tetraborate in

250 ml_ of concentrated sulfuric acid.

79

Approximately 0.034 g of freeze dried paste and approximately 0.0650 g of

freeze dried soluble solids were diluted in 50 ml_ of water. This dilution was

selected to give a galacturonic acid concentration of less than 100 (ig/mL to avoid

off scale readings on the spectrophotometer. 1 ml_ aliquots of diluted soluble

solid and paste was pipetted into a pyrex centrifuge tube and placed into an ice

bath. Then 6 ml_ of cold sodium tetraborate solution was added to the sample

dropwise. The tube was placed into a boiling water bath for exactly 6 minutes

and returned to the ice bath immediately. When cold, 0.1 ml_ of 3-phenylphenol

(0.5%) was pipetted into the hydrolyzed sample to develop the color. A similar

procedure was performed on another sample except the 0.1 ml_ of 3-

phenylphenol was replaced with a 0.5% solution of sodium hydroxide. Both

samples were vortexed and allowed to stand for 10-15 min for color formation. If

bubbles were present, the samples were centrifuged at low speed for 5 min at

room temperature. The absorbance was read at 520 nm in glass cuvettes. The

absorbance in the presence of the sodium hydroxide was subtracted from the

absorbance of the 3-phenylphenol reaction mixture. Employing the standard

curve, the concentration of galacturonic acid was determined.

2.3.5 Lycopene Determination

Numerous HPLC methods have been used to separate the various

carotenoids in fruit and vegetables. After minor modifications, the method

developed by Ishida and others (2001) was used in this study for lycopene

determination.

80


HPLC system model Waters 2690 Auto-injector, Detector model Waters 996

Photo-Diode-Array (Waters Ltd, Mississauga, ON, Canada) Column C30 (250 x

4.6mm I.D, particle diameter 3- urn) (YMC Inc., Wilmington, NC).

Spectrophotometer (Shimadzu UV-260, Tokyo, Japan). Polytetrafluoroethylene

filter (Alltech Associates, Inc., Deerfield, IL, USA), lycopene 90% from tomato

(Sigma-Aldrich, Oakville, ON, Canada )

Hexane, ethanol, methanol, acetone, ethyl acetate (EtOAc) and methyl f-butyl

ether (MTBE) were HPLC grade (Fisher Scientific, Mississauga, ON, Canada),

ethyl alcohol 95% commercial grade (Commercial Alcohol Inc., Brampton, ON,

Canada).

2.3.5.2 Methods

One gram of the diluted paste and soluble solids from the first centrifugation

prepared as detailed in sections 2.3.1.1 and 2.3.1.2 were used for lycopene

determination. The modified method used in this study was able to separate and

quantify lycopene and its geometric isomers.

Lycopene Extraction: 20 mL of hexane/acetone/methanol (2:1:1) was added to

1 g of soluble solid from the first centrifugation and diluted paste (dilutionl: 2)

and mixed until the aqueous phase was bleached and no red colour was visible

in the residue material. The hexane phase was separated by adding 10 mL of

Milli-Q water and stirring thoroughly on a vortex mixer. The top layer containing

the extracted lycopene was filtered by 0.45-um polytetrafluoro ethylene filter.

Lycopene Analysis: A HPLC system equipped with a C30 reversed phase

81

column and a thermostated column compartment was used. The system was

conditioned for 30 min at 1 mL/min with the mobile phase, methyl f-butyl ether

(MTBE): MeOH: ethyl acetate (EtOAc) (40:50:10 V/V). An Injection volume of 15

ul was used and the column temperature was set at 28°C. The absorbance was

monitored at 470 nm using a Photo-Diode Array Detector (Waters 996).

Lycopene and its cis isomers eluted within 27 min. A standard curve constructed

with known concentrations of standard all trans lycopene was used to calculate

the levels of all isomers in the tomato samples.

2.3.6 Nitrogen Determination

The total nitrogen content of the lyophilized soluble solids (from the first

centrifugation), paste and dialyzed soluble solids was determined by the Dumas

combustion method (Jung and others 2003).


Protein/Nitrogen FP-528 instrument, EDTA, Tin foil sample holder (LECO

Instruments Ltd. Mississauga, ON, Canada).

2.3.6.2 Methods

The instrument was standardized and calibrated by running at least 8 blanks

and 8 EDTA calibration standards. The same diluted paste (1:2) sample that

was subjected to pectin and lycopene measurements was freeze dried before the

nitrogen determination (described in section 2.3.1.4). Approximately 0.15 g of

freeze dried paste was compressed into the tin foil to form a ball, weighed and

subjected to the combustion procedure. The results are reported as percent total

nitrogen.

82

The total nitrogen content of the dried soluble solids and dialyzed soluble

solids prepared with the procedure outlined in section 2.3.1.3 and 2.3.1.6 were

determined as described above.The sample size was approximately 0.15- 0.20 g.

2.3.7 Gel Electrophoresis (SDS-PAGE)

SDS-PAGE was used for protein separation and identification based on size

or molecular weight. By binding to the protein, the SDS detergent gives the

protein a negative charge. The separation will be based on size as the SDS gives

the protein the same overall charge to mass ratio.

This assay was carried out to determine the source of nitrogen, which was

detected in the combustion procedure. Lyophilized soluble solids ,dialyzed

soluble solids , paste and bottom layer in ultracentrifuged soluble solids were

analyzed by the method developed by Laemmli (1970).


Acrylamide: Bis (30%). Ammonium Persulfate (10%), and TEMED (Bio-Rad

Laboratories-Canada Ltd., Missisuaga, ON, Canada),Tris-Base, SDS (Sodium

Dodecyl Sulfate), glycine, B-mercaptoethanol, bromophenol blue (1.0%) and

Commassie Brilliant Blue (Sigma-Aldrich Canada Ltd., Oakville, ON, Canada).

Ethanol, methanol, Milli-Q water, acetic acid and glycerol 75% (Fisher Scientific,

Mississauga, ON, Canada), standard protein as marker (ten protein band,

Precision Plus Protein Kaleidoscope ™,BIO-Rad Laboratories, Inc , Hercules,CA,

USA), scanner (SHARP JX-330, AmorshamBioscience, Quebec, QC, Canada).

2.3.7.2 Methods

0.0225 g freeze dried samples of soluble solids (see 2.3.1.3) and 0.0234 g

83

dialyzed soluble solids (see 2.3.1.6), 0.0211g dried paste (see 2.3.1.4) and

0.0063gr. bottom layer from ultra centrifuged sample (see 2.3.12.2.a) were

analyzed in this experiment. 200 \x\ of the extracting sample buffer (0.06g of

50mM Tris HCL (MW 121.1) and 3.0g of 5M Urea were mixed in 4 ml_ warm

water and adjusted to to pH=8.0.This solution was added to 1ml_ of 10% SDS

and 0.4ml_ of 2-mercaptoethanol) was added to each sample in Eppendorf tubes,

vortexed and heated for 10 minutes at 95°C.

Gels in 12.5% and 18% cross linking were prepared following method

described by Laemmli (1970).

5|aL and 10fal_ of each sample was loaded into the wells, along with one well

containing 10|J- of a broad range molecular weight marker (250, 150, 100, 75,

50, 37,25,20, 15, 10 KD).

The applied voltage was 200v to induce migration proteins. The gels were

placed in Coomassie working staining solution for 30 minutes, rinsed with

deionized water, and placed in destaining solution for 1 to 1.5 hours. After

destaining the gels were placed in deionized water and shaken over night. The

destained gel was scanned.

2.3.8 Fatty Acid Composition

The fatty acid composition of the lyophilized soluble solids and paste was

determined by gas liquid chromatography (GLC). This method requires

conversion of the fatty acids into their fatty acid methyl esters (FAME). The

chromatography was performed according to the method of Bannon, Craske and

Hilliker (1985).

