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The Impact of Twin—Screw Extrusion Processing on
Physical and Biochemical Properties of the
Major Components of Wheat Flour

presented by

Alfred Kojo Anderson

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tM W14

THE IMPACT OF TWIN-SCREW EXTRUSION PROCESSING ON PHYSICAL
AND BIOCHEMICAL PROPERTIES OF THE MAJOR COMPONENTS OF
WHEAT FLOUR
BY

Alfred Kojo Anderson

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Food Science and Human Nutrition

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ABSTRACT
THE IMPACT OF TWIN-SCREW EXTRUSION PROCESSING ON PHYSICAL
AND BIOCHEMICAL PROPERTIES OF THE MAJOR COMPONENTS OF
WHEAT FLOUR
By

Alfred KOJO Anderson

Extrusion processing technology has been successfully used commercially
to produce a myriad Of food products including ready-tO-eat breakfast cereal and
snack products. Yet, in spite Of this commercial success, the chemical processes
occurring among the ingredients used as raw materials in extmsion processing are
not well understood. It is desirable to gain more fundamental understanding Of the
scientific aspects of extrusion to improve upon existing extruded fOOd products as
well as in the design Of new ones. The present study looked at the effects Of
extrusion processing variables and feed raw material on some physical and
biochemical properties Of wheat flour extrudates.

Extrusion processing was achieved with a twin-screw, CO-rotating
experimental extruder. Extruded products were evaluated for physical
characteristics such as expansion ratio, bulk density, water absorption capacity,
microstructure, and specific mechanical energy inputs. Biochemically, extrudates
were analyzed for changes in protein compositions, disulfide-sulflwydryl interchange

reactions, and hydrophobic and thermal property Changes.

 

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The level Of protein content in raw feed materials Significantly affected
expansion ratio, bulk density, water absorption index, and specific mechanical
energy inputs Of extruded products.

Higher exit die temperatures resulted in increased sulfhydryl content and
Slightly reduced disulfide content Of extrudates. Both sulfhydryl and disulfide
contents of extrudates were lower than those Of the non-extruded samples.
Disulfide—sulfhydryl interchange reactions were not Significant in the extrusion
process under the conditions studied.

Fluorescence spectroscopy data indicated changes in the conformational
structure Of wheat proteins consequent to extrusion processing. All extrudates
Showed higher emission peak wavelengths than non-extruded samples. Increasing
extruder die temperatures resulted in changes in the hydrophobic environment Of
proteins as measured by fluorescence intensity and Changes in the maximum

fluorescence emission peak wavelengths.

 

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ACKNOWLEDGMENTS

This dissertation research project has been made possible by the unflinching
support and help from many people. Foremost among them is my academic advisor
and thesis director, Dr. Perry K.W. Ng. His thoughtful scientific guidance, advice,
and friendship were immeasurable in my undertaking and successful completion of
this research project, and I would like to extend my sincerest and most heartfelt
thanks to him. I would also like to express my sincere gratitude to members of my
graduate advisory committee, Drs. W.L. Chencweth, J.F. Steffe, R. W. Ward, all Of
Michigan State University, and Dr. C. T. Simmons of Kellogg Company, Battle
Creek, for their time and advice.

The generous financial support, in the form of graduate assistantships and
fellowships, from the Office of Minority and Women Graduate Fellowship Program,
the Graduate School, the College Of Agriculture and Natural Resources, the
Department Of Food Science and Human Nutrition and Dr. N9 is also acknowledged
with much thanks and appreciation. Thanks also go to my colleagues at the Cereal
Science Laboratory: VInce Rinaldi, Ling Lee, Pascal Pierre-Cesar, Monica Accerbi,
and Rita Redaelli for their immeasurable help, support, and encouragement in the
laboratory.

Finally, to my wife Adoma, and children, Kobi, Kwesi, and those yet unborn,
I say many thanks for their love, support, understanding, and sacrifice throughout

the course Of this study.

iv

TABLE OF CONTENTS

LIST OF TABLES ................................................ x

LIST OF FIGURES .............................................. xii
CHAPTER 1

INTRODUCTION ........................................... 1
CHAPTER 2

LITERATURE REVIEW ...................................... 8

2.1 Introduction ............................................ 9

2.2 Extruder Design And Components ......................... 11

2.2.1 Screw Configuration .............................. 12

2.2.2 Extruder Die .................................... 18

2.2.3 Single-Screw Extruders ........................... 19

2.2.4 Twin-Screw Extruders ............................ 20

2.3 Residence Time Distribution .............................. 22

2.4 Effects Of Extrusion Variables on Extrudate Characteristics ...... 24

2.4.1 Effects of Screw Speed and Configuration ............ 25

2.4.2 Effects Of Temperature ........................... 27

2.4.3 Raw Material Characteristics ...................... 29

2.4.3.1 Starch .................................. 30

2.4.3.2 Protein ................................. 32

2.4.4 Effects Of Moisture Content Of Raw Materials ......... 35

2.5 Biochemical Changes in Proteins During Extrusion ............. 37

2.6 Effects Of Heat and Chemical Modifications on
Cereal Protein Composition ............................... 38

2.7 Themal Transitions in Wheat Flour Components as
Measured by Differential Scanning Calorimetry ............... 40
2.8 Role Of Disulfide Bonds in Extrusion Texturization ............. 42

2.9 Effects Of Extrusion on Disulflde and Sulfhydryl Contents

Of Cereal Proteins ...................................... 43
2.10 Literature Cited ....................................... 47
CHAPTER 3

THE INFLUENCE OF TWIN-SCREW EXTRUSION PROCESSING
ON PHYSICAL AND MICROSTRUCTURAL CHARACTERISTICS

OF WHEAT FLOUR EXTRUDATES ........................... 57
3.1 Abstract .............................................. 58
3.2 Introduction ........................................... 59
3.3 Materials and Methods ................................... 61
3.3.1 Extrusion Conditions ............................. 61
3.3.2 Determination Of Physical Characteristics Of Extrudates . . 63
3.3.2.1 Expansion ratio and bulk density ............. 63

3.3.2.2 Water absorption index and specific
mechanical energy ........................ 63
3.3.2.3 Microstructure of extrudates ................. 64
3.3.3 Statistical Analysis ............................... 64
3.4 Results and Discussion .................................. 65
3.4.1 Expansion Ratio and Bulk Density ................... 65
3.4.2 Water Absorption Index (WAI) ...................... 68
3.4.3 Specific Mechanical Energy ........................ 70

3.4.4 Relationship Between Extrusion Variables

and Physical Properties ........................... 71
3.4.5 Microstructure Of Extrudates ....................... 73
3.5 Summary ............................................. 74
3.6 Literature Cited ........................................ 76

vi

 

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CHAPTER 4
CHANGES IN DISULFIDE AND SULFHYDRYL CONTENTS
AND ELECTROPHORETIC PATTERNS OF WHEAT PROTEINS

UPON EXTRUSION ........................................ 90
4.1 Abstract .............................................. 91
4.2 Introduction ........................................... 92
4.3 Materials and Methods ................................... 95
4.3.1 Extrusion ...................................... 95

4.3.2 Disulfide-Sulfhydryl Analyses ....................... 95

4.3.3 Electrophoresis ................................. 96
4.3.3.1 Extraction procedure ...................... 96

4.3.3.2 SDS-PAGE .............................. 97

4.4 Results and Discussion .................................. 98
4.4.1 Disulfide and Sulfhydryl Reactions ................... 98

4.4.2 Electrophoresis ................................ 101

4.5 Summary ............................................ 105
4.6 Literature Cited ....................................... 106

CHAPTER 5

EFFECTS OF TWIN-SCREW EXTRUSION PROCESSING

ON STRUCTURAL AND CHEMICAL PROPERTIES OF THE
MAJOR WHEAT FLOUR COMPONENTS AS MEASURED

BY FLUORESCENCE SPECTROSCOPY AND DIFFERENTIAL

SCANNING CALORIMETRY ................................ 114
5.1 Abstract ............................................. 1 15
5.2 Introduction .......................................... 1 16
5.3 Materials and Methods .................................. 117
5.3.1 Extrusion ..................................... 117
5.3.2 Differential Scanning Calorimetry (DSC) ............. 117
5.3.3 Fluorescence Spectroscopy ....................... 118
5.4 Results and Discussion ................................. 119
5.4.1 Thermal Transitions ............................. 119
5.4.2 Fluorescence Spectroscopy ....................... 122

vii

 

 

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5.5 Summary ............................................ 124

5.6 Literature Cited ....................................... 127
CHAPTER 6

GENERAL CONCLUSIONS ................................ 137
CHAPTER 7

FUTURE RESEARCH ..................................... 141
APPENDIX 1

Composition and Mixograph Data of Soft Wheat Flour (cv. Harus),
Vital Gluten and Flour-Gluten Mixture Samples Used in

Experiments ............................................. 145
APPENDIX 2

Raw Data for Extrusion Experiments .......................... 146
APPENDIX 3

Analyses of Variance Tables for Experimental Data .............. 148
APPENDIX 4

Electrophoregrams Of Non-Reduced Total Proteins in Extruded

Wheat Flour ............................................ 152
APPENDIX 5

Electrophoregrams Of Reduced Total Proteins in Extruded Wheat

Flour .................................................. 153

viii

 

 

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LIST OF TABLES

Table 3.1
Screw Configuration in Extrusion Experiment .................... 88

Table 3.2
Multiple Linear Regression Coefficients for Expansion Ratio,
Bulk Density, Water Absorption Index, and Specific Mechanical

Energy .................................................. 88
Table 3.3

Multiple Correlation Coefficients for Extrusion Variables ............ 89
Table 4.1

Effects Of Extruder Exit Die Temperature, Screw Speed and
Protein Content on Free Sulfhydryl, Disulfide and Total Cysteine
Contents Of Extruded Wheat Flour ........................... 112

Table 4.2
Statistical F-Values for Analyses Of Free Sulfhydryl, Disulfide
and Total Cysteine in Wheat Flour Extrudates .................. 113

Table 5.1
Endothermic Reaction Temperatures and Enthalpies Of Extruded
and Non-Extruded Wheat Flour Containing 9% Protein ............ 135

Table 5.2
Peak Wavelengths and Fluorescence Intensities Of Extruded
and Non-extruded Wheat Flour .............................. 136

 

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LIST OF FIGURES

Figure 2.1
Cross-section of Extruder Barrel Showing Processing Zones on
the Screw ................................................ 13
Figure 2.2
Individual Components of Extruder Screw Configuration ........... 14
(a) Agitator shaft ..................................... 15
(b) Single lead feed screw .............................. 15
(c) Single lead discharge screw ......................... 15
(d) Paddle pairs ..................................... 16
Figure 2.3
Arrangement of Screw Elements in Extruder Barrel Zones .......... 21
Figure 2.4
Classification of Twin-screw Extruders ......................... 23
Figure 3.1a
Effects Of Screw Speed and Exit Die Temperature on Expansion
Ratio Of Extrudates at 9% Feed Protein Content. ................. 80
Figure 3.1b
Effects Of Feed Protein Content Level and Extruder Screw Speed on
Expansion Ratio of Extrudates at 120°C Exit Die Temperature. ....... 81
Figure 3.2
Effects of Feed Protein Content and Extruder Exit Die Temperature
on Bulk Density Of Extrudates at Screw Speed Of 400 rpm. ......... 82
Figure 3.3

Effects Of Feed Protein Content and Extruder Screw Speed
on Water Absorption Index Of Extrudates at Exit Die Temperature
of 140°C. ................................................. 83

Figure 3.4a
Effects Of Extruder Exit Die Temperature and Screw Speed on
Specific Mechanical Energy Input Of Extrudates at Feed Protein
Content Of 9%. ............................................ 84

Figure 3.4b
Effects of Feed Protein Content and Exit Die Temperature on the
Specific Mechanical Energy Input Of Extrudates at Screw Speed
Of 400 Rpm. .............................................. 85

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Figure 3.5
Laser Scanning Micrographs Of Cross-sections Of Samples Extruded
at Exit Die Temperature of 160°C, Screw Speed Of 240 rpm, and Feed
Protein Content Levels Of: (A) 9% (B) 30% ....................... 86

Figure 3.6
Laser Scanning Micrographs of Cross-sections Of Samples Extruded
at Screw Speed Of 400 rpm, Feed Protein Content of 9%, and Exit Die
Temperature Of: (A) 140°C (B) 160°C. ........................... 87

Figure 4.2
SDS-PAGE Patterns Of Non-reduced Total Protein Of Extruded
Wheat Flour Containing 9% Protein. .......................... 110

Figure 4.3
SDS-PAGE Patterns Of Reduced Total Protein of Extruded
Wheat Flour Containing 9% Protein. .......................... 111

Figure 5.1
Endothermic Thermal Transition Energy and Temperature
Changes of Non-extruded Wheat Flour as a Function Of Protein
Content Of Flour. ......................................... 129

Figure 5.2
Representative Differential Scanning Thermograms Of Non-extruded
Wheat Flour. (A) = 9% Protein Content; (8) = 20% Protein Content;
(C) = 30% Protein Content. ................................. 130

Figure 5.3
Representative Differential Scanning Thermograms Of Wheat Flour
Extruded at Die Temperature Of 120°C and Screw Speed of 400 rpm.
(A) 9% Protein Content; (B) 20% Protein Content;
(C) 30% Protein Content ................................... 132

Figure 5.4
Effects Of Extruder Die Temperature on the Shifts in Fluorescence
Emission Peak Wavelengths of Extruded Wheat Flour Containing
9% Protein. ............................................. 134

xi

CHAPTER 1

INTRODUCTION

 

 

 

 

 

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Extrusion technology has been used extensively to produce diverse products
such as modified starches, texturized vegetable proteins, snack foods, breakfast
cereals, pasta products, biscuits, and pet foods (Harper 1981, LinkO et al 1983).
The process involves forcing a material to flow under a variety Of controlled
conditions and then passing it through a shaped die at a predetermined rate.

In extrusion, the material to be extruded is fed via a hopper intO the hollow
barrel Of an extruder where it is mixed with some determined amount Of liquid
through a metering pump. The material is then forced towards the exit by a rotating
screw (or screws). Pressure is generated through the compressed material inside
the barrel Of the extruder. This pressure increase is accompanied by a temperature
rise as a result of both transfer Of heat from the heated barrel jacket and a
conversion Of mechanical energy into heat energy.

The heated plasticized mass Of material then passes into the die section of
the extruder and into the atmosphere at lower pressure and generally lower
temperature where spontaneous evaporation Of water occurs (Ledward and Mitchell
1988), resulting in expansion Of the product. These types of products are referred
tO as direct-expanded products, and are mostly made using twin-screw extruders.
Indirect-expanded products in the form Of pellets are commonly produced by Single-
screw extruders. The pellets are expanded by baking or frying prior to consumption.
The major raw materials for both direct and indirect-expanded cereal products are
flours and starches (Wang 1997).

Proteins in cereals can be divided into two main groups based on their
biological functions. There are the biologically active enzymes and the biologically

inactive storage proteins. In wheat, the storage proteins, also called gluten proteins,

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constitute about 70% Of the total wheat flour proteins (Kasarda et al 1976), and are
dependent on the genetic background Of the plant. Thus, they are used for varietal
identification.

Osborne (1907) fractionated wheat proteins into four major groups based on
their solubilities in different solvents. The albumins are soluble in water, the
globulins are soluble in dilute salt solutions, the gliadins are soluble in aqueous
alcohol, and the glutenins are soluble in acid or alkali. Gliadins and glutenins are the
two storage proteins of wheat and make up the bulk Of the proteins in wheat
(LOOkhart 1991). The Osborne fractionation method (1907) continues to be useful
for both structural and functional studies Of cereal proteins in Spite of cross
contamination Of fractionated proteins (Bushuk 1981). Chen and Bushuk (1970)
modified this method to produce five fractions Of wheat proteins, namely, albumins,
globulins, gliadins, soluble glutenins and insoluble glutenins.

During the extrusion processing Of cereals, the combination Of shearing,
temperature, and the generation Of high pressure in the cooking zone Of the
extruder create many opportunities for physicochemical changes in the
carbohydrates, proteins, and lipids (Wen et al 1990). For example, several studies
have shown that proteins lose their native tertiary structure resulting in changes in
conformation (Harper 1986, Ledward and Mitchell 1988, Camire et al 1990, Dahl
and Villota 1991). Gelatinization of starch is also known to occur leading to
breakdown Of the starch granules and cell structure (Owusu-Ansah et al 1983,
Diosady et al 1985, Mercier and Feillet 1975, Williams et al 1977, Gomez and
Aguilera 1984, Wang 1997).

The engineering aspects Of extrusion, rheological properties, and nutritional

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quality Of extruded products have been documented (Harper 1981, Cheftel 1986,
Otun et al 1986). However, the biochemical changes that take place during
extrusion are poorly understood (Harper 1981) and have received little constructive
attention (Ledward and Mitchell 1988). There is the need, therefore, to examine the
biochemical changes that occur during the extrusion process, and how these
Changes impact the functional characteristics Of the extruded product. These
studies will lead to understanding Of the physicochemical aspects Of extrusion as
it applies to proteins, lipids, and carbohydrates Of cereals. The understanding
gained will be useful in establishing a predictable extruded end-product based on
some explained physicochemical behaviors Of product ingredients.

In the present study, therefore, the overall Objective was to investigate the
physicochemical changes in the major components Of wheat as a result Of twin-
screw extrusion processing. The study focused on the following specific Objectives:
(1) to assess the effects Of different levels Of protein content and various conditions
Of extrusion processing on some physical characteristics Of wheat flour extrudates;
(2) to evaluate the roles played by disulfide and sulflIydryl groups Of wheat storage
proteins during extrusion processing, and how Changes in the contents Of these
groups affect the physical and microstructural Characteristics Of the extruded
products; and (3) to determine the effects Of extrusion processing on the
biochemical structure Of the major components Of wheat flour.

The information Obtained in these studies will contribute to a further
understanding Of the impact Of extrusion processing on the biochemical changes
in cereals in general, and wheat storage proteins in particular, as a result Of the

extrusion process. Such understanding will lead to improvement in quality Of cereal-

4

based extruded foods by making possible the prediction Of extruded end-products
based on the known biochemical behaviors Of the components Of the raw feed
ingredients.

This document presents a number Of experiments and results that make up
the study in pursuance of the above Objectives, and is based on the format Of
Cereal Chemistry, an intematicnal journal published by the American Association
Of Cereal Chemists, St. Paul, MN. In addition to the general introduction and
general literature review, the document is organized into Chapters with each chapter
dealing with specific questions while at the same time tying in with other chapters.
The reader is, therefore, alerted to possible duplications and overlaps in issues
discussed in the general literature review as well as in the various other sections of

individual experiments.

 

 

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LITERATURE CITED

BUSHUK, W. 1981. Utilization Of cereal proteins. In Utilization Of Protein
Resources. D.W. Stanley, E.D. Murray, and DH. Lee, eds., Food and
Nutrition Press, Inc., Wesport, CT.

CAMIRE, M.E., CAMIRE, A., and KRUMHAR, K. 1990. Chemical and nutritional
changes in foods during extrusion. Crit. Rev. Food Sci. and Nutr. 29:35.

CHEFTEL, J.C. 1986. Nutrition effects Of extrusion cooking. Food Chem. 20:263.

CHEN, OH. and BUSHUK, W. 1970. Nature Of proteins in Tn'ticale and its parental
species. I. Solubility characteristics and amino acid composition of
endosperrn proteins. Can. J. Plant Sci. 50:8.

DAHL, SR. and VILLOTA, R. 1991. Effect Of thermal denaturation on the texturi-
zation of soybean protein via twin-screw extrusion. Can. Inst. Sci. Technol.
J. 24:143.

DIOSADY, L.L., PATON, D., ROSEN, N., RUBIN, L.J., and ATHANASSOULIAS, C.
1985. Degradation Of wheat starch in a single-screw extruder: mechano-
kinetic breakdown Of cooked starch. J. Food Sci. 50:1697.

GOMEZ, M.H. and AGUILERA, J.M. 1984. A physicochemical model for extrusion
Of corn starch. J. Food Sci. 49:40.

HARPER, J.M. 1981. Food Extrusion, Volumes I and II. CRC Press, Florida.
HARPER, J.M. 1986. Extrusion texturization Of foods. Food Technol. 40:70.

LEDWARD, DA. and MITCHELL, JR. 1988. Protein extrusion - More questions
than answers? In Foods Structure - Its Creation and Evaluation. J.M.
Blanshard and JR Mitchell, eds, p. 219. Butterworths, London.

KASARDA, D.D., BERNADINE, J.E., and NIMMO, CC. 1976. Wheat proteins. In
Advances in Cereal Science and Technology, Vol. 1. pp. 158. Y. Pomeranz,
ed. Am. ASSOC. Cereal Chem., St. Paul, MN.

LINKO, P., LINKO, Y.-Y., and OLKKU, J. 1983. Extrusion cooking and bio-
conversions. J. FOOd Eng. 2:243.

LOOKHART, G.L. 1991. Cereal Proteins: Composition Of their major fractions and
methods for identification. In Handbook Of Cereal Science and Technology.
K.J. Lorenz and K. Kulp, edS., p. 441. Marcel Dekker, New York.

 

 

 

WII

MERCIER, C. and FEILLET, P. 1975. Modification Of carbohydrate components by
extrusion-cooking Of cereal products. Cereal Chem. 522283

OSBORNE, TE. 1907. The Proteins Of the Wheat Kernel. Carnegie Inst. Pub. NO.
84.

OTUN, E.L., CRAWSHAW, A., and FRAZIER, JP. 1986. Flow behavior and
structure Of proteins and starches during extrusion cooking. In Fundamentals
Of Dough Rheology. H. Faridi and J.M. Faubion, eds., p. 37. American
Association Of Cereal Chemists, St. Paul, Minnesota.

OWUSU-ANSAH, J., VAN DE VOORT, PR, and STANLEY, D.W. 1983. Physica-
Chemical changes in cornstarch as a function Of extrusion variables. Cereal
Chem. 60:319.

WANG, SW. 1997. Starches and starch derivatives in expanded snacks. Cereal
Foods World. 42:743.

WEN, L., RODIS, P., and WASSERMAN, BR 1990. Starch fragmentation and
protein insolubilizaticn during twin-screw extrusion Of corn meal. Cereal
Chem. 67:268.

WILLIAMS, M.A., HORN, RE, and RUGULA, RP. 1977. Extrusion: An in-depth
look at a versatile process. J. Food Eng. 49:99.

CHAPTER 2

LITERATURE REVIEW

2.1 INTRODUCTION

Extrusion technology evolved from the metallurgical industry in 1797 when
it was first used to manufacture seamless lead pipes (Rhodes and Olbertz 1985).
The technology was first applied to fOOds in the 1800's for the production Of
sausage and processed meats (Harper 1980), and General Mills Inc. (Minneapolis,
MN) was the first to employ extrusion processing for the production of ready-tO-eat
(RTE) cereal products in the late 1930's (Harper 1981).

At present, extrusion processing iS one of several texturization processes,
which include flaking, puffing, and baking, used extensively in the food industry to
produce diverse products such as ready-tO-eat (RTE) breakfast cereals made from
wheat, corn, rice and oats; snacks from corn and wheat, and baked goods mainly
from wheat (Clark 1986). Biscuits, pasta, crackers, crisp breads, baby foods,
confectionery items, chewing gum, texturized vegetable proteins, modified starches,
and beverage mixes are all items which can be produced through extrusion
processing (Linko et al 1983). Extrusion cooking is a versatile process that also
improves organoleptic and nutritional qualities in foods (O’Connor 1987). The cost-
tO-benefit ratio Of extrusion technology gives producers, processors, and consumers
more Choices by increasing the variety of ingredients used in cereal-based products
(Rayas-Duarte et al 1998).

The US. snack food industry has a total retail value Of $16 billion Of which
about 5%, or $810 million, is from extruded products. In all, about 5.7 billion pounds
(2.5 million metric tons) Of products are produced from extrusion annually (Purvis

1998). Extrusion can also be used for several different functions including mixing,

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cooking, forming, conveying, puffing, or drying, depending on the extruder design
and the process conditions.

FOOd extruders are generally divided into two Classifications: Single-screw
and twin-screw (Huber 1991). Single-screw extruders are by far the more commonly
used. However, because Of its improved pumping action, the co-rotating twin-screw
extruder is finding increased use for producing products requiring precise process
control (Hauck 1989).

Extrusion processing variables that directly control product quality attributes
are called independent variables. These include the raw material formulation, raw
material feed rate, configuration Of the screws, the die restriction, moisture content
Of raw materials, screw speed, and temperature Of the various zones Of the extruder
barrel (Huber 1991). Dependent variables change as a result of Changes made to
the independent variables, and they include product temperature, product moisture
content, residence time in the extruder, pressure within the extruder, and moisture
content Of the extruded product. Responses are measured by the final product
qualities that result from changes made to independent or dependent variables.
These qualities include moisture content of extrudates, expansion, solubility,
absorption, texture, color, and flavor (Huber 1991, Purvis 1998).

Extrusion cooking of cereals and starches requires the control Of processing
variables such as moisture content Of the ingredients, extrusion cooking
temperature, screw speed, and extruder barrel and die configurations (Owusu-
Ansah et al 1983, Meuser et al 1982, Guy and Home 1988, Chinnaswamy and
Hanna 1988). Of these variables, screw design and its elements, the moisture

content Of the ingredients prior tO extrusion cooking, and extrusion temperature are

10

all known to drastically affect the quality attributes Of the products (Mercier and
Feillet 1975, Owusu-Ansah et al 1983; 1984, Chinnaswamy and Hanna 1988).

One Of the important characteristics Of extruders is the possibility of high
temperature short time (HTST) cooking (Harper 1978). Smith and Ben-Gera (1980)
described HTST extrusion cooking as the most versatile and most economical
thermal processing system due to the fact that many conversions could be carried
out at lower moisture contents which lowers the drying costs for gelatinized or
texturized products at shorter residence times. In HTST cocking, controlled cooking
conditions are Obtained by maintaining a lower processing temperature during
processing Of the dough and then elevating the product temperature during the last
few seconds Of residence time. Johnston (1979) has discussed the technical
fundamentals Of HTST extrusion cooking equipment, and Smith and Ben-Gera
(1980) have discussed the methodologies Of HTST extrusion cooking.

The following sections will review key extruder design and operating features
which influence the textural and other qualities of the finished extruded products.
The effects Of extrusion on the biochemical components of the raw feed ingredients

will also be considered.

2.2 EXTRUDER DESIGN AND COMPONENTS

There are generally three categories Of extruders, namely, piston extruders,
roller extruders, and screw extruders (T horz 1986). Piston extruders are mainly
used for forming, and consist Of pistons which deposit precise amount Of materials

onto a wide conveyor. These extruders are commonly used in the confectionery

11

industry, for example, to deposit the center fillings of Chocolates. Roller extruders
consist of two counter-rotating drums that turn at similar or different speeds. The
gap between the rolls can be Closed to compress the material passing through the
unit. Screw extruders have single, twin, or multiple screws within a stationary barrel
to push the material forward and through a specially designed orifice called a die.

The Single- and twin-screw extruders are the most commonly used in the
extrusion processing of cereal products. The mechanical actions of both types of
machines are affected by the configuration of the screw and its rotational speed, the
back-pressure requirements of the die, and the characteristics of the food material
being extruded (Harper 1986). Figure 2.1 shows the cross-section of a typical

extruder barrel.

2.2.1 Screw Configuration

The screw is the key element of an extruder because its configuration
influences the unit operation of the extruder (Harper 1978). It consists of a helical
flight wound around a metal shaft enclosed within a cylindrical barrel. Individual
screw elements and paddles may be arranged on a Shaft in the barrel of the
extruder to obtain different extruder screw configurations. Figure 2.2 Shows
schematic diagrams of different types Of screw elements and the permissible
orientation of paddles on the shaft.

Shaft: The shaft serves both as the mounting on which the slip-on agitator
parts are assembled and as a means to transmit the mechanical energy from the
drive to the components where it is converted to work (heat and mixing) on the feed

materials.

12

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13

Figure 2.2

Individual components of extruder screw configuration (from APV Baker, Grand
Rapids, MI).

14

 

 

 

 

 

 

 

 

 

 

 

 

 

(d) Paddle Pairs - Must always be at 90°

Feed Screws: The function of the feed screw components are to convey the
materials downstream within the extruder. The feed screws will tend to push most
materials through paddle sections.

Discharge Screws: The discharge screws have multiple functions made
possible by their design. In addition to conveying the final product from the extruder
against high die exit pressures, they also function as the downstream bearer of the
agitator assembly.

Paddles: Paddles are the primary working components of an agitator
assembly. It is the motion of the paddles relative to each other and the barrel that
causes the cooking of the product and distribution of the ingredients. They provide
very good mixing and generate the friction within the product which converts the
mechanical energy into heat. Paddle pairs must always be oriented 900 from each
other as Shown in Figure 2.2(d).

The changes in molecular conformation of raw or preconditioned food
ingredients, which ultimately affect the texture of extruded products, are primarily
due to extrusion screw design and configuration (Harper 1986). The action of both
single screw and twin-screw extmders is affected by the configuration of the screw
and its rotational speed, among other variables. Mechanical energy dissipation in
the screws causes the temperature of the ingredients to increase rapidly,
transforming the mass from a granular state into a continuous plasticized mass. The
shear in this process is very high and causes mechanical damage to food
molecules. Damage to starch or protein molecules will reduce their capability of
forming an elastic matrix capable of expanding and retaining texture upon passing

through the die (Harper 1986).