84


Shimadzu GC-8A equipped with a flame ionization detector and Shimadzu C-

R3A Chromatopac integrator, glass column packed with 10% Silar 9CP

Chromosorb W- AW, 80/100 (Chromatographic Specialties Inc. Brokville, ON,

Canada). Potassium hydroxide, methanol, iso-octane, hydrochloric acid (Fisher

Scientific, Mississauga, ON, Canada), standard fatty acid methyl esters (Nu

Check Prep supplier, Elysian.MN, USA ).

2.3.8.2 Methods

Fatty acid methyl esters (FAME) were prepared by dissolving 50 mg of freeze

dried paste and soluble solids in 2 ml_ of iso-octane. 200 .̂L of KOH (2N) in

methanol was added to the solution and vortexed for 1 min. The prepared

sample was held for 5 minutes to allow the reaction to proceed. Two drops of

methyl orange indicator was added and the reaction mixture neutralized with 2 N

HCI according to the method of Bannon and others (1985).

1 nl of the top layer was injected into a Shimadzu GC-8A equipped with a

flame ionization detector (FID) and Shimadzu C-R3A Chromatopac integrator.

The carrier gas was nitrogen, the injector and detector temperatures were 230°C

and the column was temperature programmed from 60-210 °C at 6°C/min. The

FAME were identified by comparison of their retention time with those of

authentic compounds previously analyzed.

2.3.9 Enzymatic Treatment of Soluble Solids

The red colour of the soluble solid fraction is a good indication of the

presence of carotenoids and in particular lycopene. This was supported by the

85

HPLC analysis of the soluble solids. The possible interactions and association

between lycopene and other components in the soluble fraction was investigated

with an enzyme procedure reported by Choudhari and Ananthanarayan (2007).


Cellulase from Aspergillus sp.activity >1000 U/g, protease from Aspergillus

oryzae activity >500 U/g (Novozyme Corp, Franklinton, NC, USA), pectinase

from Rhizospus sp. activity 448 U/g, (SigmaAldrich, st.Louis, MO, USA),

hemicellulase from Aspergillus niger contaminated with galacto-mannanase,

polygalacturonase and acid protease (Enzyco®, New York,NY,USA ).

2.3.9.2 Methods

5 ml_ of dilute soluble solid (7.57± 0.05 % dried solids) was treated with

different concentrations of enzymes (Pectinase, Protease, Cellulase,

Hemicellulase) and different pHs (4.5, 5.0, 5.5, 6.0, 6.5, 7.0 and 7,5). These

reaction mixtures were held for 24 h at different temperatures (-8 °C, 21 °C, 40,

50, and 60 °C). The schematic diagram (Figure 2.6) shows the various conditions

used for the enzyme treatments.

Moreover various combinations of enzymes were practiced. In this case

suitable condition for pectinase and cellulase was determined based on previous

step in which the reaction of enzyme and substrate took place (0.02g enzymes

added to 5 ml_ sample at 20°C, pH 5). The optimum condition for cellulase and

protease determined based on the information from provider company (50|aL at

40-50°C, pH 7.5). For each enzyme the optimum condition in sample was

provided and after 24h samples were combined starting from combination of two

86

followed by the combination that 3 enzymes were involved and eventually the

last combination when all enzymatic treated samples were mixed together (Table

2.5).

Cellu

lase

Pectinase

25,

50,

75,

100

fiL

0.02, 0.05 g

pH 4.5, 5.5, 6.5, 7.5

Temp-8, 21,40, 50, 60 °C

0.02, 0.05 g

at

en o •>i

©

o •p r-

Hemicellulase

/

Protease

Figure 2.6 Enzymatic Treatment of Soluble Solids in Terms of Concentration,

Temperature and pH.

Table 2.5 Combination of Enzymes in Their Optimum Conditions.

Enzyme Combination 1 2 3 4 5 6 7 8 9 10

Pectin

* * * * *

*

Hemicellulase

*

*

* * *

*

Cellulase \ Protease

* *

. 1 __ * l * *

*

* * *

The designed combination is illustrated in Figure 2.7, while each overlapping

area demonstrates the combination of samples.

87

Pecf inase 0.02gr, pH 5 Temp 21CC

HemiceJutas€ 0,02gr, pH 5 Temp 21 -C

Cel fu lase SO pL, pH 7.5 Temp 4 0 ':Q.,-

Figure 2.7 Combinations of Enzymes in Their Optimum Conditions. Each

Overlapping Areas Signifies Single Combination.

2.3.10 Ions Determination (Ca+2, Fe +2, Mg +2, K+, Na +, P-5) by ICP-OES

The main ions present in soluble solids (filtered and dialyzed) were measured

by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) as

reported by McKinstry and others (1999). The theory behind this measurement is

based on the excitation of atoms with a high energy source, which promotes the

electrons in the atom to specific higher energy levels. When these electrons

return to their ground state(s), they emit characteristic wavelengths of radiation.

By determining what wavelengths are being emitted, the analyst can determine

what elements are present in the sample. By measuring the intensities of these

wavelengths and comparing them to those generated by known standards, the

concentrations of the different atoms can be determined.


Microwave CEM corp. (MARXpress), Inductively Coupled Plasma Optical

Emission Spectrophotometer (Varian, Vistapro), Nitric acid >90% Reagent A.C.S,

Protease 50ML,pH 7.5 ) Temp SO ! : C , /

88

HPLC grade submicron filtered water W5-4 (Fisher Scientific, Mississauga, ON,

Canada).

2.3.10.2 Methods

The soluble solid samples were prepared following the method described in

section 2.3.1.2. Dialyzed soluble solid prepared following method detailed at

2.3.1.5. A third aliquot of the same sample of soluble solid was filtered through

0.45 u filter and subjected to the same procedure.

2 g of each sample were microwave digested in 5 ml_ of concentrated nitric

acid. The digested sample was diluted to 25 mL final volume with HPLC grade

water and filtered (submicron filtered W5-4). This solution was analyzed using a

radial ICP-OES. The intensity of light was compared to a calibration curve

prepared with known concentrations of each element.

2.3.11 Transmission Electron Microscopy (TEM) Analysis

TEM was employed to determine the microstructure of three soluble solids

fraction.


LEO 912 ab TEM (Carl Zeiss SMT, Oberkochen, Germany) at 100kv

equipped with camera SIS/Olympus Cantega 2K CCD employing iTEM software

(Soft Imaging System, Munster, Germany), ultracentrifuge model Optima LE-80K

(Beckman Coulter, Rotor 70.1 Tl Mississauga, ON,Canada).

2.3.12.2 Methods

Sample preparation: The soluble solid fraction from the first centrifugation

(red coloured liquid) was collected and subjected to ultracentrifugation at

89

~171,000 x g for 30 min at 20 °C. Three distinct layers were obtained under

these high centrifugal forces. Each layer was collected by carefully pipetting out

the fractions.

Analysis: Each layer was subjected to EM following the procedure

described by Jacob and Paliyath (2008). A drop of each layer of sample was

mounted on a 200 mesh fomvar-coated nickel grid for 1 min. After removing the

excess sample, the grid sample was blotted dry and stained for 30 s with 1%

uranyl acetate. After removing excess stain the grid was monitored in the

transmission electron microscope.

90

2.4 Results

2.4.1 Schematic Diagram of Sample Replications

Measurements of total solids, total soluble solids, soluble solid (1st

centrifugation of diluted paste), pectin, lycopene and nitrogen were replicated 3

times, and in the case of pectin, lycopene and nitrogen 3 readings were obtained.

The mean value for three readings was considered as the result for that

replication. Figure 2.8 shows the assay and the number of replications performed

on each sample. The complete sequence is shown only for rep 2. The samples

that were examined only for their qualitative attributes were not replicated.

These analyses were electrophoresis (SDS-PAGE), and transmission electron

microscopy (TEM).

PASTE

Replication 1 Replication 2 1

Replication 3

Soluble Solid 2 (1st centrifugation)

4 Paste 2

| Spectrophotometer

Pectin

{ T I

JHPLC

Lycopene

I I I Inject 1 Inject 2 Inject 3

, Dumas

Nitrogen

T 1 Absorb 1 Absorb 2 Absorb 3 Combust 1 Combust 2 Combust 3

Figure 2.8 Schematic Illustration of Sample Replications.

91

2.4.2 Total Solids and Total Soluble Solids

Total solids in the tomato paste were 27.07% while the soluble solids was

21.99%. These values indicated that the main constituent of tomato paste, after

water are the soluble solids which account for approximately 81% of the total

solids (Table 2.6).

Table 2.6 Total and Soluble Solids Content of Tomato Paste

Sample

Replication 1 Replication 2 Replication 3 Mean

Total Solids (%) 27.11 27.01 27.09 27.07±0.05

Total Soluble Solids (%) 22.05 21.99 21.96 21.99±0.05

Ratio Solids Soluble /Total (%) 81.33 81.27 81.03 81.30±0.03

2.4.3 Soluble Solid Dry Weight (1s t centrifugation)

Some measurements such as lycopene concentration were performed on the

liquid soluble solids, but the final results were reported on a dry weight basis.

For these samples a value for total solids in the liquid soluble solids fraction was

required.

Table 2.7 Solids in Soluble Solids Fraction (1s t centrifugation)

Sample

Replication 1 Replication 2 Replication 3 Mean

Soluble Solid (1st Centrifuge) (% total solids)

7.54 7.54 7.64

7.57±0.05

2.4.4 Pectin Determination

Pectin measurements were conducted in triplicate on paste and soluble solids.

Results from this experiment showed that 69-76% of the pectin present in the

92

tomato paste was soluble. Although these values were higher than most

reported values (60-70%), it should be noted that the samples in this study were

concentrated using a severe heat treatment that could have induced the

solubilization of more insoluble pectin.

Table 2.8 Pectin Content in Paste and Soluble Solid (|ag/g dry wt)

Sample

Replication 1

Replication 2

Replication 3

Mean

Pectin |ag/g

Soluble Solid

933.85±50.63

940.77±14.14

878.46±13.68

917.69±34.15

Pectin |ng/g

Paste

994.37±36.58

1107.29±34.75

943.32±35.54

1014.90±83.75

2.4.5 Lycopene Determination

Lycopene measurements were performed on paste and soluble solids

samples. The sample was extracted three times and each extract was injected

three times into the HPLC. The mean value from three injections was reported

as the lycopene content. The determined lycopene content is reported in table

2.9.

Table 2.9 Lycopene Content in Paste and Soluble Solid (jag/g dry weight)

Sample

Replication 1

Replication 2

Replication 3

Mean

Lycopene ^g/g

Soluble Solid

82.96l1.264

84.98±0.949

82,30±0.55

83.41±1.40

Lycopene jag/g

Paste

190.042±1.730

175.001±4.428

167:418±3.1

177.487±11.52

93

http://82.96l1.264

These results demonstrate that the soluble solids contain approximately 35-39%

of the paste's lycopene content.

2.4.6 Nitrogen Determination

This procedure was used to determine the protein content of the paste and

soluble solids. However, the procedure measures total nitrogen and not just the

nitrogen from protein. Therefore, the results are reported as % total nitrogen

and not converted to % protein.

Table 2.10 Nitrogen Content of Soluble Solid, Paste and Dialyzed Soluble Solid

(% dry wt.)

Sample

Replication 1

Replication 2

Replication 3

Mean

N%

Soluble Solid

2.42±0.02

2.44±0.03

2.44±0.02

2.43±0.01

N%

Paste

2.62±0.01

2.63±0.02

2.68±0.03

2.64±0.03

N%

Dialyzed

0.89±0.33

0.88±0.30

0.88±0.31

0.88±0.01

The results indicate that the main source of nitrogen in the paste is associated

with the soluble solids and that most of soluble nitrogen compounds have a Mw

of <3500 Da as they passed through the dialysis tubing.

2.4.7 Gel Electrophoresis (SDS-PAGE)

No distinctive protein bands were seen in the soluble solids or the dialyzed

soluble solid, tomato paste samples. Similarly no distinctive band was recognized

in the precipitate from ultracentrifugation. In general, the presence of a distinct

smear indicated proteolytic products of varying molecular weight. Figure 2.9

shows the migration of samples in the SDS-PAGE.

94

250 KD

150

•i nr\ IUU

75

50

37

25

20

250 KD

150

100 T^ to

50

37

25

?n

15

1 f

• Z

18% Gel

2 3 4 5 6 7

12.5% Gel

Figure 2.9 SDS-PAGE of Tomato Paste and Its Different Fraction in Gel Cross

Linking of 18% and 12.5%. Marker (1), Tomato Paste(2), Soluble

Solids (3) , Bottom Layer of Ultracentrifuge Soluble Solids(4) and

Dialyzed Soluble Solids (5) (10(il lane 2-5, 5^1 lane 6-9).

These results suggest that the proteins in the soluble solids did not migrate

like typical proteins or they did not have the same affinity for Coomassie Brilliant

95

Blue and did not stain. This could be an indication that glycoproteins or fibrous

proteins are present. A more likely reason for the lack of protein bands on the

SDS gels despite the high nitrogen levels is the presence of non protein nitrogen

compounds, small peptides and amino acids.

2.4.8 Fatty Acids Determination

There was a small 18:2 peak in the lyophilized tomato paste sample indicating

the presence of linoleic acid. The absence of other fatty acids could be due to

low concentrations, which were below the detection level of the method. Most of

the fat would have been removed when the seeds and skins were separated

from the pulp.

The soluble solid samples showed no fatty acids. Again the levels of fatty

acids could be below the sensitivity of the instrumentation.

2.4.9 Enzymatic Treatment of Soluble Solids

The presence of lycopene in the soluble solids fraction suggests the possibility

of its interaction with other components such as cellulose, hemicelluloses, pectin

and/or peptides to form a soluble complex. According to Linder (1984) this is

possible through processing and disintegration of tomato components into

submicron particles which do not precipitate when subjected to high shear rates

(27000xg).

A systematic set of experiments was carried out to determine the effects of

enzymatically removing each component and observing the effect on the

remaining compounds. The enzyme used were pectinase, hemicellulase,

cellulase and protease.

96

Before the enzyme treatment, 5 ml_ sample of soluble solids appears as a red

colored solution (Figure 2.10). After the pectinase treatment (0.02 g) of this

solution at its natural phi of 4.5 to 4.6, a red precipitate forms and separates from

a clear yellowish liquid. Centrifugation of this treated sample produced a red

pellet and a yellowish supernatant. The precipitate formation was more effective

when 0.05 g of pectinase was add to that 5 ml_ of soluble solids. There was a

similar effect with samples treated with hemicellulase. However this behaviour

was not solely due to the hemicellulase as the commercially available enzyme

contained appreciable amounts of galacto-mannanase, polygalactronase and

acid protease activities.

After Treatment >

Before treatment p^>

. Figure 2.10 Enzyme Treatment of Soluble Solids. 0.02g of Pectinase (label 2)

and 0.02g Hemicellulase (label 3) in 5 mL of Soluble Solids.

Contrary to the effects observed with pectinase and hemicellulase treatments,

the addition of protease and cellulase did not destabilized the colloidal particles

when tested at specific temperatures, concentrations and various pH.

In another set of experiments, the effects of enzyme combinations were

tested. In these experiments, no precipitate was formed in the absence of

pectinase or hemicellulase

97

The hydrophobic nature of lycopene would suggest that it would float to the

surface of an aqueous solution if it existed in the free form. The precipitation of

red particles following pectinase treatment suggests that there is an association

of lycopene with compounds other than pectin that is preventing its release in the

free form. It appears that a complex of lycopene with pectin and other solids is

being formed as a soluble complex that is able to suspend itself in an aqueous

solution that cannot be precipitated by centrifugation but can be precipitated with

pectinase treatment. Removing pectin makes the complex insoluble but the

remaining complex can still hold the lycopene molecules.