17

2.2.2 Extruder Dle

The die effects of extruders can be considered independently from the type
of extruder used to cook materials. Die shape influences finished piece shape and
texture. Harper (1986) has reviewed the effect of die Shapes on extruded product
shape and quality. A tapered die reduces back pressure requirements, creates a
smoother product surface, and causes less mechanical damage to the extruded
ingredients. On the other hand, a die insert having an abrupt cross-sectional change
will cause greater mechanical damage to food ingredients and lead to a finer cell
structure and softer texture.

The laminar-flow shear environment through the die can be characterized by

the Shear rate:

4Ql 4th,
RR 3 rtR 3p

 

where Q,, the volumetric rate of flow through individual die holes, is the mass rate
of flow In divided by density p of cooked material, and R is the radius of the die
hole. Dies having a high Shear rate have a potentially greater effect on product
texture by causing greater Shear-induced damage and reduced molecular size,
creating softer-textured products with smaller pores and increased solubility (Harper
1986). In high temperature extrusion, sudden release of pressure through the die

allows expansion of steam with development of a puffed product (Miller 1994).

18

2.2.3 Single-Screw Extruders

Single-screw extruders may be regarded as friction pumps, since they rely
entirely on friction between the material being processed and the barrel wall to
convey materials (Purvis 1987, Starer 1996). Thus, they rely on drag flow for
conveyance. The mass of material being processed must stick to the barrel wall in
order to be forwarded, and the higher the frictional forces, the more efficient the
conveyance is.

The operational features Of Single-screw food extruders were first described
by Rossen and Miller (1973). The initial feed section of the screw receives and
conveys the food materials into the barrel of the extruder. Decreasing flight height
or other types of restriction cause the flights to become completely filled. The
relationship between the reduced area for flow in later sections of the barrel to that
in the feed section is defined as the compression ratio (Harper 1986).

The screw is the key element of the Single-screw extruder because its
geometry influences the unit operation of the extruder (Harper 1978). It consists of
a helical flight wound around a metal shaft enclosed within a cylindrical barrel. A
number Of different screw configurations may be placed on an extruder shaft, the
purpose being to convey raw materials, knead, create back-flow, input high
mechanical energy turbulence, and increase or decrease residence time of
materials in the extruder (Sunderland 1996). As the material is moved along the

screw in extrusion cooking, it is transformed under conditions of heat, pressure and

mechanical shear into a viscous mass and finally into a product with specific 1

properties. The changes in the product correspond to specific zones of the extruder

screw as Shown in Figure 2.1, namely the feed zone, the kneading zone, and the

19

 

 

 

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final cooking or metering zone (Matson 1982, Hauck 1985).

The feed zone of the screw generally has deep Channels which receive and
mix the feed ingredients, and Slightly compress the feed into a more homogenous
state between the screw flights. Water may be added at this point to help develop
a uniform dough and to improve the heat transfer in the extruder barrel (IFT 1989).
The kneading zone applies compression, mild shear, and thermal energy to the
feed, producing a visco-amorphic mass at or above 100°C (Faubion et al 1982).
The final cooking zone has the shallowest Channel depths and functions to
compress and pump the material in the form of a plasticized mass to the die.

Throughout the extrusion process, heat input to the product comes from
several sources. These include steam injection into the extruder barrel, frictional
heat developed at barrel wall and in the die area during rotation of the screw, or
heat transfer from a steam jacket encasing the barrel (Clark 1978). By the end of
the extrusion process, the thermal processing conditions have produced changes
in the physical properties, chemical properties and/or chemical composition of the

feed ingredients.

2.2.4 Twin-Screw Extruders

Twin-screw extruders consist of two screws of equal length placed inside the
same barrel for its entire length (Martelli 1983). An example of arrangement Of
screw elements in a twin-screw extruder is shown in Figure 2.3. These extruders are
generally categorized according to the position of the screws in relation to one
another and to the direction of screw rotation (IFT 1989). Thus, twin-screw extruders

can be: (a) intenneshing, in which the flights of one screw engage or penetrate the

20

 

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Figure 2.3

Arrangement of screw elements in extruder barrel zones (from Sunderiand
1 996).

21

Channels of the other; (b) non-intermeshing, in which each screw turns without
interference from the other; (c) co-rotating, in which the screws turn in the same
direction with their crests matching up with opposing troughs; or (d) counter-rotating,
in which the screws turn in Opposite directions, as illustrated in Figure 2.4.

The fully interrneshing, CO-rotating twin-screw extruders are being widely
used in the food industry (Harper 1986) because of their ability to provide better
process control and versatility, their flexible design permitting easy Cleaning and
rapid product Changeover, their ability to handle difficult formulations, and their cost
(IFT 1989). In twin-screw extruders, the screws can be configured to enhance
conveying, kneading, Shearing, pressure development, and filling of the barrel.
Conveying sections are usually only filled partially with product and therefore impart
relatively little energy and shear. Kneading elements create a significant Shearing

and mixing action and dissipate large amounts of mechanical energy (Harper 1986).

2.3 RESIDENCE TIME DISTRIBUTION

Residence time is defined as the length of time the process material spends
in the barrel Of the extruder during extrusion. Residence Time Distribution (RTD)
reveals information about flow patterns, degree of mixing, design of equipment,
processing conditions, and retention time of the material in the processing device.
Twin-screw extruders are characterized as having a narrow residence time
distribution.

Although the study of RTD for extrusion processes has received considerable

attention, no generally satisfactory studies of residence time distribution and mixing

22

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 2.4

Classification of twin-screw extruders (from IFT 1989).

23

in interrneshing, co-rotating twin-screw extruders have been done (White 1991).
Jager et al (1988) and van Zullichem et al (1988) have presented a numerical RTD
model to translate the RTD into a mass flow pattern for CO- and counter-rotating

twin-screw extruders.

2.4 EFFECTS OF EXTRUSION VARIABLES ON EXTRUDATE
CHARACTERISTICS

Extrusion variables directly control product quality attributes. Such variables
may include screw speed, the configuration of the screws, temperature in the
extruder barrel or at the die, moisture content of the feed ingredients, and the raw
material formulation and feed rate. These variables, through interactions with
themselves and/or with other variables, act to directly affect the quality
Characteristics of the extruded products which are observed in terms of responses
in the extrudates. Such measured responses include extrudate expansion, bulk
density, water absorption and solubility, texture, color, and several other
measurable responses (Huber 1991 ). This section will review the effects of screw
Speed, barrel/die temperature, and raw ingredients characteristics on some
measured responses which include expansion, bulk density, water absorption and
solubility, and texture of extrudates.

Expansion is usually expressed as a ratio between the diameters Of the
extruded product and the exit die of the extruder, and is called the expansion ratio.
The bulk density of extrudates is the weight Of extruded material that fills a unit

volume Of space. It therefore follows that a well-expanded product with a high

24

expansion ratio will have less bulk density and vice-versa. Water absorption index
(WAI) is the weight of gel Obtained per gram of dry extrudate at room temperature.
Because damaged starch granules absorb water at room temperature and swell,
creating increased viscosity, water absorption index is found to correlate well with
cold-paste viscosity (Anderson et al 1969). After reaching a maximum, related to
the degree of starch damage, WAI decreases with the onset of dextrinization. Water
solubility index (WSI) is the percentage of dry matter recovered from the
evaporation of supernatant Obtained from the WAI determination. WSI depends on
the quantity of soluble molecules which is related to the degradation of starch (Jin
et al 1 995).

2.4.1 Effects of Screw Speed and Configuration

Extruder screw type and configuration can affect the specific mechanical
energy (SME) input to the material being extruded and consequently affect
expansion ratio and other physical properties of the extrudate (Sokhey et al 1994).
The screw Speed has been reported to have Significant effects on extrudate
properties. Jin et al (1995) reported that increasing the screw speed during twin-
screw extrusion produced a lower overall water absorption index (WAI) and higher
overall water solubility index (WSI) in corn meal extrudates. It was also observed
earlier (Jin et al 1994) that increasing the screw speed from 150 to 350 rpm in a
twin-screw extruder increased the product temperature by about 15°C when corn
meal was extruded with soy fiber, salt and sugar. The combined effect of increased
temperature and increased degradation of corn starch due to increased screw

Speed, enhanced the amount of soluble materials and produced increased water

25

solubility of the extrudates. The researchers concluded that screw speed appeared
to be the most important factor influencing the extrusion process variables.

According to Diosady et al (1985), the lower WAI and higher WSI of
extrudates with increasing screw speed might be related to the increased Shear
rate, resulting in the structural modification of starch. At low screw speed (i.e., low
Shear rate) more undamaged polymer chains exist and a greater availability of
hydrophilic groups bind more water molecules resulting in higher values of WAI
(Gomez and Aguilera 1983). In contrast, WSI depends on the quantity of soluble
molecules which is related to the degradation of starch. Wen et al (1990) indicated
that screw speed had a direct effect on polysaccharide size distribution. A higher
screw speed resulted in more fragmentation than a lower screw speed.

Chen et al (1991) investigated the effects of extrusion conditions on sensory
properties of corn meal extrudates and reported that screw speed had no significant
effects on the color of extrudates. However, the surface texture of the extrudates
was affected by the screw speed at low temperatures and high moisture. Decreased
screw speed led to increased surface texture scores because it led to longer
exposure.

The configuration of the screw elements during extrusion is as important as
the speed of the screw. Sokhey et al (1994) studied the effect of screw configuration
on corn starch expansion during single-screw extrusion. Three different extruder
screw configurations were used: one with no mixing element; one with a Single
stage mixing element; and one with a dual stage mixing element. Evaluation of the
bulk density, expansion, and specific mechanical energy values indicated that

expansion ratios and bulk density differences were not significant. However, upon

26

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re-extrusion Of the samples, it was observed that the different screw configurations
Significantly affected all variables studied, except overall expansion ratio.

The relation between extnlder screw Speed and mechanical energy has been
established. Jin et al (1994) reported that increasing the screw Speed in a twin-
screw extmder decreased the extruder torque in part due to Changes in the length
of the filled flights in the extruder barrel. An increase in screw Speed decreased the
length of filled flights at a constant feed rate (Martelli 1983) and thus decreased the
load on the screw shaft motor. Thus the extruder torque was reduced. Additionally,
increased screw speed produced higher product temperature which indicated the
temperature of the dough in the extruder barrel was greater and hence viscosity
was less (Harper et al 1971, Colonna et al 1989). A reduced dough viscosity also
decreased the load on the extruder motor, and therefore the torque. The specific
energy might increase or decrease with increasing screw speed, depending on the
relative contribution of the screw speed and dough mass viscosity (Hsieh et al

1991)

2.4.2 Effects of Temperature
Temperature plays a Significant role on extrusion and product characteristics
(Bhattacharya and Choudhury 1994). During extrusion processing, heat is needed
to texturize the products. Although heat may need to be added, much Of the final
heat energy necessary to achieve high discharge temperatures comes from the
dissipation of the mechanical energy input used to turn the screw (Harper 1986).
The apparent viscosity of the dough in an extruder barrel is affected by

temperature or by residence time distribution (RTD). An empirical model for cooked

27

cereal dough has been derived (Harper et al 1971) as follows:
‘I = C1 ,Yc, ecyr eC‘M

where n is the apparent viscosity, ”y is the shear rate, T is temperature, M is
moisture content, 9 is exponential factor, and C,, CZ, C , and C4 are constants.
Since C3 is positive, at a constant shear rate and moisture content, an increase in
Tdecreases n (Levine 1983). Changes in dough mass viscosity due to variations
in extrusion temperature lead to changes in the load on the extruder motor, and
therefore the torque.

The viscosity of cereal flour dough is also affected by the extent of
gelatinization (Bhattacharya and Choudhury 1994). At a low temperature,
gelatinization of starch is incomplete and shows lower viscosity. However, at the
beginning of gelatinization, increased extrusion temperature increases apparent
viscosity. Apparent viscosity drops again after the completion Of starch gelatinization
with further increases in temperature.

The control of temperature has a profound effect on the condition of the
dough just behind the exit die and on the final expansion of the product. Harper
(1986) reported that the vapor pressure of the water in the dough, which is related
to temperature, provides most of the force which causes expansion once the
product is released to ambient temperature and pressure. Bhattacharya and
Choudhury (1994) studied the effect of extruder Iength-to-diameter ratio and barrel
temperature on extrusion parameters and product characteristics in a twin-screw
extrusion of rice flour. Increases in barrel temperature increased both the extent of

gelatinization and the content Of superheated steam, causing the extrudate to

28

expanc
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extent

values

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expand more and yielding a low density product. Starch gelatinization was
incomplete at low temperatures. Hence, inadequate binding in the product resulted
in low values for maximum breaking force. With increased barrel temperature, the
extent of gelatinization increased which caused better binding, as reflected in higher
values for maximum breaking force.

It has also been demonstrated by several researchers that the expansion of
extruded starch-based materials depended on the degree of gelatinization, which
in tum was determined by process temperature, Shear rate and moisture content of
the feed material (Lawton et al 1972, Chiang and Johnson 1977, Guy and Home

1 988).

2.4.3 Raw Material Characteristics

The composition of the feed raw material into an extruder greatly influences
the texture of the extruded food product (Harper 1986, Jin et al 1995). It is well
established that starch is the predominant functional ingredient in extruded snack
and ready-to-eat (RTE) cereals and other extruded products (Harper 1986, Guy and
Home 1988, Colonna et al 1989). However, defatted soy protein containing about
50% protein, or protein concentrates in which a large fraction of the carbohydrate
has been removed to increase protein concentration, can also be extruded to
produce a layered meat-like structure (Harper 1986). Secondly, in most cases,
degenned cereal grits are used as an extrusion feed material instead of a purified
starch, hence, some protein, fat, and fiber are all part of the extrusion feed mixture.

The following will briefly review the role of starch and protein in extruded products.

29

2.4.3.1 Starch

Carbohydrates are a main constituent in food products, and are generally
used at a 70% level or more (Huber 1991). They are known to give functionality to
extruded products by serving as binding agents, viscosity builders, suspending
agents, and emulsifiers. They also influence textural and esthetic characteristics of
food such as expansion, bulk density, mouth feel, gelation, Clarity, and flavor (Huber
1991). Generally, carbohydrates can be grouped into four categories: fibers,
starches, gums, and sugars. Starches form the bulk Of the carbohydrate component
in extruded cereal products in which they are responsible for adhesion, cohesion,
and expansion.

Several studies have elucidated the role of starch as a feed ingredient during
the extrusion process (Mercier and Feillet 1975, Gomez and Aguilera 1983; Owusu-
Ansah et al 1984; Meuser and van Lengerich 1984). Native starch is reported to
undergo substantial Changes leading to greater molecular disorganization. In the
process, starch losses its crystallinity, undergoes molecular degradation, and often
forms complexes with lipids in the feed mixture. In the case of wheat starch, for
example, the degree of molecular fragmentation is evidenced by decreased
viscosity (Owusu-Ansah et al 1983, Diosady et al 1985), decreased water
absorption index (Mercier and Feillet 1975, Williams et al 1977, Owusu-Ansah et
al 1983), increased amylase susceptibility (Gomez and Aguilera 1984), and
increased degree of gelatinization (Gomez and Aguilera 1984). Gel filtration
chromatography studies on extruded corn and wheat starches (Colonna et al 1984,
Davidson et al 1984) have also shown that starch fragmentation results in a

decrease in high molecular weight materials and a corresponding increase in lower

30

molecular weight polysaccharides.