Another possibility could be the protective effect of components such as

pectin, which prevents the protease and cellulase enzymes from reaching their

specific substrates. The enzyme combination experiment aimed to remove these

protective components and provide substrates accessible for protease and

cellulase. However, the results demonstrated that protease and cellulase were

still unable to free lycopene from the complex.

2.4.10 Determination of ions (Ca+2, Fe +2, Mg +2, K\ Na \ P-5 ) by ICP-OES

Both negative and positive ions passed through the 0.45(j. filter which indicated

that they are not tightly associated with the lycopene complex. Dialysis removed

a significant amount of Ca, Mg, and K ions from the soluble solid solution but low

amounts of Ca and Mg still remained. These positively charged divalent ions may

be involved in balancing the negative charges on the compounds in the soluble

solids solution. The concentration of ions in various fractions is reported in table

2.11.

98

Table 2.11 Ions in Filtrated, Dialyzed and Original Soluble Solids.

Elements

Calcium Iron

Magnesium Phosphorous

Potassium Sodium Sulphur

Original \xglg Soluble Solid

81 <50 170 240 3900 <48 130

Filtered |ag/g Soluble Solid

81 <50 170 240 3900 <48 130

Dialyzed pg/g Soluble Solid

25 <50 38 <12 <19 <48 <15

2.4.11 Transmission Electron Microscopy (TEM) Analysis

The soluble solids sample for TEM analysis was prepared by high speed

ultracentrifugation. After centrifugation, the sample separated into three layers.

The top layer was deep red in colour and would quickly go back into solution if

shaken. The second layer or the middle layer was light yellow in colour and was

the largest layer, while the third layer formed a precipitate at the bottom of the

centrifuge tube. This precipitate consisted of orange to reddish coloured particles.

Figure 2.11 shows three distinct layer induced by ultracentrifugation.

TEM was also used to visualize ultra structural detail of the three layers.

The top layer consisted mainly of lycopene in crystalline form (LC) with

dimensions of approximately 10 ^m in length and 1 jim in width (Figure 2.13

panel A). In addition, the top layer showed the presence of cell wall casting and

cell wall envelops (ghosts, G) devoid of cytoplasm. As well, the presences of tiny

vesicular structures (V) were also observable. The middle layer was free of

lycopene crystal and containing particulate matrix (Figure 2.14 panels A, B). The

bottom precipitate showed crystalline fibrous material (cellulose yield) along with

99

file:///xglg

micron size granules (Figure 2.15 panels A, B). It should be noted that there is

the possibility that each layer may have been contaminated with the adjoining

layer during the isolation and pipetting procedure. The whole soluble solids

solution is shown in Figure 2.12.

Transmission electron microscopy of soluble solids has several fractional

structures include lycopene crystals (Figure 2.12 panel A, LC), carbohydrate lipid

complexes which stained dark (Figure 2.12 panel A, CM) and formed a matrix. In

addition, remnants of broken cell wall ghosts were also observed (Figure 2.12

panel B, G).

Figure 2.11 Ultracentrifuged Soluble Solid Samples Showing Three Distinct

Layers and an Ultracentrifuged Sample after Sitting for 1h.

100

Figure 2.12 Transmission Electron Micrograph of Total Soluble Solids.

LC: Lycopene Crystalline. G: Ghosts CM: Carbohydrate Lipid

complexes, Which Stained Dark.

Figure 2.13 Transmission Electron Micrograph of Top layer after

Ultracentrifuge. LC: Lycopene Crystalline. G: Ghosts. V: Vesicle

CM: Carbohydrate-Lipid complexes, Which Stained Dark.

101

B

PM

Figure 2.14 Transmission Electron Micrograph of Middle layer after

Ultracentrifugation. LSPM: Light Staining Particulate Matter

V: Vesicles, PM: Particulate Matter

%.

DSM

y # :

B

i

H Cellulose Fiber

Figure 2.15 Transmission Electron Micrograph of Bottom layer after

Ultracentrifugation. DSM: Dark Staining Matrix

102

2.6 Discussion

The present study was employed to determine chemical and structural factors

affecting the consistency of tomato products. One of these methods is known as

precipitate weight ratio, which refers to the ratio of precipitate weight to the initial

weight of sample (Takada and Nelson, 1983). The precipitate is obtained by

centrifugation at 12,880* g for 30min at 4°C. Takada and Nelson (1983)

recommended this g-force to avoid producing a loose and less packed precipitate

and to cleanly separate insoluble solids from soluble solids. However in our

experiment, we applied a centrifugation force of 25,000* g for 18 min, which was

more effective at separating the soluble and insoluble fractions.

The collected soluble solids appeared as a distinctively red liquid, which was

shown by further analysis to contain considerable amounts of lycopene.

Lycopene is present in nature as trans/cis (90/10) crystals, insoluble in water and

with limited solubility in oil or fat. It has a dark red colour when dispersed in

water and a particle size ranging frornl to 10 urn.

In order to better understand how a highly lipophilic compound such as

lycopene can exist as a soluble complex in an aqueous environment, an

investigation into its composition and properties was undertaken.

The present study showed that a large amount of pectin was present in the

soluble solids fraction. Pectin is a high molecular weight charged hydrocolloid

that can exist as a soluble molecule, a colloidal particle form or as a gel. The

association of pectin and lycopene in the soluble complex was investigated by

digesting the pectin polymer with pectinase. The enzymatic breakdown of the

103

pectinases material resulted in the destabilization of the soluble complex and the

formation of a distinctively red precipitate when left standing or after

centrifugation. The hydrolysis of the pectinases material resulted in a dramatic

loss in solubility and indicated the importance of pectin in maintaining the

colloidal complex in solution. Lycopene was not free to float to the surface but

was mostly recovered with the insoluble material, suggesting that lycopene is

present in a complex interacting not only with pectin but also with other

components. Treatment with other enzymes (protease, cellullase) did not induce

any changes in the behavior of the soluble complex except for hemicellulase,

most likely because this enzyme contained some pectinase as well. It could be

hypothesised that proteins and cellulose are not directly responsible for

maintaining the solubility of the complex. A combination of protease and cellulase

also showed no effects on the soluble complex, which may indicate the possibility

that a pectin layer is surrounding the complex, preventing the protease and

cellulase enzymes from reaching their respective substrates.

Another interesting behavior of the soluble solid fraction was its precipitation

following dialysis. A major change brought about by dialysis was the reduction in

divalent ion levels, especially Ca ions. The loss of Ca ions could disrupt

electrostatic cross linkages of the pectin-protein complexes or induce swelling of

the pectin colloidal particles. These changes may lead to the breakdown of

pectin's interactions in the soluble complex and result in the precipitation of the

complex. It was reported that the heat degradation rate of crystalline lycopene

was much greater (Lee and Chen, 2002) than the rate of degradation of lycopene

104

in tomato pulp (Sharma and Le Maguer, 1996). This is further evidence that

macromolecules in tomato pulp, such as pectin are associated with lycopene and

in some way protect lycopene from heat degradation. This same protective

structure may also be involved with the stabiliziation of the soluble complex and

maintaining its solubility.

The direct complexation of pectin and lycopene is highly unlikely due to the

hydrophobic nature of lycopene and the hydrophilic properties of the charged

pectin molecule. In order for a complex to form between pectin and lycopene,

there needs to be an intermediate compound with both hydrophilic and

hydrophobic properties. Proteins are good candidates for that can associate with

the polar pectin molecule through electrostatic interactions or cross linkages with

Ca ions and to interact with non polar lycopene molecules through hydrophobic

interactions if sufficient hydrophobic amino acids such as valine, leucine

isoleucine and methionine are present. The combustion experiment that

determined the nitrogen content in paste and soluble solids, indicated the

presence of more than 2% N. However, the absence of protein bands on the

SDS-PAGE gel appears to exclude proteins with molecular weights between 6.5-

200 KDa. There is however the possibility that these proteins are mainly

glycoproteins (Hansen 2005) and do not migrate normally on the SDS-PAGE

gels or that the source of the nitrogen are amino acids and small peptides. This

speculation is supported by the dialyses results on the soluble solid samples that

showed a reduction in the amount of nitrogen when the soluble solids were

dialyzed in a 3500 MW cut off membrane dialysis tube. The presence of non-

105

protein N in the soluble solid has also been reported by Under and others (1984).

However it seems a little unrealistic to exclude proteins at this time since a high

amount of glycoproteins are present in the plastids (chromoplasts) where

lycopene is synthesized and deposited in plant cells (Hansen 2005).

Assuming that the nitrogen source in the soluble solids is peptides and

amino acids would explain why the protease treatment did not cause changes in

the lycopene complex. The enzymes are specific for certain chemical bonds

within the substrate molecule in much the same way as a key fits into a lock.

Without this specific bonding, no reaction will occur. In our research, this

addresses the reason why employing a protease did not have an effect on the

suspension behavior of lycopene.

In an industrial patent, micronized lycopene was coated with a thin film of

amphiphilic biopolymers such as proteins forming an insoluble network in water.

This water insoluble complex was then further treated with a colloidal material

such as pectin. By introducing lycopene (particle size of 1-10 urn) into an

aqueous solution of proteins, a protein coated lycopene complex can be

produced which can then be further treated in a colloidal water system to form a

second colloidal coat over the protein. The TEM experiment demonstrated the

presence of lycopene in a size smaller than 10 urn suitable for coating. The

protein present in the soluble fraction mostly as small peptides may be suitable

for this coating process. The presence of hydrophobic amino acids in the

peptides or free hydrophobic amino acids in the soluble solids would facilitate

such absorption.

106

Amino acids such as Valine, leucine and isoleucine have aliphatic

hydrocarbon side chains and these side chains are incapable of forming

hydrogen bonds with water as they have no, or very small, electrical charges

associated with their structure. In an aqueous solution, these side chains tend to

stable themselves by disrupting the hydrogen bonding structure between water

molecules as well as joining together through hydrophobic interaction, thus

minimizing the ordering of water molecules around hydrophobic pockets.

Lycopene is an oil soluble compound (highly hydrophobic) having along a long

aliphatic hydrocarbon chain structure which can undergo hydrophobic

interactions with other aliphatic or aromatic side chains of proteins or other super

molecular structures by packing together in order to exclude water (i.e. the lowest

energy conformation). If the side chains of leucine, isoleucine or valine can form

an aliphatic hydrophobic pocket, it may be possible for lycopene to become a

part of conformation. These interactions (hydrophobic) are to an important extent,

solvent (water) dependent, as the hydrogen bond network form by the water

molecules keep the hydrophobic groups together.

The hydrophilic amino acids face away from the hydrophobic core of proteins

and are conformationally more mobile in the water surrounding them. Amino

acids with fairly strong acidic or basic side chains are more or less fully ionized at

the measured pH (4.5-4.6) and therefore carry an electrical charge. Those amino

acids with acidic side chains carry a negative charge, while those with basic side

chains, are positively charged. Among this group of charged amino acids, the

most recognized ones are lysine and arginine. Lysine provides a positive charge

107

on the protein, as it is strongly basic (pKa=10.8). Arginine is more strongly basic

than lysine (pKa=12.5) and also provides a positive charge. The tomato products

have a relatively large amount of lysine that may facilitate such association.

A well-defined dissociation constant is not known for pectin but a pKa range of

3.0-5.0 has been suggested by Deuel (1958). In this experiment, the sample had

a pH of 4.5-4.6, where pectin carries a negative charge on its backbone and

peptides that are part of the peptide structure will have a positive charge on the

amino acids. Under these conditions it is possible to have electrostatic

interactions between positively charged side chains on the peptide molecule with

the negative charges on the backbone on the pectin molecule. These are the

interactions suggested in the model by Takada and Nelson (1983).

It is hypothesized that the hydrophobic lycopene molecules can interact with

hydrophobic patches on the peptide molecules to form complexes. These

complexes are not completely soluble in an aqueous medium and will form a

precipitate with time. However, in the presence of pectin, a soluble complex can

form where the positively charged groups on the peptide could bind to the

negatively charged pectin. The charge interactions between peptide and pectin

could stabilize the complex and at the same time protect embedded lycopene

from the aqueous medium.

The removal of pectin by pectinase treatment could destabilize the pectin-

peptide-lycopene complex and result in the precipitation of lycopene, which is still

attached to the peptides.

108

2.7 Conclusion

The objectives of this research were to investigate some of the important

properties of tomato paste. The initial study focussed on the methodologies used

to determine percent total solids (%TS), percent soluble solids (%SS) and

percent water insoluble solids (%WIS) in paste by two methods. The solids were

determined by the direct vacuum over method and the indirect microwave oven

method. The repeatability of each method was tested on 20 paste samples and

the values produced by the two methods (%TS, %SS, and %SSF) were

compared by the paired t-test, linear regression, regression of exact equality and

the average difference between the means. In the last chapter, the properties of

the soluble solids fraction were examined.

The repeatability of the vacuum oven and microwave oven methods were

determined by calculating the average standard deviation (ASD). The ASD were

0.067% and 0.045% based on 20 samples for the vacuum and microwave

methods respectively. The microwave method showed slightly better results but

overall, the two methods showed good repeatability. The paired t-test indicated

that the mean (27.17%) for total solids by vacuum oven was significantly smaller

than the mean (27.36%) determined by microwave oven. The same trend was

seen with the %WIS results. The mean (5.71%) for water insoluble solids was

significantly smaller than the mean (5.86%) determined by the microwave oven

method. The %SS however was just the reverse. The paired t-test indicated that

the mean (21.63%) for the soluble solids was significantly greater than the mean

(21.50%) for the microwave oven method. The slightly greater means obtained

109

with the microwave method for %TS and %WIS may have been caused by

incomplete drying in the case of %TS or incomplete extraction of soluble solids

in the case of %WIS. Because the %SS value is calculated with the %TS

and %WIS values, any error in the two latter values will be expressed in the %SS

value. These differences, although significant may not be large enough to be a

factor in the commercial processing of paste and that both methods can be used

to determine the solids content of paste. Supporting evidence that the two

methods were able to give essentially the same values for %TS, %WIS and %SS

was gleaned from the results obtained by simple linear regression, by regression

of exact quality, and root mean square error.

The soluble solids fraction was shown to contain considerable levels of

lycopene which in its native state is insoluble in water and has only limited

solubility in oil. The lycopene in the soluble solids fraction was dispersed in water

as a soluble complex. The properties of this soluble complex were shown to

contain large amounts of pectin. The hydrolysis of the pectin with pectinase

resulted in the destabilization of the soluble complex and the formation of a red

precipitate. The dramatic loss of solubility after pectinase treatment is one

indication of pectins involvement in the structure of the soluble complex. It

appears that lycopene is complex with other components since the hydrolysis of

pectin did not release free lycopene.