The roles of shear, temperature, moisture, and feed composition Significantly
affect the starch transformation process. Gomez and Aguilera (1984) described a
process in which Shear causes mechanical damage to the starch while heat and
moisture are responsible for the loss in crystallinity. Lower moisture content causes
increased viscosity and more mechanical damage. Davidson et al (1984) have
reported that the bulkiness of the amylopectin fraction of starch prevents it from
aligning itself in the direction of flow in the screw and exit die, resulting in greater
mechanical damage and reduced molecular size. These damaged starches are less
cohesive than gelatinized undamaged starch and therefore expand less in the
longitudinal direction, creating products with smaller pores, softer textures, greater
solubility, and a sticky Character.

Amylose is less susceptible than amylopectin to mechanical damage in the
flow environment within the extruder. Thus, high amylose products are denser,
harder, and less radially expanded when extruded (Harper 1986). Mercier (1980)
has also shown that amylose forms a complex with lipids during the extrusion
process which helps maintain the native structure of amylase. However, the
formation of amylose-lipid complexes reduces the digestibility and water solubility
of cooked starches (Galloway et al 1989), and as starch in the presence of lipids is
exposed to increasing heat and pressure during extrusion, the amount of amylose-

lipid complexing increases.

31

2.4.3.2 Protein

Plant protein, particularly from soy meal protein, is the only commercially
important protein material used in extrusion. However, interest has been shown in
the improvement of utilization of both dairy (van de Voort 1984) and animal (Areas
1986) proteins through extrusion processing. Thermal extrusion of proteinaceous
ingredients is a relatively recent industrial process. The first patent for extrusion
processing of plant protein was issued in 1966 (Linko 1983).

Defatted soy protein can be extruded to create a layered meat-like structure
commonly called meat analogs or extenders. It has been Shown that the globular
protein contained in defatted soy meal is not homogeneously distributed, but exists
as nearly spherical structures known as protein bodies (Kinsella 1978, Bair and
Snyder 1980, Daniels 1981). In the extrusion process, the protein molecules are
restructured into a layered cross-linked mass which is further heated and sheared
in the extrusion screw. The proteins unfold upon the disruption of the ionic, disulfide,
and hydrogen bonds which make up the native tertiary structure of the protein
molecule (Ramsen and Clark 1978). The results are denaturation, association, and
coagulation involving reduction or oxidation. Extrusion screws which turn rapidly, or
are configured to increase residence time, will have the most disruptive effect on the
molecules’ native structures. These effects include the denaturation of the protein
and a loss of functionality which may lead to reduced solubility and decreased
extrusion effectiveness (Harper 1981, 1986).

The quality characteristics of extruded products have been related to the
structural integrity of the protein matrix. Proteins in expanded products are important

for elasticity, gas retention and cellular structure, adhesiveness, stretchability,

32

extensibility, water absorption, binding and expansion. Kazemzadeh et al (1982)
examined extrudates using differential staining light microscopy which revealed
carbohydrate inclusions and steam-generated voids enclosed within a protein
matrix. Frazier and Crawshaw (1984) also associated well-textured soy with a
smooth, homogenous protein matrix containing small, evenly distributed, insoluble
carbohydrate inclusions.

Several studies have revealed the major role of protein during extrusion
texturization. Sheard et al (1985), for example, found that an extrudate from soy
isolate containing 91% protein had a larger diameter and higher Shear values than
one from soy flour containing 50% protein, although these data are in contrast to
previously reported data (Sheard et al 1984), and the data reported in the current
study. Increasing protein levels significantly improved rheological properties Of
extruded soy, according to Rhee et al 1981. Maurice and Stanley (1978) used three
levels of protein, through the addition of soy isolate, to Show that protein level and
second-order protein effects accounted for most of the variation in product shear
values.

A number of proteinaceous materials have been used to fortify ready-to-eat
breakfast cereals to improve their protein content and its quality. Cereal grains are
moderately low in protein and the protein is of poor biological quality because the
essential amino acid, lysine, is present in limited amounts (Harper 1981). In order
to produce nutritionally improved cereal, therefore, some cereals are fortified with
protein, commonly in the form of soy protein concentrate and sodium caseinate. It
has been reported that the addition of 10 to 15% of these ingredients will increase

the protein content Of the entire blend by 12 to 20% (Harper 1981). However, the

33

addition of high protein ingredients tends to reduce the puffing of the extruded
product and also results in the dissipation of more mechanical energy in the
extrusion process and higher back pressures at the exit die of the extruder.

Vital gluten is the gluten protein of wheat flour Obtained after the starch and
soluble components have been removed by a washing process (Wang and Ponte
1995). It contains about 80% protein on a dry basis. The unique viscoelastic,
adhesive, and cohesive properties of vital gluten improve dough strength, mixing
tolerance, and handling properties, and also permit its use as an ingredient in meat,
fish, and poultry products (Czuchajowska and Smolinski 1997). Its water-absorption
capacity improves baked product yield, softness, and Shelf-life. Even though vital
gluten is commonly used in foods to improve their handling properties and to modify
their texture, its use as a protein source for extrusion processing has not been
explored.

The effect of gluten on texture has been mostly studied in relation to baked
products Of flour fortified by gluten. Dreese et al (1988a,b) tested the rheological
properties of doughs made from blends of commercial gluten and starch by
measuring the rheological properties as affected by moisture, starch content, and
heating rate. In a recent study (Wang and Ponte 1994), the baking Characteristics
and freeze-thaw tolerance of frozen dough were found to be improved significantly
by the addition of vital wheat gluten to relatively weak flour. The study showed that
the beneficial effect provided by vital wheat gluten was due to the increase in
dough strength that could overcome weakening effects caused by freezing.

In view of the success in the application of extrusion processing to produce

meat analogs from soy protein sources, and given the fact that vital wheat gluten

34

is in abundant supply as a source of cereal protein, it would be beneficial to study
the role of vital wheat gluten as a source of protein in the extrusion texturization of

cereal products.

2.4.4 Effects of Moisture Content of Raw Materials

The moisture content of raw feed ingredients going into an extruder as well
as any amount of liquid added to ingredients during the extrusion process directly
affects the viscosity of the dough in the extruder barrel and therefore the energy
requirements of the process. Bhattacharya and Hanna (1987) have concluded that
any variable affecting the dough mass viscosity would also affect the extruder
torque.

Water inside the extruder exists as superheated steam due to high
temperature and pressure (Park et al 1993). When the extrudate exits the die, water
vaporizes until the water temperature within the product reaches 100°C (Williams
et al 1977). The moisture remaining in the product depends on the pressure
differential between the extruder and the atmosphere (Conway 1971a). According
to Park et al (1993), it is difficult to generalize the effect of feed moisture on the
texture of extrudates. However, Lawton et al (1972) have shown that water in the
extruder acts as a heat trap and lubricant to reduce the Shear strength of
extrudates. A low feed moisture can increase the power requirements of the
extruder and can also result in excessive browning and stoppage of extrudate at the
exit die nozzle opening (Conway 1971 b).

Starch is the predominant ingredient in the extrusion of cereals, and the

gelatinization of starch is a necessary condition for the expansion of cereal

35

extrudates. It is well documented that water content in combination with temperature
has a significant effect on the conversion of starch. This is seen from the fact that
the transition temperature as well as the enthalpy of transition in differential
scanning calorimetry (DSC) increases with decreasing water concentration for a
given starch type (Lai and Kokini 1991). In a DSC therrnogram of potato starch,
Donovan (1979) observed a single peak when the moisture content was 81%.
When moisture content was dropped to 64%, a second endotherm was observed
at a higher temperature, and became predominant when water concentration was
even lower than 51%. The responses at low water concentrations have been
attributed to melting of crystallites. Under excess water all the crystallites in starch
might be pulled apart by swelling, leaving none to be melted at higher temperatures.
However, in the limited water environment characteristic of extrusion processing,
the swelling forces are much less Significant, and the crystallites melt at a
temperature much higher than the gelatinization temperature in excess water
(Donovan 1979).

In a study to investigate the effects of raw material composition and process
temperature on physical and rheological properties of high protein extrudates, Park
et al (1993) found that feed moisture had a significant linear effect on extrudate
shear force and water absorption, and a quadratic effect on expansion ratio and
bulk density. It was concluded that an insufficient amount of water available for
evaporation appeared to be the main cause of low expansion ratio and high bulk

density.

36

2.5 BIOCHEMICAL CHANGES IN PROTEINS DURING EXTRUSION

Studies elucidating the biochemical changes that occur in proteins during
extrusion are limited (Strecker et al 1995). Therefore, understanding the chemical
and physical Changes that proteins undergo during extrusion conditions of high
temperature, pressure and Shear, and low moisture iS limited (Ledward and Mitchell
1988). Nevertheless, it has been established that during the extrusion process,
proteins become denatured and the forces that stabilize the tertiary and quaternary
structures of the proteins are weakened by a combination of increased heat and
Shear within the extruder (Camire 1991). The result is that the solubility of the
proteins decreases as a result of extrusion processing.

Racicot et al (1981) reported a significant decrease in the solubility of corn
meal protein in ethanol and alkali after extrusion. Cumming et al (1973) had earlier
suggested that the exposure to heat and pressure during extrusion affects free
sulfhydryl groups and disulfide bonds in soy protein, which results in lowering the
solubility of the protein. Their study also observed that the solubility of water-soluble
proteins, which broke into subunits of smaller molecular weights during the
extrusion process, also decreased, and that the degree of insolubilizaticn was
different in different proteins according to gel electrophoresis analyses.

Wheat proteins can generally be fractionated into four groups based on the
original classification by Osborne (1907). Albumins are the fractions soluble in
water, globulins are soluble in salt solutions; gliadins are soluble in aqueous alcohol;
and the glutenin fractions are soluble in dilute acid or alkali. Chen and Bushuk

(1970) later modified this classification to include insoluble glutenins. Recently,

37

Ummadi et al (1995) investigated the Changes in solubility and distribution Of
semolina proteins due to extrusion processing at two different temperatures and
observed a marked decrease in the percentage of total protein present in the
albumin, globulin, gliadin, and glutenin fractions with a corresponding increase in
the insoluble residue fraction. It was suggested that polypeptides of albumins,
globulins, and glutenins of semolina most likely aggregated after extrusion, leading
to decreases in solubility of these proteins. In contrast, molecular weight distribution
of gliadin proteins seemed to have been unaffected by extrusion at the experimental

temperatures used in the study.

2.6 EFFECTS OF HEAT AND CHEMICAL MODIFICATIONS ON CEREAL
PROTEIN COMPOSITION

Native protein structure can be described on four levels. The primary
structure, which is indicated by the amino acid sequence Of the protein, determines
the chemical and biological properties of the protein; the secondary structure refers
to the regular arrangement Of the polypeptide backbone of which or-helix and 8-
sheet structures are examples; tertiary structure refers to the three dimensional
structure of the protein molecule; and the quaternary structure is the assembly of
individual protein molecules to form a functional protein aggregate (T atham et al
1990). A combination of hydrogen bonds, hydrophobic interactions, electrostatic
forces, and disulfide bonds act together to stabilize these protein structures (T atham
et al 1990).

Hydrophobic and hydrophilic interactions play an important role in the

38

structure-function relationships of proteins (Weegels et al 1994). Generally, the
surface hydrophobicities of proteins are increased by heating (Tanford 1980,
Matsudomi et al 1982, Voutsinas et al 1983), which can be explained by an
increased exposure of hydrophobic core groups at the outer surface of the molecule
(Tanford 1980). Surface hydrophobicity is generally determined by measuring the
interaction of the protein with hydrophobic fluorescent ligands, e.g., 1-anilinO-8—
naphthalenesulfonic acid (ANS). Since surface hydrophobicity is related to protein
structures, conformations and functionality (Aktan and Khan 1992), fluorescence
may be able to reveal denatured or structurally altered proteins.

Proteins also possess native fluorescence due to the presence of the
aromatic amino acids tryptophan, tyrosine and phenylalanine; however, the
fluorescence contributions of tyrosine and phenylalanine are low. The fluorescence
properties of these amino acid residues reflect the environment of the protein and
can, therefore, be used to indicate changes in conformation and/or stability of the
protein (Yeboah et al 1994).

Studies on the effects of extrusion processing on native protein
conformations, especially with respect to wheat storage proteins, are limited.
Weegels et al (1994) reported a study on the changes in the extractability of
glutenin aggregates differing, among other things, in secondary structures of
glutenins, gliadins, albumins and globulins after heating of gluten at different
moisture contents. The hydrophobicity of the heated gluten was determined by
interaction Of the protein with ANS. The study Observed that the number of binding
sites of ANS decreased when gluten with a moisture content of 13% or higher was

heated, indicating a decrease in hydrophobicity. Similarly, Yeboah et al (1994) used

39

fluorescence Spectroscopy to study the conformation and dynamics of two y-gliadin
fractions.

Lakkis and Wlota (1992) studied the effect of succinylation, acetylation, and
reductive alkylation on substructural properties of casein, bovine serum albumin
(BSA), and whey proteins. Hydrophobicities of the native and modified proteins
were determined by using ANS to react with the proteins and measuring the
fluorescence intensities. Data from the study revealed that surface hydrophobicity
of the three proteins increased in parallel with the degree of Chemical modification,
indicating a Change in the conformational structure of the proteins and a subsequent
exposure of their hydrophobic core to the ANS probe.

It is evident from the foregoing that fluorescence spectroscopy has been
extensively used to study Changes in conformation of protein structure, yet such
studies have not been adequately applied to the conformational changes in cereal
proteins as a result of extrusion. In view of the possible applications of extrusion to
both existing and future food products, it will be necessary to understand the
changes in the conformation of proteins and other ingredients in order to better
understand the relationship between the extrusion process and end-product
Characteristics.

2.7 THERMAL TRANSITIONS IN WHEAT FLOUR COMPONENTS AS
MEASURED BY DIFFERENTIAL SCANNING CALORIMETRY

Wheat flour is a multi component system containing about 80% starch (by

dry weight), proteins, lipids and other minor components. The protein content may

vary from 9-18%, depending on the wheat variety. It has been found that the

40

quantity of the protein component may be too small to influence the phase transition
properties of the whole system (Brent et al 1997). Thus, it may not be possible to
measure the thermal properties for the protein component of the system. Secondly,
on heating in water, protein denaturation reactions may occur at temperatures very
close to the range associated with starch gelatinization. Wheat flour samples
examined by DSC may therefore Show an endothermic peak due to protein
denaturation superimposed on the starch gelatinization peak (Stevens and Elton
1971). Even the major wheat proteins - gliadins and glutenins - have been reported
to Show little or no calorimetric response upon heating (Eliasson 1983, Ma 1990),
and vital gluten containing 77.3% protein did not exhibit any obvious thennogram
(Arntfield and Murray 1981). The DSC therrnogram of vital gluten does not Show
any definite denaturation peak, indicating that there is no ordered structure in the
gluten proteins (Hoseney 1994).