A second interesting behaviour of the soluble solid fraction was its

precipitation when dialyzed against water. There was a significant reduction in

the Ca+2 ions. Concentration after dialysis. The loss of Ca+2 ions could disrupt

110

the electrostatic cross linkages of pectin-protein complexes. These disruptions

could lead to the breakdown of pectin's interactions in the soluble complex and

result in the precipitation of the complex. It is unlikely that pectin and lycopene

are able to form a soluble complex without the aid of an intermediate compound

that can bond to both lycopene and pectin. Protein may be able to serve as that

intermediate compound through electrostatic interactions or crosslinkages with

Ca ions and through hydrophobic interactions with hydrophobic amino acids

and peptides. Based on the Dumas analysis, there is more than 2% nitrogen in

the soluble solids fraction which is enough to account for 12-13% proteins,

peptides or amino acids. It is therefore possible for the hydrophobic lycopene

molecules to interact with hydrophobic patches on the peptide/protein molecules

to form complexes. These complexes are not completely soluble in an aqueous

medium and will precipitate with time. To form the soluble complex, the pectin

molecule binds to the positively charged groups on the peptide/protein with its

negatively charged carboxyl groups. The charge interactions between

peptide/protein and pectin stabilize the complex and at the same time protect

lycopene from the aqueous medium. It is therefore hypothesized that the soluble

complex has a structure with a peptide/protein-lycopene core stabilized with a

pectin coat.

I l l

2.8 Future Study

To explore the accuracy of the microwave oven/formula method, it is

suggested that a study be undertaken to utilize Equation 3 to calculate (%WIS)

but determine (%TS) and (%SSF) by the vacuum oven. That is the (%TS) will be

determined by the procedure in section 1.3.2.1.a and (%SSF) by procedure in

section 1.3.2.2.C on the supernatant from the first centrifugation. The WIS values

from the vacuum method can then be compared to the value obtained by

calculation but without the microwave drying steps. This will give a better

indication if the microwave drying is causing the difference between vacuum and

microwave drying or if the supernatant from the first centrifugation is or is not a

true representation of the soluble solids in the paste.

A collaborative study of solids measurements employing the

microwave/formula method among laboratories is suggested to determine the

reproducibility and robustness of the microwave/formula method.

More work should be done to further characterize the soluble lycopene

complex and determine its composition and perhaps its structure. Since this

complex is able to suspend the very hydrophobic lycopene molecule in an

aqueous environment, it would be worth while to investigate the possibility that it

may have some other unique properties. For example, pure lycopene in an

organic solvent is very unstable and will rapidly oxidize and isomerize. The

structure of the complex may protect lycopene from these reactions.

Experiments can be conducted to determine the stability of the lycopene

complex against light exposure (photooxidation), high temperatures, pH, salts

112

and other environmental variables. Because the lycopene can be suspended in

an aqueous medium, the complex has the potential to be used as a delivery

system for lycopene in aqueous food systems. For this application to be viable,

the stability of the lycopene complex in the food system and its bioavailability has

to be investigated.

113

REFERENCES Ahmed, A. E. R., Labavitch, J. M. 1977. A simplified method for accurate

determination of cell wall uronide content. J. Food Biochem. 1: 361-365.

Anguelova T, Warthesen J. 2000. Lycopene stability in tomato powders. J. Food Sci. 65: 67-70.

AOAC . 2000 .Official Method of Analysis .17th ed. Solids (Insoluble) in Canned Vegetables .Gravimetric Method, sec 42.1.06.

AOAC . 2000 .Official Method of Analysis .17th ed. Solids (Soluble) in Canned Vegetables.Refractive index Method.sec 42.1.10.

AOAC . 2000 .Official Method of Analysis .17th ed. Solids (Total) in Canned Vegetables .Gravimetric Method.sec 42.1.05.

AOAC . 2000 .Official Method of Analysis .17th ed. Solids (Total) in processed tomato products.Microwave Oven drying Method.sec 42.1.09.

AOAC.1965. Official Methods of Analysis. 10th ed.Total Solids (1).30.003.

AOAC. 1980. Official Method of Analysis .13th ed. Total Solids (2). 32.010.

Axelos, M.A.V., Thibault, J.F., 1991.The chemistry of low-methoxyl pectingelation. In: Walter, R.H. (Ed.), The Chemistry and Technology of Pectin. Academic Press, San Diego, pp. 109-118.

Bannon, C. D.,Craske, J. D., Hilliker, A. E. 1985. Analysis of fatty acid methyl esters with high accuracy and reliability.IV. Fats with fatty acids containing four or more carbon atoms. J Am Oil Chem Soc. 62: 1501-1507.

Bigelow, W. D. Smith, H.R. and Green leaf ,C.A. 1950.Tomato products, National Canners Association, Bulletin 27-L,Revised.

Blanch, G.P., Ruiz del Castillo, M.L., Del Mar Caja,M., Perez-Mendez, M., Sanchez-Cortes, S. 2007. Stabilization of all-trans-Lycopene from tomato by encapsulation using cyclodextrins. Food Chem. 105:1335-1341.

Blumenkrantz, N., Asboe-Hansen, G. 1973. New method for quantitative determination of uronic acids. J. Biochem. 54: 200-201.

Bohart, G.S. 1940. Studies of western tomatoes. J. Food Sci. 5(5): 469-486.

114

Boileau, A. C , Merchen, N. R., Wasson, K., Atkinson, C. A., and Erdman, J. W. 1999. C/s-lycopene is more bioavailable than trans lycopene in vitro and in vivo in lymph-cannulated ferrests. J. Nut. 129: 1176-1181.

Brady, J. D.; Sadler, I. H.; Fry, S. C. Di-isodityrosine, a novel tetrameric derivative of tyrosine in plant cell wall proteins: a new potential cross-link 1996. Biochem. J.:315, 323-327.

Buss, CD. 1961. Refractometric tests on tomato products. Food technol. 15: 327-328.

Cantarelli, P.R., Regitano-D'arce, M.A.B. and Palma, E.R. 1993.Physicochemical characteristics and fatty acid composition of tomato seed oils from processing wastes. Sci. agric. (Piracicaba, Braz.) 50(1): 117-120.

Cheftel ,H.,Frichet.P. and Estang, J. 1951. Die Bestimmung der Troekensubstanz in Lebensmitteln. J.Ann.Fals.Fraudes 44, 1-16.

Chin, H. B.,Kimball, J.R., Hung, J. and Allen, B. 1985 . Microwave drying determination of total solids in processed tomato products: Collaborative study. J.Assoc.off Anal.Chem., 68(6), 1081-1083.

Choudhari, S.M., Ananthanarayan, L. 2007. Enzyme aided extraction of lycopene from tomato tissues, Food Chem. 102: 77-78.

Clark, A.H., Farrer, D.B., 1996. Shear modulus-concentration relationships for low DE pectin-calcium gels in the temperature range 20-85 °C. Food Hydrocolloids. 10, 31-39.

Clinton, S. K. 1998. Lycopene: Chemistry, biology, and implications for human health and disease. Nutrition Reviews. 56: 35-51.

Davies, J. N. 1964. Effect of nitrogen, phosphorous and potassium fertilizers on the non-volatile organic acids of tomato fruit. J. Sci. Food Agric. 15: 665-673.

Davies, J.N. and Hobson, G.E. 1981 .Constituent of tomato fruit-the influence of environment nutrition and genotype.C.R.C. Crit.Rev.Food Sci.Nutr. 15: 205-280.

Davis,A.B. and Lai,C.S. 1984. Microwave utilization in the rapid determination of flour moisture .Cereal chemistry,61(1) :1-4.

Denny C: technical editor. 1997 Tomato Products. 7th ed. NFPA Bulletin 27-L . 197p.

115

Di Mascio, P. Murphy, M. C. Sies, H. 1991. Anti-oxidant defense system, the role

of carotenoids, tocopherol and thiols. Am. J. Clin. Nutr. Suppl. 53: 194-200.

DiLorenzo, C. Williams, CM. Hajnal, F. Valenzuela J.E.1988. Pectin delays gastric emptying and increases satiety in obese subjects. Gastroenterology. 95:1211-5.

Dougherty. R.H. and Nelson, P.E. 1974. Effects of pH on quality of stored tomato juice. J. Food Sci. 39: 254.

Dzurec, D.J. and Baptie,P. 1989. Rapid,inexpensive microwave oven method for total solids determination in fluid dairy products. J.Dairy Sci.72:2777'-27'81.