In view of the foregoing, therefore, the main effect of the presence of protein
on wheat starch endotherms may be merely that of a diluent. It would therefore
seem permissible to measure the endotherm of a flour sample and then assign Tm
and AH values to starch (Stevens and Elton 1971).

Two thermally competing processes that can occur in starch DSC are
endothermic melting of starch crystallites and exothermic formation of amylose-lipid
complexes (Biliaderis 1990). The presence and disorganization of amylase-lipid
complexes in starch systems have been reported to be responsible for the high
temperature endotherms of 100-140°C Observed in starch, which is well above the
melting endotherm of starch crystallites (Kugimiya et al 1980, Donovan et al 1983,

Biliaderis et al 1986). Bae and Lim (1998) have reported that transition

41

temperatures in the range of 85-125°C found during thermal analysis of extruded
normal and high-amylase corn starch is typical for a lipid complex with amylose.
Wheat starch is reported to contain about 1.12% lipid (Acker and Schmitz 1967),
and Mercier (1980) has also Shown that amylose forms a complex with lipids during

the extrusion process which helps maintain the native structure of amylose.

2.8 ROLE OF DISULFIDE BONDS IN EXTRUSION TEXTURIZATION

Protein texturization involves a restructuring of the protein molecules into a
layered cross-linked mass which is resistant to disruption (Harper 1986). Different
researchers have attributed the stability of the structure of extrudates to different
forces; thus the subject of structural stabilizing forces in extrudates is a subject of
debate.

Cumming et al (1973) suggested that exposure to heat and pressure during
extrusion texturization of soy proteins affected free sulfhydryl groups and disulfide
bonds in the soy protein, which resulted in insolubilizaticn of the protein. Recently,
Li and Lee (1994) studied the effect of extrusion temperature on quantitative
Changes in extractable protein, disulfide bonds and sulfliydryl groups in wheat flour
and a mixture of wheat flour and egg white. Three solvents [1% sodium dodecyl
sulfate (SDS), 1% 2-mercaptoethanol (2-ME), and 1% SDS+2-ME] were used to
investigate the types of bonding forces of protein interactions in extrudates. Sodium
dodecyl sulfate can disrupt the hydrogen bonds and the hydrophobic interactions,
and 2-ME can reduce the disulfide bonds to sulfl'Iydryl groups. Their results Showed

a synergistic effect between $08 and 2-ME for protein solubility in all extrudates.

42

The disulfide and sulfhydryl group contents in the extracts of extrudates Changed
with the various extrusion temperatures. This indicated that the Changes in solubility
of protein in the extrudates were due to wheat proteins and/or egg protein
aggregated by disulfide bonds and to hydrophobic interactions that occurred during
the extrusion process (Li and Lee 1994). Disulfide bond formation has also been
suggested as being responsible for reduced protein solubility of extrusion-texturized
legume flours (Pham and Del Rosario 1984).

Burgess and Stanley (1976), in contrast, suggested that disulflde bonds
played only a minor role in extrusion texturization and proposed that intermolecular
amide bonds are responsible for texture formation in extruded products. However,
Hager (1984) and Neumann et al (1984) later showed that the intermolecular
disulfide bonding in soy is important and found no evidence for appreciable
intermolecular peptide bond formation. Hager (1984) used different solvents to
study the non-covalent forces that produced insolubility in extruded soy concentrate
and concluded that at extrusion temperatures below 150°C, the formation of new
disulfide bridges as well as changes in hydrogen bonding dominate in the protein
conformation, but at higher temperatures intermolecular peptide bonds may also be
formed.

2.9 EFFECTS OF EXTRUSION ON DISULFIDE AND SULFHYDRYL
CONTENTS OF CEREAL PROTEINS

Sulfhydryl and disulfide groups are major contributors to the stability of

native conformations of proteins (T hannhauser et al 1987) and are thus important

in maintaining structure and functional properties of native proteins (Synowiecki and

43

Shahidi 1991). They are also thought to play an important role in the texture of
cereal-based products (Chan and Wasserrnan 1993). The determination of the
number of disulfide bonds per molecule is therefore crucial to structural studies of
proteins (T hannhauser et al 1987).

Several studies have attempted to relate the disulfide and suIflTydryl contents
of extrudates to some functionality of the extrudates. The exposure to heat and
pressure during extrusion, for example, has been Shown by Cumming et al (1973)
to affect free sulfhydryl groups and disulfide bonds in soy protein, resulting in
reduced solubility of the proteins. Hager (1984) and Neumann (1984) have also
shown the importance of inter-molecular disulfide bonds in soy proteins to the extent
that they contribute to the new and extended protein networks produced by
extrusion of soy concentrate. Ummadi et al (1995) reported differences between
SDS-PAGE patterns of reduced and non-reduced globulin fractions from raw and
extruded semolina and attributed these differences to the role played by disulflde
bonds in the extruded products.

Gluten, the storage protein of wheat which consists of gliadins and glutenins,
exhibits decreased extractability when heated to 75-80°C (Booth et al 1980,
Schofield et al 1983). This decreased extractability has been attributed to a
decrease in extractability of the glutenin fraction (Booth et al 1980, Schofleld et al
1983) which is accompanied by a disulfide-sulfliydryl interchange (Schofield et al
1983). Similariy, disulfide-sulfliydryl interchange has also resulted in the decreased
extractability of heated gliadins (Booth et al 1980, Wrigley et al 1980, Feillet et al
1989).

The interchanges occurring between disulfide and sulfliydryl contents of

44

cereal proteins during extrusion processing have been related to thermal
polymerization of gluten proteins. Strecker et al (1995) evaluated the polymerization
and mechanical degradation kinetics of wheat gluten and glutenin during extrusion
by analyzing protein solubility and disulfide bond content. Soluble protein and
disulfide bond concentrations Of gluten and glutenin extrudates were used as an
index of extent of reaction. The results of the study showed the disappearance of
soluble protein and the formation of disulfide bonds. The authors concluded that the
reaction mechanism in gluten during extrusion was dominated by polymerization
reactions contributed mainly by the formation of disulfide bonds. Schofield et al
(1983), after analyzing the effects Of heating on sulfliydryI-disulfide interchange
reactions, concluded that the formation of protein network upon therrnosetting was
primarily due to interchanges between sulflnydryl-disulfide bonds. Several
researchers (Beckwith and Wall 1966, Graveland et al 1978, Kaczkowski and
Mieleszko 1980) have supported the disulfide bond hypothesis.

Chan and Wasserrnan (1993) also studied the effect of twin-screw extrusion
on the free sulfhydryl and disulfide contents of corn meal and reported that
extrusion resulted in an increase in free sulfhydryl content and a decrease in
disulfide groups, suggesting an extrusion-induced rupturing Of disulfide bonds. In
an experiment to study extrusion effects on sulfhydryl groups of extruded wheat
flour proteins, Koh et al (1996) added cysteine to hard red winter wheat flour. After
extrusion, there was a marked increase in protein sulfl'Iydryl content and a decrease
in protein disulfide content. According to Koh et al (1996), this increase in protein
sulfl'IydryI content is a marker for sulfl'IydryI-disulfide interchange reactions during

extrusion. Physical and chemical properties of the wheat flour were also markedly

45

affected by the cysteine addition. For example, the expansion ratio of samples
decreased with increasing levels of cysteine. Cysteine addition also resulted in
smaller cells with finer and more even structure (Koh et al 1996).

The foregoing review of the literature highlights some aspects of progress
made in extrusion processing, especially as it relates to the extrusion processing of
cereals. Several conclusions have been drawn by the published literature, stating
that Changes in physicochemical properties of cereal proteins as a result of twin-
screw extrusion have not been adequately documented in spite of the commercial
success of extrusion processing. Research in this area will therefore be helpful to
the improvement of existing extruded products as well as in the design of new ones.

The present study was designed to seek further understanding with regard
to physicochemical changes in wheat flour, with an emphasis on protein, during

twin-screw extrusion.

46

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RAMSEN, CH and CLARK, JP. 1978. A viscosity model for a cooking dough. J.
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CHAPTER 3
THE INFLUENCE OF TWIN-SCREW EXTRUSION PROCESSING ON

PHYSICAL AND MICROSTRUCTURAL CHARACTERISTICS OF WHEAT
FLOUR EXTRUDATES

57

 

3.1 ABSTRACT

This study was conducted to assess the effects of extrusion processing
parameters on the extmdate quality of wheat flour (at 9% protein content) and two
wheat flour blends. Blends were made by mixing vital gluten containing 70% protein
with the wheat flour (9% protein) to Obtain 20 and 30% protein levels. Samples
were extruded in a twin-screw extruder with full factorial combinations of die exit
temperature of 120, 140, and 160°C; screw speed of 240, 320, and 400 rpm; and
the three protein content levels of 9, 20, and 30%. Protein level of raw feed material
significantly affected expansion ratio, bulk density, water absorption index, and
specific mechanical energy. Extruder exit die temperature also affected bulk density
and specific mechanical energy Significantly. Overall, the linear effects Of extrusion
variables on expansion ratio and bulk density were not as pronounced (R2 = 0.15,
0.44, respectively) as the effect on water absorption index (WAI) (R2 = 0.86), and
specific mechanical energy (R2 = 0.94). Correlation data showed that screw speed
and specific mechanical energy were positively correlated (r = 0.95, ps 0.001 ), while
die temperature was negatively correlated with bulk density (r = -0.38, ps 0.01 ).
Protein content of feed negatively correlated with expansion ratio, water absorption
index, and specific mechanical energy (SME), and positively with bulk density, while
an inverse relationship existed between expansion ratio and bulk density. The
protein content level of feed material was the only factor which Significantly affected

all extrusion dependent variables.

58

 

3.2 INTRODUCTION

Extrusion technology has had wide applications in the food industry for the
manufacture of a wide range of snack foods, ready-to-eat cereals and other
breakfast cereals using an energy-efficient, rapid and continuous system with
numerous ingredients and processing conditions (Harper 1981). Texture is very
important as a quality parameter in extruded products. Stanley (1986) has
suggested that the texture of a food results from microstructure, which depends on
the influence of physical forces on chemical components.

The texture and mouthfeel of most extruded snack foods depend on their
expansion volume (Owusu-Ansah et al 1984), and several studies have reported
the role of extrusion processing variables on the expansion volume Of cereals
(Owusu-Ansah et al 1983, Meuser et al 1982, Chinnaswamy and Hanna 1988). It
has been Shown that among the several extrusion variables that affect extrudate
properties, the barrel temperature and moisture content of the raw feed material
most prominently control expansion volume and hence texture Of extrudates
(Mercier and Feillet 1975, Owusu-Ansah et al 1983,1984, Chinnaswamy and Hanna
1988).

In recent years, extrusion research has focused on how changes in extrusion
variables such as extruder screw Speed, feed moisture content, extrusion
temperature, etc., affect the texture, microstructure, gelatinization (Case et al 1992),
and other physico-chemical properties of the extruded products. Most related
research has been concerned with optimization of conditions, and effect of changes

on physical characteristics and microstructure of extrudate (Czamecki et al 1993).

59

 

Chauhan and Bains (1988) investigated the effect of some extruder variables on the
physico-Chemical properties of extruded rice-legume blends and reported that
expansion ratio of extruded products increased with increase in extruder exit
temperature while bulk density decreased.

The texture of extruded food products is greatly influenced by the
composition of the extrusion feed material. Jin et al (1995) studied the effects of soy
fiber, salt, sugar, and screw speed on physical properties and microstructure of com
meal extrudates. The findings Of the research indicated that increasing screw speed
lowered water absorption index. Studies on soybean flour/Oil and wheat bran effects
on characteristics of cassava (Manihot esculenta Crantz) flour extrudates by Badrie
and MelloweS (1992) showed that the crude protein content of extrudate correlated
negatively with expansion and water solubility while the correlation with bulk density
and water absorption was positive. Other studies have also reported that the
qualities of raw materials such as contents of protein, lipid, and starch and their
composition and type are also important in controlling physical properties of the
extruded products (Faubion et al 1982, Chinnaswamy and Hanna 1988).

The effect of higher protein content of wheat flour on the physical
Characteristics of extrudates is not well documented. Proteins from different
sources, varying in composition, molecular weight and structure would presumably
affect the product upon texturization (Czamecki et al 1993). This study was
therefore designed to assess the effects of different levels of protein content and
various conditions of extrusion processing (screw Speed and exit die temperature)

on some physical characteristics of wheat flour extrudates.

6O

 

3.3 MATERIALS AND METHODS

Whole soft white wheat grain (cv. Harus) produced in Michigan in the 1994
crop year was milled to Obtain straight grade flour with a 67% extraction rate and
9% protein content (14% moisture basis) on a Buhler MLU-202 Automatic Mill
(Buhler AG, Uzwil, Switzeriand). The wheat grain was tempered overnight to 14%
moisture prior to milling. \fital gluten (70% protein, 14% moisture basis) was
obtained from Midwest Grain Products (Atkinson, KS). Properties of wheat flour and
vital gluten are given in Appendix 1.

Appropriate amounts of vital gluten were added to the wheat flour to obtain
flour-gluten samples containing 20% or 30% protein. Moisture and Kjeldahl nitrogen
contents of wheat flour, vital gluten and flour—gluten samples were determined
according to AACC (1983) Approved Methods 44—19 and 46-11A, respectively.

Nitrogen was converted to percent protein by using a factor of 5.75.

3.3.1 Extrusion Conditions

Each sample was extruded in duplicate on an APV Baker MP19TC-25 CO-
rotating and intenneshing twin screw extruder (APV Baker, Grand Rapids, MI), with
barrel diameter of 19 mm and length-to-diameter (LID) ratio of 25:1. The extruder
barrel is divided into five zones including the die, and each zone is equipped with
a temperature controller which reads the actual temperature of the barrel via a
thermocouple. Discharge melt pressure developed by the screws just prior to
entering the exit die is monitored with a transducer, and indicators are provided to

monitor feeder speeds, screw speeds, pressure, barrel temperature of each zone,

61

 

and torque. An exit die drilled with a tapered hole of 2 mm in diameter was used in
the experiment. The screw configuration used in the extrusion experiment is Shown
in Table 3.1. This screw configuration was shown in preliminary experiments to
produce expanded cereal products.

Samples were fed into the extruder with a K-Tron K2M twin-screw volumetric
feeder (K—Tron Corp., Pitman, NJ) equipped with safety hopper, variable speed
drive and digital control. The feed rate was maintained constant throughout
experimentation at 2 kg per hr. Deionized water was injected with an E2 Metripump
positive displacement metering pump (Bran & Luebbe, Northampton, UK.) at the
rate of 0.21 kg per hrto Obtain a constant feed ingredient moisture of 19%.

A full factorial arrangement with two replications was used. Extrusion
variables were: (1) screw speed (240, 320, and 400 rpm), (2) extruder exit die
temperature (120, 140, and 160°C), and (3) feed sample protein content (9, 20 and
30%). Temperatures at the four extruder barrel zones preceding the exit die were
maintained constant at 40, 60, 80, and 100°C, respectively.

The extruder was run for at least 5 min after attaining a constant torque and
pressure. Percent torque readings were taken, and the extrudates were collected
for about a 5 min time period. After extrusion, samples were dried in a convection
fan oven at 40°C to approximately 8% moisture and cooled to room temperature.
Samples requiring grinding for subsequent analyses were ground in a Udy Cyclone
Sample Mill (Udy Corp., Fort Collins, CO) through a 0.5 mm screen. Samples were

then stored in airtight plastic freezer bags at 4°C till further analysis.

62

3.3.2 Detenninatlon of Physical Characteristics of Extrudates

3.3.2.1 Expansion ratio and bulk density

The expansion ratio of dried extrudates was determined as the cross-
sectional diameter of the extrudate divided by the diameter of the die opening of the
extruder (Harper 1981). The diameter of the die used in this study was 2.0 mm.
Data for each sample was Obtained by averaging caliper measurements from 5
random samples with five observations on each sample. Bulk density was
determined according to the method of Park et al (1993). Pieces of extrudates,
about 1 cm in length, were placed in a 250 ml graduated cylinder. The bottom of the
cylinder was then repeatedly tapped gently on a laboratory bench until there was
no further reduction in sample volume. Bulk density was calculated as the weight

of sample per unit volume (g/ml).

3.3.2.2 Water absorption index and specific mechanical energy

The water absorption index (WAI) is the weight of gel Obtained per gram of
dry ground sample at room temperature, and was determined according to AACC
method 56-20 (AACC 1983). The specific mechanical energy (SME), defined as the
mechanical energy input required to Obtain 1 kg of extrudate (Bhattacharya and

Choudhury 1994), was calculated using the formula according to Brent et al 1997:

SME= rpm (test) x % torque x horsepower
rpm (rated) 100 feed rate

 

 

where rpm (test) is the screw speed for a particular test, rpm (rated) is the maximum

63

 

speed of the extruder shaft, horsepower is the extruder drive power input. The
extruder drive power input and the maximum screw speed of the extruder Shaft
were provided by the manufacturer as being 2 kW and 500 rpm, respectively. The

% torque of each experimental run was read directly from the controller unit.

3.3.2.3 Microstructure of extrudates

Microstructure of extrudates was studied using a Zeiss 10 Laser Confocal
Microscope (Carl Zeiss, InC., Thomwood, NY.) equipped with digital image
acquisition capabilities. About 1 mm cross-section of a dried extrudate was cut and
placed directly on the microscope slide. The Slide was then placed on the viewing
platform of the microscope and directly viewed through the Objective lens. Images
were acquired in the transmission, non-confocal mode. After acquisition, images

were processed using the Adobe Photoshop Version 4.0 software.

3.3.3 Statistical Analysis
Data was analyzed using the General Linear Models (GLM) procedure of
SAS Version 6.0 (SAS Institute, Cary, NC 1989). Pearson’s correlation coefficient

and LSMeans procedures were used to analyze relationships among variables.

3.4 RESULTS AND DISCUSSION

3.4.1 Expansion Ratio and Bulk Denslty

The expansion ratio of extrudate was negatively affected by increasing
extruder die temperature and screw Speed (Figure 3.1a), and protein content
(Figure 3.1b) of raw feed material. Analysis of variance (Appendix 3) shows that the
main effects as well as the interaction effects were all Significant (ps0.001).

The decreasing effect of protein content on expansion ratio has also been
noted by Badrie and Mellowes (1992) who observed that increasing the crude
protein content in the extrusion of cassava (Manihot esculenta Crantz) by adding
soybean flour decreased expansion of extrudate. Park et al (1993) also noted that
increasing protein level in the feed for the extrusion of soy flour, corn starch and raw
beef blends decreased the expansion ratio of extrudates. ln work done by Faubion
et al (1982), it was shown that adding wheat gluten to wheat starch in extrusion
cooking caused a reduction in expansion.

One of the possible explanations of such observations is that the addition of
protein material reduced the percentage Of total carbohydrate available which
ultimately led to reduced water absorption (Badrie and Mellowes 1992). A reduction
in the ability to absorb water inhibits the propensity to expand when the extrudate
exits the die to the atmosphere. A second potential explanation is that one or more
non-starch components were inhibitory to expansion (Badrie and Mellowes 1992).
Eliasson (1983) had earlier observed that the lack of protein in raw extruding
materials can make starch gelatinization easier because there is no material to

compete with starch for water absorption. Proteins, lipids, bran and other

65

 

components in starch may also act as a heat sink, absorbing the heat that is
required to gelatinize the starch as well as limiting the water available for
gelatinization (Case et al 1992). These components, therefore, protect the starch
and result in a lower extent of gelatinization and lower expansion.

The overall negative effect of die temperature on expansion ratio Observed
in this study is in contrast to some earlier reports in the literature. Bhattacharya and
Choudhury (1994) observed that product expansion increased markedly with
increasing barrel temperature when rice flour was extruded in a twin-screw extruder.
Bhattacharya and Hanna (1987) had also noted previously that as barrel
temperature was increased during extrusion of corn starch products, there was
more expansion and reduced density of extrudates. In an earlier study on the
effects of starch gelatinization on physical properties of extruded wheat and corn
products, Case et al (1992) noted that products extruded at lower temperatures
were thin and that expansion was greatest at highest extrusion temperatures which
also corresponded with higher gelatinization.

However, in a single-screw extrusion of defatted soy flour, corn starch and
raw beef blends, Park et al (1993) reported that increasing the process temperature
increased expansion ratio up to a point and decreased thereafter with further
increases in temperature. It is possible that at high temperatures, such as used in
the present study, excessive starch damage occurred. Damaged starch granules
could be more easily penetrated by water thereby decreasing the temperature and
degree of starch gelatinization (Chang and Lii 1992). The reduced ability of starch
to gelatinize reduces its ability to expand.

Increasing screw Speed had overall negative effect on expansion ratio at all

66

 

protein levels. Even though the effects were not significant (p>0.05), the
combinations of lower die temperature and lower screw Speed (Figure 3.1a), and
lower protein level and lower screw speed (Figure 3.1b), appeared to have
produced well-expanded products with higher expansion ratios in extrudates.

The decreasing effect of screw speed on expansion ratio of extrudates
indicates that increasing the screw speed increased the shear rate of raw materials
and hence produced more damage to molecules. Jin et al (1995) have reported that
damaged starches are less cohesive than gelatinized undamaged starches and
therefore expand less and create products with smaller pores. Several researchers
have demonstrated that the expansion of extruded cereals or starch-based
materials depended on the degree Of gelatinization, which in turn was determined
by process temperature, shear rate, and moisture content Of the feed material
(Lawton et al 1972, Chiang and Johnson 1977, Guy and Home 1988).

Increasing temperature at the extruder exit die had an overall negative effect
on bulk density, while an increasing protein content of feed ingredient positively
correlated with the bulk density of extruded samples (Figure 3.2, Table 3.2).
Overall, exthdateS with highest bulk densities were produced by combinations Of
low die temperature and high protein content level within the range of extruder
variables used in the present study.

A decrease in the bulk density of extrudates as a result Of increasing screw
speed has been noted by several researchers. Onwulata et al (1992) found that the
bulk density of an extrudate varied as a function of the screw speed and was
dependent on the final temperature of the dough behind the die. At a higher screw

speed, there is a greater amount of mechanical work and frictional heat which

67

causes an increase in product temperature. As shear force and product temperature
increase, more starch granules are dispersed into a polymer phase, increasing the
extrudate expansion and decreasing bulk density. However, in a study of the effects
of dietary fiber and screw speed on corn meal extrusion processing, Hsieh et al
(1989, 1991) reported that increasing either fiber content or screw speed increased
the axial expansion but decreased the radial expansion. The net result was an
increase in bulk density and a decrease in specific volume Of extrudate. Rayas-
Duarte et al (1998) have reported a Significant negative correlation between
expansion ratio and bulk density of buckwheat flour extrudates, which is Similar to

the findings reported in Table 3.3 Of the present study.

3.4.2 Water Absorption Index (WAI)

Results of correlation analyses listed in Table 3.2 indicate that extruder screw
Speed and feed protein content level have significant effects on WAI. Increasing
protein content and extruder screw speed both decreased WAI (Figure 3.3). Overall
die temperature effects were not Significant (p>0.05). Water absorption of extruded
products can be attributed to the dispersion of starch in excess water which is
increased by the degree of starch damage due to gelatinization and extrusion-
induced fragmentation (Rayas-Duarte et al 1998). The increasing protein levels in
raw feed materials mean that increasing amounts of protein material competed with
decreasing amounts Of starch for water absorption. Starch gelatinization is
incomplete when starch is present in relatively low amounts thereby decreasing
WAI. Similar observation was made by Eliasson (1983) who, working on wheat-

starch-gluten mixtures, reported that the lack of protein in raw materials made

68

starch gelatinization easier since there was no material which could compete with
starch for water absorption.

Increasing screw speed significantly reduced WAI. This finding is in
agreement with the observation of Jin et al (1995). Increasing screw speed results
in an increase in the shear rate of molecules which produces structural
modifications of starch (Diosady et al 1985). High screw Speeds produce more
damaged polymer chains and reduce the availability of hydrophilic groups; the
ability of starch molecules to bind more water molecules is thus reduced, which is
reflected in lower WAI values. Wen et al (1990) have reported that screw speed had
a direct effect on polysaccharide size distribution. Higher screw speed resulted in
more fragmentation than a lower screw speed.

In an extrusion of manioc starch, Colonna and Mercier (1983) reported that
extrusion resulted in the degradation of macro-molecular amylose and amylopectin
through Chain splitting. Similar effect on wheat starch degradation has been
observed in a single screw extrusion in which starch degradation was attributed to
Shear forces generated by increase in screw speed (Davidson et al 1984).

Water absorption correlated positively with expansion (Table 3.3), even
though the correlation was not significant (ps0.05). This indicates that high
expansion was associated with high water absorptions, meaning that extrudates
with higher expansion ratios would also be more porous, according to Rayas-Duarte
et al (1998), and as indicated by the Significant (r=-0.54, ps0.001) negative
correlation with bulk density. The amount of water absorbed by ground extrudate
has been indirectly used to indicate the porosity of the material (Colonna et al

1989). Hence, as the porosity of the extrudate material increases, the water

69

absorption would also increase. According to Rayas-Duarte et al (1998), a slow rate
of water absorption in extruded breakfast cereals is desirable to maintain crispness.
The absorption of water in extruded products can be explained on the basis
of starch-water-protein interactions that govern the
solid phase structure (RayaS-Duarte et al 1998). Generally, it has been attributed
to the dispersion of starch in excess water. The dispersion is increased by the
degree of starch damage due to gelatinization and extrusion-induced fragmentation
of amylose and amylopectin molecules (Colonna et al 1989). Other factors that
affect water absorption include the type of proteins, degree of denaturation, and

amount of fiber present (Gujska and Khan 1990).

3.4.3 Specific Mechanical Energy

The specific mechanical energy (SME) of extrudates increased with screw
Speed and decreased with increasing extruder die temperature (Figure 3.4a). The
protein content levels of the feed material also had a negative effect on the SME as
Shown in Figure 3.4b and Table 3.2. On the whole, increasing screw speed was the
only factor that positively affected SME as indicated by the regression and
correlation coefficients in Tables 3.2 and 3.3, respectively. It has been reported that
the mechanical energy input in an extruder can be Changed using the screw speed
or feed rate (Blenford 1994).

In a study of extrusion cooking of corn meal with soy fiber, salt , and sugar,
Jin et al (1994) found that increasing screw speed reduced extruder torque due to
changes in the length of filled flights in the extruder barrel. Whalen et al (1997) also

reported that increasing the barrel temperature in a twin-screw extrusion

70

significantly decreased the torque due to the lowering of apparent viscosity. The
SME is usually calculated from the percent torque of the extruder motor and its
Speed (Levine 1997). In a review, Weidmann (1990) observed a decrease in SME
with an increase in barrel temperature during extrusion of wheat flour which agrees
with the findings in the present study.

The viscosity of cereal flour dough is affected by the extent of gelatinization
(Bhattacharya and Choudhury 1994). At a low temperature and/or high screw
speed, gelatinization of starch is incomplete and Shows lower viscosity. However,
at the beginning of gelatinization, increasing extrusion temperature or decreasing
screw speed increases apparent viscosity. After the completion of starch
gelatinization, apparent viscosity drops again with further increases in temperature.

Consequently, the torque decreases resulting in lower SME values.

3.4.4 Relationship Between Extrusion Variables and Physical Properties
Correlation data in Table 3.3 shows that there was a Significant positive
correlation between screw speed and specific mechanical energy (r = 0.95, ps
0.001) while die temperature was negatively correlated with bulk density. Protein
content of feed negatively correlated with expansion ratio and water absorption
index, and positively with bulk density, while an inverse relationship existed between
expansion ratio and bulk density. Park et al (1993) found a similar negative
correlation between expansion ratio and bulk density and noted that the degree of
puffing of an extrudate as it exits the die nozzle of an extruder is governed by the
expansion ratio and bulk density. However, these two properties are not always the

same, according to previous studies. Phillips et al (1984) and Falcone and Phillips

71

 

 

(1988) explained that the expansion ratio considers expansion only in the direction
perpendicular to extrudate flow, whereas bulk density is concerned with expansion
in all directions.

The relationship between and among extrusion variables and physical
properties of extrudates indicates that water absorption index, protein content of raw
material, and bulk density are all related to the expansion Characterisfics of extruded
products. Thus, extrudates with high protein content tended to have low expansion
and higher bulk density characteristics and reduced ability to absorb water, as

indicated by correlation analysis.

72

 

3.4.5 Microstructure of Extrudates

The laser scanning micrographs revealed the microstructure of the extruded
products, and are shown in Figures 3.5 and 3.6. Even though it was difficult to
discern any quantitative relationship between extrusion variables and microstructure
of extrudates, the most visible features of the microstructure of the extrudates
observed were the number and sizes of air bubbles formed due to extrusion. Figure
3.5 shows that protein level effects were Shown mainly on the size and presence of
air vacuoles as well as the cell wall thickness between air vacuoles. The extrudates
containing 9% protein (Figure 3.5a) had a well-defined air bubble Shape (arrow)
which seemed to disappear in the higher protein content sample. Physical
examination of extrudates showed that the higher protein content products had
reduced expansion and increased bulk density.

In the extrudates produced at higher exit die temperature (Figure 3.6b), air
cells were present, but the cell wall thickness was much smaller than in samples
extruded at lower exit die temperature (Figure 3.6a). Thin cell walls collapse upon
evaporation of superheated water when extrudate exits the die into ambient
pressure, thus producing an insufficiently expanded product. Correlation analysis
showed that higher temperatures produced less expanded products.