El Miladi. S.S., Gould, W.A. and Clements, R.L.1969. Heat processing effect on starch,sugar, proteins, amino acids and organic acids of tomato juice. Food Technol. 23:93.

Ellis, G. H., Hammer, K. C. 1943. The carotene content of tomatoes as influenced by various factors. J. Nutr. 25: 539-553.

Erdman, J.J.W., Biere, T.L., Gugger, E.T. 1993. Absorption and transport of carotenoids. Ann. N. Y. Acad. Sci. 691:76-85.

Foda, Y.H., McCollum, J.P. 1970. Viscosity as affected by various constituents of tomato juice. J. Food Sci. 35 (4): 333-338.

Galili, G. 1995. Regulation of Lysine and threonine biosynthesis.Plant Cell.7:899-906.

Garti, N., Zelkha. M, Sedlov, T. 2003. Carotenoid formulation,Patent No. WO/2003/045167.

Gamier, C, Axelos, M.A.V.Thibault, J.-F.1993. Phase diagrams of pectin-calcium systems: influence of pH, ionic strength, and temperature on the gelation of pectins with different degrees of methylation. Carbohydrate Research 240,219-232.

Gartner, C. Stahl, W. and Sies H. 1997. Lycopene is more bioavailable from tomato paste than from fresh tomatoes. Am J Clin Nutr .66:116-122.

Goose, P.G., Binsted, R. 1964. Tomato paste. In Tomato paste, puree, juice and powder, 1s t ed.; Food Trade Press: London. 62-77.

Gould, W.A. 1983. Tomato Production, processing and quality evaluation, 2nd ed., Westport, CT.AVI Publishing Co. Inc. 445p.

116

Gould, W.A. 1992. Tomato Production, Processing & Technology, 3 ed., Baltimore, MD. CTI Publication lnc.550 p.

Grant, G.T., Morris, E.R., Rees, D.A., Smith, P.J.C., Thorn, D.1973. Biological interactions between polysaccharides and divalent cations: the egg-box model. FEBS Letter. 32: 195-198.

Green, W.C and Park,K.K. 1980. Comparison of AOAC microwave and vacuum oven methods for determination total solids in milk. J.Food Prot. 43:782.

Hadley, C.W., Clinton, S.K., Schwartz, S.J. 2003. The consumption of processed tomato products enhances plasma lycopene concentrations in association with a reduced lipoprotein sensitivity to oxidative damage. J. Nutr. 133:727-732.

Hague, A., Manning, A. M., Hanlon, K. A., Huschtscha, L. I., Hart, D., Paraskeva, C. 1993. Sodium butyrate induces apoptosis in human colonic cell lines in a p53 independent pathway. Implications for a possible role of dietary fiber in the prevention of large-bowel cancer. Int. J. Cancer. 55: 498.

Hansen, L. U., Chiu, M.M. 2005. Isolation and composition of chromoplasts from tomatoes. J.Agric.Food Chem. 53: 6678-6682.

Hart, D. J., Scott, K. J. 1995. Development and evaluation of an HPLC method for the analysis of carotenoids in foods, and the measurement of the carotenoid content of vegetables and fruits commonly consumed in the UK. Food Chem. 54: 101-111.

Hayes, W.A., Smith P.G. and Morris A.E.J. 1998 . The production and quality of tomato concentrates. Crit.Rev.Food Sci.Nutr .38(7): 537-564.

Health protection Branch Ottawa. 1981. Determination of tomato solids. Official Method FO-19 October 15.

Heutink R. 1986 Tomato Juices and tomato juice concentrates:a study of factores contribution to their gross viscosity . WAU dissertation no.1095.Nageninge Publisher, p 200.

Heinonen, M. I., Ollilainen, V., Linkola, E. K.,Varo, P. T., and Koivistoinen, P. E. 1989.Carotenoids in Finnish foods, vegetables,fruits, and berries. J. Agric. Food Chem. 37,655-659.

Hileman, G.A., Upadrashta, S.M., Neau, S.H., 1997. Drug solubility effects on predicting optimum conditions for extrusion and spheronization of pellets. Pharmaceutical Development and Technology 2, 43-52.

117

Hogans,SA, McNamess.B.F., 0'Riordan,E.D., O'Sullivan, M. 2001. Microencapsulating Properties of Sodium Casein. J.Agric.Food Chem. 49: 1934-1938.

Hurmann K.1979.Ubersicht Uberdie In haltstoffe dertomaten.Z.Lebensm.Unters. u.-Forsch.169:179-200.

Ishida, B.K., Ma, J.C., Chan B.G., Bartley, G.E., Grossman, J.N. 2001. Modified method for simple, rapid hplc analysis of lycopene isomers. Acta Hort ISHS, 235-249.

Jackson, M. J. 1997. The assessment of bioavailibility of micronutrients: introduction. Eur. J. Clin. Nutr. 51 (Supple 1): S1-S2.

Jung, S. Rickert, D.A., Deak, N.A., Aldin , E.D ., Recknor, J., Johnson, L.A., Murphy, P.A. 2003. Comparison of Kjeldahl and Dumas methods for determining protein contents of soybean products. J. Am. Oil Chem. Soc. 80:1169-1173.

Kieliszewski, M.J and Lamport D.T.A.7994. Extensin: repetitive motifs, functional sites, post-translational codes, and phylogeny, Plant J. 5(2): 157-172.

Kimball, L.B., Lillian, B., Kertesz, Z.I. 1952. Practical determination of size distribution of suspended particles in macerated tomato products. Food Technol. 6: 68-71.

Krinsky, N. I., Russett, M. D., Handeman.G. J., and Snodderly, D. M. 1990. Structural and germetrical isomers of carotenoids in human plasma. J. Nutr., 120, 1654-1662.

Laemmli U.K. 1970, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Mature.221 .$ 680-685.

Lamb F. C. 1969. Collaborative Study of the determination of soluble Solids in tomato products by refractive Index Expressed as Per Cent Sucrose. J. AOAC. 52(5) : 1050-1054.

Lamb, F.C. 1977. Tomato Product. Bulletin 27-L National Canners' Assoc, Washington. DC.

Lamb. F.C. 1964. Processed vegetable products: Total solids in processed vegetable. J.Assoc.Off. Agric.Chem. 47,492-500.

Lee M.T., Chen B. H. 2002. Stability of Lycopene during heating and illumination in model system. Food Chem. 78: 425-432.

118

Lindner, P., Shomer, I., Vasiliver, R. 1984. Distribution of Protein, Lycopene and the Elements Ca, Mg, P and N among various fractions of tomato juice. J. FoodSci. 49(4): 1214-1215.

Liu, Y. K., Luh, B. S. 1979. Effect of harvest maturity on free amino acids, pectins, ascorbic acid, total nitrogen and minerals in tomato pastes. J. Food Sci. 44(2):425-428.

Livney,Y. Dalgleish, D. 2007. Casein micelles for nanoencapsulation of hydrophobic compounds. Patent no. WO/2007/122613.

Lootens, D., Capel, F., Durand, D., Nicolai, T., Boulenguer, P., Langendorff, V., 2003. Influence of pH, Ca-concentration, temperature and amidation on the gelation of low methoxyl pectin. Food Hydrocolloids. 17, 237-244.

Lurie, S. Handros, A. Fallik, E. and Shapira.1996. Reversible inhibition of tomato fruit gene expression at high temperature. Plant Physiol. 110: 1207-1214.

MacDougall, A.J., Brett, G.M., Morris, V.J., Rigby, N.M., Ridout, M.J. and Ring, S.G. 2001. The effect of peptide-pectin interactions on the gelation behaviour of a plant cell wall pectin. Carbohydr. Res. 335: 115-126.

Madhayi, D.L.Salunkhe, D.K.1998. Tomato.ln: Salunkhe ,D.K. and Kadam.S.S. Handbook of vegetable science and technology: Production, composition, storage, and processing.CRC Publisher, p 171-203.