The overall effect of screw speed on the expansion ratio and bulk density of
extrudates was negative and not significant. The micrographs depicting the effects
of screw Speed on microstructure (data not shown) did not Show any Obvious
differences among the various screw speed levels used in this study, except that
air cell Sizes and wall thickness between the vacuoles appeared to be well-defined

in the higher screw speed sample.

73

3.5 SUMMARY

This study assessed the impact of heat processing on the functionality of
wheat flour. In particular, the study focused on the effects of twin-screw extrusion
processing on the physical and microstructural Characteristics Of wheat flour
extrudates.

The role of increased protein level in extrusion raw material and how this
protein augmentation, together with other extrusion variables, influenced the
physical characteristics of the extruded product were investigated. Like many other
studies in this area, this study has established that extrusion is a complex process
which involves several variables each Of which contributes differently under different
sets of extrusion conditions to the overall quality characteristics of the extrudates.
The interactions among variables are complex and make it Challenging to
differentiate among the individual variables as to Changes in the Characteristics of
the final product.

The functional properties Of extruded products result from both the structural
modification and changes in physical properties resulting from the mechanical and
thermal stresses applied during extrusion processing. The various interactions
among constituents of the extruding feed material all have Significant impact on
quality attributes of extrudates.

In this study, expansion ratio correlated negatively with bulk density and had
a non-significant but positive correlation with water absorption index. Similarly, bulk
density correlated negatively with WAI. Protein level negatively correlated with

expansion ratio and WAI, but positively with bulk density. These results suggest

74

that WAI, protein level, and bulk density are all related to the expansion
characteristics Of extrudates.

Increasing protein levels inhibit the ability of starch to absorb more water due
to competition, hence the high negative correlation coefficient with WAI. The
reduced ability of starch for more water absorption inhibits the expansion capability
of extrudates Since the flashing Of water at the die of the extruder (which influences
expansion), is significantly reduced. Lower expansion Of extrudates corresponded
with high bulk density as is seen in the negative correlation between expansion ratio
and bulk density.

The results of this study provide evidence that higher protein content of raw

material may not be beneficial in the extrusion processing of wheat flour.

75

3.6 LITERATURE CITED

AACC. 1983. Approved Methods of the American Association of Cereal Chemists.
Eighth ed. The Association, St. Paul. MN.

BADRIE, N. and MELLOWES, WA 1992. Soybean flour/Oil and wheat bran effects
on characteristics Of cassava (Manihot esculenta Crantz) flour extrudate. J.
Food Sc. 57:108.

BHATTACHARYA, M. and HANNA, MA. 1987. Textural properties of extrusion-
cooked corn starch. Lebensm. Wiss. Technol. 20:195.

BHATTACHARYA, S. and CHOUDHURY, GS. 1994. Twin-screw extrusion of rice
flour: effect of extruder length-to—diameter ratio and barrel temperature on
extrusion parameters and product characteristics. J. Food Proc. Pres.
18:389.

BLENFORD, D. 1994. The impact of moisture on extrudate quality. Extrus. Comm.
3(Suppl.):4.

BRENT, J.L. Jr., MULVANEY, S.J., COHEN, C., and BARTSCH, J.A. 1997.
Thermomechanical grass transition of extruded cereal melts. J. Cereal Sci.
26:301.

CASE, S.E., HAMANN, DD, and SCHWARTZ, SJ. 1992. Effect of starch
gelatinization on physical properties Of extruded wheat- and com-based
products. Cereal Chem. 69:401.

CHANG, Y.-H. and Lil, C.-Y. 1992. Preparation of starch phosphates by extrusion.
J. Food Sci. 57:203.

CHAUHAN, GS. and BAINS, GS. 1988. Effect of some extruder variables on
physico-Chemical properties of extruded rice-legume blends. Food Chem.
27:213.

CHIANG, B.-Y. and JOHNSON, J.A. 1977. Gelatinization of starch in extruded
products. Cereal Chem. 54:436.

CHINNASWAMY, R., and HANNA, MA. 1988. Relationship between amylose
content and extrusion-expansion properties of corn starches. Cereal Chem.
65:138.

COLONNA, P. and MERCIER, C. 1983. Macromolecular modification of manioc

starch components by extrusion cooking with and without lipids.
Carbohydrate Polymers 3:87.

76

COLONNA, P., TAYEB, J., and MERCIER, C. 1989. Extrusion cooking of starch
and starchy products. pages 247-319. In Extmsion Cooking. C. Mercier, P.
Linko, and J.M. Harper, eds. Am. Assoc. Cereal Chem. St. Paul, MN.

CZARNECKI, Z., GUJSKA, Z., and KHAN, K. 1993. Enzyme-pretreatment of pinto
bean high protein fraction and time of blending corn meal affects extrudate
properties. J. Food Sci. 5821404.

DAVIDSON, V.J., PATON, D., DIOSADY, LL, and LAROCQUE, G. 1984.
Degradation Of wheat starch in a Single-screw extruder: Characteristics of
extruded starch polymers. J. Food Sci. 49:453.

DIOSADY, L.L., PATON, D., ROSEN, N., RUBIN, L.J., and ATHANASSOULIAS,
C. 1985. Degradation of wheat starch in a Single-screw extruder: mechano-
kinetic breakdown of cooked starch. J. Food Sci. 5021697.

ELIASSON, AC. 1983. Differential scanning calorimetry studies on wheat starch-
gluten mixtures. Effect on gluten in gelatinization Of wheat starch. J. Cereal
Starch 1:199.

FALCONE, RG. and PHILLIPS, RD. 1988. Effects of feed composition, feed
moisture, and barrel temperature on the physical and rheological properties
of snack-like products prepared from cowpea and sorghum flours by
extrusion. J. Food Sci. 53:1464.

FAUBION, J.M., HOSENEY, RC, and SEIB, PA 1982. Functionality of grain
components in extrusion. Cereal Foods World 27:212.

GUJSKA, E. and KHAN, K. 1990. Effect of temperature properties of extrudates
from high starch fractions of navy, pinto and garbanzo beans. J. Food Sci.
55:366.

GUY, R.C.E. and HORNE, AW. 1988. Extrusion and co-extrusion of cereals. In
Food Structures - Its Creation and Evaluation. pp. 331-349. M.V. Blanshard
and JR. Mitchell, eds. Butterworths, London.

HARPER, J.M. 1981. Extrusion of Foods. Vol. 1. CRC Press, Inc., Boca Raton, FL.

HOLAY, SH. and HARPER J.M. 1982. Influence of extrusion shear environment
on plant protein texturization. J. Food Sci. 4721869.

HSIEH, F., HUFF, H.E., LUE, S., and STRINGER, L. 1991. Twin-screw extrusion
of sugar beet fiber and corn meal. Lebensm. Wiss. Technol. 24:495.

HSIEH, F., MULVANEY, S.J., HUFF, H.E., LUE, S., and BRENT, J.R. Jr. 1989.
Effects of dietary fiber and screw speed on some extrusion processing and
product variables. Lebensm. Wiss. Technol. 22:204.

77

JIN, Z, HSIEH, F., and HUFF, HE, 1994. Extrusion cooking of corn meal with soy
fiber, salt, and sugar. Cereal Chem. 71 :227.

JIN, Z., HSIEH, F., and HUFF, HE, 1995. Effects of soy fiber, salt, sugar, and
screw speed on physical properties and microstructure of corn meal
extrudate. J. Cereal Sci. 22:185.

LAWTON, B.T., HENDERSON, GA, and DERLATKE, E.J. 1972. The effects of
extruder variables on the gelatinization of corn starch. Can. J. Chem Eng.
50:168.

LEVINE, L. 1997. Further discussion of extrusion temperatures and energy
balances. Cereal Foods World 42:485.

MERCIER, C. and F EILLET, P. 1975. Modification of carbohydrate components by
extrusion-cooking of cereal products. Cereal Chem. 52:283.

MEUSER, F., LENGERICH, B.V., and KOEHLER, F. 1982. The influence of
extrusion parameters on the functional properties of wheat starch.
Starch/Staerke 34:366.

ONWULATA, C.l., MULVANEY, S.J., HSIEH, F. and HEYMANN, H. 1992. Step
changes in screw speed affect extrusion temperature and pressure and
extrudate characteristics. J. Food Sci. 52:512

DWUSU-ANSAH, J., VAN DE VOORT, F.R., and STANLEY, D.W. 1983. Physico-
chemical changes in cornstarch as a function of extrusion variables. Cereal
Chem. 60:319.

OWUSU-ANSAH, J., VAN de VOORT, PR, and STANLEY, D.W. 1984. Textural
and microstructural changes in corn starch as a function of extrusion
variables. J. Can. Inst. Food Sci. Technol. 17:65.

PARK, J., RHEE, K.S., KIM, SK, and RHEE, KC. 1993. Single-screw extrusion of
defatted soy flour, corn starch and raw beef blends. J. Food Sci. 58:9.

PHILLIPS, R.D., CHINNAN, MS, and KENNEDY, MB. 1984. The effects of feed
moisture and barrel temperature on the physical properties of extruded
cowpea meal. J. Food Sci. 49:916.

RAYAS-DUARTE, P., MAJEWSKA, K.,' and DOETKOTT, C. 1998. Effect of
extrusion parameters on the quality of buckwheat flour mixes. Cereal Chem.
75:338.

SHEARD, P.R., MITCHELL, JR. and LEDWARD, DA. 1985. Comparison of the

extrusion cooking of a soya isolate and a soya flour. J. Food Technol.
20:763.

78

STANLEY, D.W. 1986. Chemical and structural determinants of texture of
fabricated foods. Food Technol. 40(3):65.

WHALEN, P.J., BASON, M.L., BOOTH, R.I., WALKER, CE, and WILLIAMS, P.J.
1997. Measurement of extrusion effects by viscosity profile using the rapid
viscoanalyser. Cereal Foods World. 42:469.

WEIDMANN, W. 1990. Control of extrusion cooking. In Processing and Quality of
Foods. Vol. 1. High Temperature/Short Time (H TST) Processing. P. Zeuthen,
J.C. Cheftel, C. Eriksson, T.R. Gonnley, P. Linko, and K. Paulus, eds. pp.
237-248. Elsevier Applied Science, London.

WEN, L.-F., RODIS, P., and WASSERMAN, B.P. 1990. Starch fragmentation and
protein insolubilizaticn during twin-screw extrusion of corn meal. Cereal
Chem. 67:268.

79

N 5»
(TI 0‘!

Expansion Ratio
'0:

 

P
01

 
 

240 0 Die Temperaque,°C

Screw Speed, rpm

Figure 3.1a

Effects of screw speed and exit die temperature on expansion ratio of
extrudates at 9% feed protein content.

80

 

go 9)
o :3

Expansion Ratio
b

 

p
C)

Screw Speed, rpm

  

20
Protein Content, % 30

Figure 3.1b

Effects of feed protein content level and extruder screw speed on
expansion ratio of extrudates at 120°C exit die temperature.

81

P
on

 

120

 
 

Bulk Density, g/ml
.0 P
A N

140

Die Temperature,°C
1 60

    

Protein Content, % 30

Figure 3.2

Effects of feed protein content and extruder exit die temperature on bulk
density of extrudates at screw speed of 400 rpm.

82

 

‘J

E”
o

p)
C)

N
o

    
 

320
Screw Speed, rpm

Water Absorption Index, gig
5: A
o o

400

   

30
Protein Content, %

Figure 3.3

Effects of feed protein content and extruder screw speed on water
absorption index of extrudates at exit die temperature of 140°C.

83

 

240

_L
o
o

   
     
 

320
Screw Speed, rpm

 

400

Specific Mechanical Energy, W hlkg

Die Temperamrefléso

Figure 3.4a

Effects of extruder exit die temperature and screw speed on specific
mechanical energy input of extrudates at feed protein content of 9%.

 

Specific Mechanical Energy, W hlkg

30
Protein Content, %

Figure 3.4b

Effects of feed protein content and exit die temperature on the specific
mechanical energy input of extrudates at screw speed of 400 rpm.

85

 

Figure 3.5

Laser sunning micrographs of cross-sections of samples extruded at exit die
temperature of 160°C, screw speed of 240 rpm, and feed protein content levels
of: (a) 9% (b) 30%.

86

 

Figure 3.6

Laser scanning micrographs of cross-sections of samples extruded at screw
speed of 400 rpm, feed protein content of 9%, and exit die temperature of:
(a) 140°C (b) 160°C.

87

Table 3.1

Screw configuration in extrusion experiment

 

 

 

Feed Zone Exit Die Zone
Length (8D) (1.750) (80) (0.750) (0.750) (2D) (1D) (0.75D) (20)
(mm) 152 33.25 152 14.25 14.25 38 19 14.25 38
Screw TL FP TL FP RP SL FP RP SL
elements
Degrees -- 30 — 60 - 30 — 60 - 30 —

 

 

 

 

 

 

 

 

 

 

 

 

TL = twin lead; FP = forward paddle; RP = reverse paddle; SL = single lead.
D = Extruder barrel diameter.

Table 3.2

Multiple Linear Regression Coefficients for Expansion Ratio, Bulk Density, Water
Absorption Index, and Specific Mechanical Energy

 

 

Water Specific
Bulk Absorption Mechanical
Expansion Density, Index, Energy,
Ratio g/ml glg W hlkg .
Intercept 3.6542 0.3250 6.8261 68.4593
Screw Speed ns ns -0.0013* 0.6069“
Die Temperature ns -0.0013*** ns -0.4031***
Protein Level -0.0124* 0.0032*** -0.0718*** -0.9327**
R2 0.15 0.44 0.86 0.94

 

*ps0.05; “ps0.01; *“ps0.001
ns - not significant at 5% level.

88

Table 3.3

Multiple Correlation Coefficients for Extrusion Variables

 

 

Water Specific
Expansion Bulk Absorption Mechanical
Ratio Density Index Energy
Screw Speed -0. 14'“ -0. 1 7'“ -0. 1 2"“ 095*”
Die Temperature -0.23"’ -0.38** 0.05"8 -0.16"‘
Protein Level 028* 0.51*** -0.92*** -0.19"'
Expansion Ratio -0.54*** 0.18"3 -0.02"‘
8qu Density -0.35** -0.23"‘
Water Absorption 001'“
Index

 

*ps0.05; **ps0.01; ***ps0.001
ns - not significant at 5% level.