Makarchuck, M. J., Hill, A. R.,and Szijarto, L. F. 1991. Accuracy and repeatability of milk total solids analysis by microwave moisture oven. Can. Inst. Sci. Technol. J., 24(3/4): 172-175.

May B. 2003 Handbook of Vegetable Preservation and Processing : Hui Y.H, Ghazala S., Graham D.M., Murrell K.D. ,Nip Wai-Kit. 2003 ..CRC Press.752 p.

McColloch, R J., Nielsen, B.W, Beavens, E.A. 1950. Factors influencing the quality of tomato paste. II. Pectic changes during processing. Food Technol. 4: 339.

McCollum, J. P. 1955. Distribution of carotenoids in the tomato. Food Res. 20: 55-59.

McKinstrya, P. J., Indyka, H. E., and Kim, N. D.1999. The determination of major and minor elements in milk and infant formula by slurry nebulisation and inductively coupled plasma - optical emission spectrometry (ICP-OES). Food Chem. 65 . 245-252.

119

Morris, E.R., Powell, D.A., Gidley, M.J., Rees, D.A.1982. Conformations and interactions of pectins. I. between gel and solid states of calcium polygalacturonate. J. Mol. Biol. 155: 507-516.

Nguyen, M. L, Schwartz, S. J. Lycopene 1999: chemical and biological propertiesn. Food Tech 53: 38-45.

Oomah, B. D., and Mazza, G. 1992. Microwave oven drying for moisture determination in flax, canola and yellow mustard seeds. Lebensm. -Wiss. U.-Technol.25: 523-526.

Parker, R.S.1996. Absorption, metabolism, ad transport of carotenoids. FABS J. 10:542-51.

Powell, D.A. 1982. Conformations and interactions of pectins II.Influence of residue sequence on association in calcium pectate gels. J. Mol. Biol. 155:517-531.

Qi, X.; Behrens, B. X.; West, P. R.; Mort, A. E. 1995. solubilization and partial characterization of extensin fragments from cell walls of cotton suspension cultures (Evidence for a covalent cross-link between extensin and pectin). PlantPhysiol. 108, 1691-1701.

Qiu, W., Jiang, H., Wang, H , Gao, Y. 2006. Effect of high hydrostatic pressure on lycopene stability. Food Chem. 97: 516-523.

Reitsma, J.C.E., Thibault, J.F., Pilnik, W., 1986. Properties of amidated pectins. I. Preparation and characterization of amidated pectins and amidated pectin acids. Food Hydrocolloids. 1:121-127.

Ribayo-Mercado, J. D., Garmyn, M., Gilchrest, B. A., Russell, R. M. 1995. Skin lycopene is destroyed preferentially over betacarotene during ultraviolet irradiation in humans. J. Nutr. 125: 1854-1859.

Ribeiro HS, Ax K, Schubert H. 2003. Stability of lycopene emulsions in food systems. J Food Sci .68(9):2730-2740.

Riso, P. & Porrini, M. (1997) Determination of carotenoids in vegetable foods and plasma. Int. J. Vitam. Nutr. Res. 67: 47-54.

Rock, C.L. and Swendseid,M. F. 1992. Plasma -carotene response in human after meals, supplemented with dietary pectin. Am. J. Clin. Nutr. 55: 96-99.

120

Sa' nchez M.C., Valencia C, Ciruelos A., Latorre A. and Gallegos C. (2002). Influence of processing on the rheological properties of tomato paste. J. Sci. FoodAgric .82: 990-997.

Sainju, U. M. and Dris R. 2006. Sustainable production of tomato. In: Dris.R. editor, Crops: Quality, growth, and biotechnology. Helsinki, Finland: WFL Publisher, p. 190-216.

Saywell.L . G., and Cruess, W .V. 1932. Composition of canning tomatoes. Calif. Agr.Expt. Sta. Bull. 545.

Schierle, J., Bretzel, W., Buhler, I., Faccin.N., Hess, D., Steiner, K., and Schuep, W. 1996. Content and isomeric ratio of lycopene in food and human blood plasma. Food Chem. 59(3): 459^65.

Semo, E.,Kesselman, E., Danino, D., Livney, Y.D. 2007.Casein micelle as a natural nano-capsular vehicle for netraceuticals. Food Hydrocolloids. 21:936-942.

Sharma, S.K. and Le Maguer, M. 1996. Lycopene in tomatoes and tomato pulp fractions. J. Food Sci., 2, 107-113.

Sharma, S.K. and Le Maguer, M. 1996. Kinetics of lycopene degradation in tomato pulp solids under different processing and storage conditions. Food Res Int. 29: 309-315.

Shi, J., Le Maguer, M., Kakuda, Y., Liptay, A.1999. Lycopene degradation and isomerization in tomato dehydration. Food Res. Intl. 32(1): 15-21.

Showalter, A. M. 1993. Structure and function of plant cell Wall protein, of Plant cell 5: 9-23.

Smith, J. G., Yokoyama, W. H., German, J. B. 1998. Butyric acid from the diet: actions at the level of gene expression. Crit. ReV. Food Sci. 38 (4): 259-297.

Sommer-Knudsen, J., Bacic A., Clarke A.E., 1998. Hydroxyproline-rich plant glycoproteins. Phytochemistry, 47(4), pp. 483-497.

Stahl, W., Sies, H. 1992. Uptake of lycopene and its geometrical isomers is greater from heat-processed than from unprocessed tomato juice in humans. J. Nutr. 122: 2161-2166.

Stahl, W., Sies, H. 1996. Perspectives in biochemistry and biophysics. Arch. Biochem. Biophys. 336(1): 1-9.

121

Stein, W.H. and Mohr, S. 1949. Amino acid composition of-Lactogobulin and bovine serum albumin. J. Biol. Chem. 178: 79.

Takada, N. Nelson P. E. 1983. Pectine protein Interaction in tomato products. J. Food Sci. 48: 1408-1411.

Tavares, C. A., Rodriguez-Amaya D. B. 1994. Carotenoid composition of Brazilian tomatoes and tomato products. Lebensmittel - Wissenschaft Technologie.27 (33): 219-224.

Tho, I., Sande S.A., Kleinebudde P. 2005. Cross-linking of amidated low-methoxylated pectin with calcium during extrusion/spheronisation: effect on particle size and shape Chem. Eng. Sci. 60, 3899-3907.

U.S. Department of Agriculture (2000-01). Standards for Grades of Canned Tomatoes. Quoted in: The Almanac,2000-01. P. 642. Westminster, MD: E.E. Judge and Sons, Inc.

U.S. Supreme Court. MX v. HEDDEN, 149 U.S. 304. 1893. Collector. No. 137. May 10.

Wang,S. L. 1987.Microwave drying method for total solids determination in tomatoes: collaborative study. J.Assoc.offAnal.Chem.,70(4),758-759.

Weisburger, J.H. 2002. Lycopene and tomato products in health promotion. Exp Biol Med. 227: 924-927.

Whittenberger, R.T., Nutting, G.C. 1958. High viscosity of cell wall suspensions prepared from tomato juice. Food Technol. 12: 420-424.

Yoo,B. and Rao M.A. (1994). Effect of unimodal particle size and pulp content on the rheological propertiesof the tomato puree. Journal Texture Studies 25:421^36.

Young. E.G. 1963. Occurrence, classification, preparation and analysis of proteins. In "Comprehensive Biochemistry", Vol.7: 1. Elsevier Publishing Co.. New York, NY.MS received 3131183; revised 5127183: accepted 613183.Volume48.

Zechmeister L, Lerosen A.L, Schroeder W.A, Polgar A, Pauling L. 1943. Spectral characteristics and configuration of some stereoisomeric carotenoids including prolycopene and pro-y-carotene. J. Am Chem Soc .65:1940-1945.

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