89

CHAPTER 4

CHANGES IN DISULFIDE AND SULFHYDRYL CONTENTS AND
ELECTROPHORETIC PATTERNS OF WHEAT PROTEINS UPON EXTRUSION

90

4.1 ABSTRACT

Vital gluten containing 70% protein (14% moisture basis) was used to
increase the protein content of wheat flour from 9% to 20 and 30%. The three
samples with 9, 20, and 30% protein contents, respectively, were extruded with a
twin-screw extruder and analyzed for changes in disulfide and sulfhydryl contents
as well as the electrophoretic patterns of the total proteins during extrusion.
Increasing extruder exit die temperatures resulted in increased sulfhyde content
of 9 and 20% protein content samples, but did not appear to have any effect on the
30% protein content sample. Similarly, disulfide content decreased slightly following
the same trend. Both sulfl'tydryl and disulfide contents of extrudates were lower
than those of the non-extruded samples. Total cysteine content of extruded samples
decreased by about 16% relative to non-extruded samples, but otherwise remained
almost unchanged among all extruded samples. Polyacrylamide gel electrophoresis
patterns of total proteins showed marked differences between reduced and non-
reduced proteins with a shift in the molecular weights of certain protein bands. In
all, data from this study suggest that disulfide—sulfhydryl interchange reactions were
not significant in the extrusion process under the conditions studied, and that the
changes in the disulfide and sulfhydryl contents of the total proteins might have
predominantly resulted from the scission of disulfide bonds rather than the

interchange between thiol and disulfide contents.

91

4.2 INTRODUCTION

Wheat flour is an important raw material in the manufacture of breakfast
cereals, infant foods, snack foods, and pasta products through extrusion
processing. The extrusion process has the potential to change protein structure,
solubility and digestibility through a combination of shearing, heat and pressure
(Phillips 1989, Wen et al 1990).

The formation of covalent bonds between polypeptide chains, or between
polypeptide chains and other constituents, is considered to be the chemical basis
of extrusion texturization (Jeunink and Cheftel 1979). Several studies have
concluded that during the extrusion process, proteins are denatured and the forces
that stabilize the tertiary and quaternary structures are weakened through a
combination of increased heat and shear within the extruder, resulting in change in
protein conformation (Harper 1986, Ledward and Mitchell 1988, Camire et al 1990,
Camire 1991, Dahl and \fillota 1991).

The cross-linking of proteins during extrusion is known to be responsible for
the textural characteristics of extruded products, and disulfide bonds are one
obvious source of cross-links (Koh et al 1996). Disulfide cross-links have been
reported in spun soy fibers as well as in heated soy protein systems. The texture of
extruded soy granules has been improved through the addition of elemental sulfur
or potassium or sodium sulfites which are believed to increase disulfide cross-links
(Jenkins 1970).

Sulfhyde groups and disulfide bonds are also major contributors to the

stability of the native conformations of proteins and are thus important in

92

maintaining structure and functional properties of native proteins (Thannhauser et
al 1987, Synowiecki and Shahidi 1991). Furthermore, they are thought to play an
important role in the texture of cereal-based products (Chan and Wassennan 1993).

Several studies have attempted to relate the disulfide and sulfhydryl contents
of extrudates to some properties of the extrudates. Cumming et al (1973) have
shown that during extrusion texturization of soy proteins, there is dissociation of
water-soluble proteins followed by aggregation, and that a sulfi'tydryI-disulfide
interchange reaction is involved in the insolubilizaticn of the soy protein. Hager
(1984) and Neumann et al (1984) have also shown that inter-molecular disulfide
bonds in soy proteins contribute to the new and extended protein networks
produced by extrusion of soy concentrate. Ummadi et al (1995) reported differences
between sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
patterns of reduced and non-reduced fractions of albumins, globulins, and glutenins
from raw and extruded semolina, and attributed these differences to the role played
by disulfide linkages during extrusion.

Disulfide-sulfhydryl interchanges during extrusion processing have been
related to the thermal polymerization of gluten proteins during extrusion. Strecker
et al (1995) evaluated the polymerization reactions of wheat gluten proteins during
extrusion through analysis of solubility and disulfide bond content and concluded
that the reaction mechanism in wheat gluten during extrusion was dominated by
polymerization reactions contributed mainly by disulfide bond formation. In contrast,
however, Chan and Wassennan (1993) had earlier reported that extrusion of corn
meal caused an increase in free sulfhydryl content and a decrease in disulfide

groups relative to native samples, suggesting an extrusion-induced rupturing of

93

 

disulfide bonds.

In an experiment to study effects on sulfhydryl groups of extruded wheat flour
proteins, Koh et al (1996) added cysteine to hard red winter wheat flour. After
extrusion, there was a marked increase in protein sulfhydryl content and a decrease
in protein disulfide content indicating a disulfide-sulfhydryl interchange as a result
of the extrusion process. Physical and chemical properties of the extrudates were
significantly affected by the interchange.

In order for extrusion processing to be more controllable and predictable,
there is the need to gain further information on the changes in wheat proteins as a
result of extrusion processing. In the present study, the effects of twin-screw
extrusion processing on the sulfhydryl and disulfide contents and the electrophoretic

patterns of the total wheat flour proteins were examined.

94

4.3 MATERIALS AND METHODS

4.3.1 Extrusion
Materials and extrusion processing were described in Chapter 3 of this

document.

4.3.2 Disulfide-Sulfhydryl Analyses

Disulfide (SS) and sulfhydryl (SH) contents of non-extruded and extruded
samples were determined according to a modified solid-phase colorimetric assay
method of Chan and Wassennan (1993). For free sulfhydryl (free and buried SH)
content determination, about 30 mg samples were suspended in 1.0 ml of reaction
buffer consisting of 8 M urea, 3 mM EDTA, 1% SDS and 0.2 M Tris-HCI (pH 8.0)
(Buffer A). Samples were vortexed for 30 sec and placed on a constant agitation
shaker at room temperature. After 1 hr, 0.1 ml of 10 mM DTNB in 0.2 M Tris-HCI
(pH 8.0) (Buffer B) was added to each sample and shaking continued for another
1 hr. Samples were then centrifuged at 13,600 x g for 10 min at room temperature,
and the absorbance of the supernatant was read at 412 nm against a blank
consisting of 1.0 ml of buffer A and 0.1 ml of buffer B. It was not necessary to dilute
these samples before taking spectrophotometer readings.

For determination of total sulfhyde (SH + reduced SS) content, reaction
buffer consisted of Buffer A containing 0.1 M Na.‘,SO3 and 0.5 mM NTSBZ'
synthesized from DTNB according to Thannhauser et al (1987). The pH of the
buffer was adjusted to 9.5. One ml of the reaction buffer was added to 30 mg of

each sample and vortexed for about 30 sec at room temperature. Samples were

95

then shaken in the dark for 1 hr and then centrifuged at 13,600 x g for 10 min at
room temperature. A 0.1 ml aliquot of the supernatant was diluted with 0.9 ml of
reaction buffer and the absorbance read at 412 nm.

Free SH and total SH contents were calculated from the absorption readings
using a molar absorption coefficient of 13,600 M"cm'1 (Chan and Wasserman,
1993) as follows:

A = ebc,
where A is the absorbance readings, e is the molar extinction coefficient, b is the
cell thickness, and c is the concentration. Disulfide group content was calculated as
the difference between total SH and free SH contents, using the formula:
SS = (TS-SH)/2,
where SS is disulfide content, TS is total sulfhydryl content (free SH + reduced SS),

and SH is free sulfhydryl content.

4.3.3 Electrophoresis

4.3.3.1 Extraction procedure

Total reduced proteins were extracted from ground extrudates and native
flour according to Pogna et al (1990). Each sample was stirred in 1 ml of extraction
buffer consisting of 0.67 ml distilled water, 0.05 ml of 2-mercaptoethanol (2-ME),
and 0.28 ml of stock solution containing 0.2 M Tris-HCI (pH 6.8), 7% (w/v) SDS,
30% (v/v) glycerol, and 0.04% (w/v) Pyronin Y. For non-reduced total proteins,
extraction buffer did not contain 2-ME.

The extraction mixture was vortexed for 30 sec and placed on a constant

agitation shaker at room temperature for 2 hr. Samples were then heated at 80°C

96

for 30 min and allowed to cool before aliquots of the clear solution were loaded onto

gels for SDS-PAGE.

4.3.3.2 SDS-PAGE

Aliquots (25 pl) of extracted samples were loaded onto 15% (T =15.1%,
C=0.58%) SDS separating gels. For each gel, the solution mixture consisted of 15
ml of 30% acrylamide, 1.74 ml of 1.5% bis-acrylamide, 11.25 ml of 1 M Tris-HCI (pH
8.4), 0.96 ml water, 0.3 ml of 10% (wlv) SDS, 0.75 ml of 1% (wlv) ammonium
persulfate and 20 pl N,N,N’,N’-tetramethylethylenediamene (T EMED). The stacking
gels were 4.5% (T =4.5%, C=1.3%) and consisted of 1.575 ml of 30% acrylamide,
0.433 ml of 1.5% bis-acrylamide, 1.25 ml of 1M Tris-HCI (pH 6.8), 6.142 ml water,
0.1 ml 10% (wlv) SDS, 0.5 ml of 1% (wlv) ammonium persulfate, and 10 pl TEMED.
The gels were run at room temperature for 18 hr at a constant current of 12.5 mA
per gel.

After the run, gels were stained according to the procedure of Redaelli et al
(1995). Each gel was placed overnight in a staining solution consisting of 15 ml
Coomassie Brilliant Blue R-250 (4 9 dissolved in 1 liter of 95% ethanol), 25 ml of
60% trichloroacetic acid (T CA), and 210 ml distilled water. Gels were then washed

several times with deionized water and photographed.

97

 

4.4 RESULTS AND DISCUSSION

4.4.1 Dlsulflde and Sulfltydryl Reactions

The relationships between the protein content and sulfhydryl, disulfide and
total cysteine contents of non-extruded flour samples are shown in Figure 4.1.
Increasing the total protein content of flour resulted in decreases in free sulfhydryl,
disulfide and total cysteine contents of the non-extruded samples. The albumin and
globulin fractions of wheat flour are known to contain higher amounts of total
cysteine (6.2 and 5.4 91169 of nitrogen, respectively) than the gluten proteins of
gliadin and glutenin (2.7 and 2.2 g/16g of nitrogen, respectively) (Bushuk and
Wrigley 1974). Thus, it is reasonable to expect that increasing the flour protein
content from 9 to 30% using vital gluten is essentially equivalent to dilution of the
total SH content per unit weight of sample. The effect of this is a declining content
of free SH, disulfide content and total cysteine content as protein content is
increased in the non-extruded flour.

Table 4.1 shows the results of analysis of sulfhydryl, disulfide, and total
cysteine contents of non-extruded and extruded samples from flour samples with
9, 20 and 30% protein content. Analysis of variance (Table 4.2) indicates that both
linear and interaction effects of variables on free sulfhydryl, disulfide, and total
cysteine contents were significant (ps0.001). At a screw speed of 240 rpm,
increasing the die temperature from 120 to 160°C increased free SH content from
6.95 to 10.98 pM/mg protein, an increase of about 58%. Similarly, increases of 40
and 67% in free SH were noted over the same temperature ranges at constant

screw speeds of 320 and 400 rpm, respectively, for the extrudates containing 9%

98

protein. There were much higher increases in free SH for the 20% protein content
extrudates, and relatively much smaller increases for the 30% protein content
sample, as exit die temperature was increased. It is evident from the data that
temperature was positively correlated with free SH content of all extruded samples.

When non-extruded samples are compared to extruded samples, Table 4.1
shows that at all screw speed levels, free SH contents in non-extruded samples
were higher than in samples extruded at 120 and 140°C for the 9 and 20% protein
content samples, and at all three temperature levels for the 30% protein samples.
However, within and among all extruded samples, increasing die temperature also
increased free SH at all screw speed levels, while correspondingly, SS contents
decreased. This was particularly evident for the 9 and 20% protein content samples.

The decrease in free SH content of extrudates relative to non-extruded
samples, and the opposite increases with increasing die temperatures among all
extruded samples concomitant with decreases in SS, are in agreement with the
findings reported by Chan and Wassennan (1993). These authors showed a
decrease in the SH group content of extruded corn meal proteins, relative to that of
non-extrudates. However, among extrudates, samples extruded at a higher
temperature showed an increase in free SH content relative to those extruded at a
lower temperature, and this was accompanied by slight decreases in SS group
content. Burgess and Stanley (1976) had previously found slight decreases in SS
and an increase in SH contents on extrusion of soy meal.

However, the results of the present study contrast the findings of Synowiecki
and Shahidi (1991) who reported a decrease in the content of sulfhydryl groups and

an increase in the content of disulfide bonds on heating seal meat proteins.

99

Together, these results suggest that the process of sulfhydryl-disulfide changes
occurring in seal meat proteins, as described by Synowiecki and Shahidi (1991),
may be essentially one of disulfide cross-linking of protein molecules due to
oxidation of sulfhydryl to disulfide groups, while the extrusion of wheat proteins, as
reported in the present study, appeared to result in the reduction of disulfide groups
to sulfhydryls. Secondly, the involvement of shear force in extrusion, as in the
present study, as opposed to ordinary heating alone, may be significant in the
differences between the results. In general, the apparent increases in free SH group
content accompanied by small decreases in disulfide group content in the present
study, with increasing temperatures, may be indicative of the inter-conversion of
disulfide and sulfhydryl groups during the extrusion processing of wheat proteins.

Data in Table 4.1 also show that the total cysteine contents of all extrudates
were lower than that of the non-extruded flour in the 9% protein sample, and varied
between 81.82 and 89.57 pM/mg protein compared to 102.51 pM/mg protein for the
non-extruded sample. Thus, on average, extrusion reduced total cysteine content
by approximately 16%.

It can also be concluded from the data in Table 4.1 that the sample
containing 30% protein was virtually unaffected by extrusion in terms of changes in
total cysteine content. This sample contains much higher vital gluten, as compared
to the 9 and 20% protein content samples, and the protein content is composed
mainly of the gluten proteins (gliadins and glutenins). Schofield et al (1983) reported
that the level of total cysteine groups in gluten essentially remained constant
irrespective of temperature and was unaffected by heating to 100°C.

The decrease of 16% in total cysteine content in extruded 9% protein content

100

 

samples relative to the non-extruded samples, and the negligible variations in total
cysteine contents for the 20 and 30% protein samples, suggest that some cysteine
was lost during extrusion. According to Koh et al (1996), this could represent
cysteine loss at the extruder die as hydrogen sulfide or volatile organic compounds
such as those found as flavor compounds in the extrudates. Extruded cereals are
generally believed to have less flavor than those prepared by traditional baking, and
this is attributed to loss of produced or added aroma volatiles when the melt leaves
the extruder die and becomes an expanded solid material (Bredie et al 1997). Chan
and Wasserman (1993) and Weegels et al (1994) have also suggested that
conformational changes in the protein structure during extrusion might inhibit access
of colorimetric reagent in the presence of SDS. Alternatively, the cysteine in the
extrudates is more prone to oxidation after extrusion (Chan and Wassennan 1993),
leading to the formation of more non-covalent disulfide bonds. The results of the
present study did not support this theory since there were no noticeable increases

in disulfide group content after extrusion.

4.4.2 Electrophoresis

Figure 4.2 shows the electrophoretic patterns of non-reduced total proteins
of samples containing 9% protein. For both non-extruded and extruded samples,
bands in the high molecular weight protein region (labeled i) and several bands in
the low molecular weight region (labeled iii) were not visible. There was no major
difference in the intensity of visible bands in the mid-region (labeled ii) among all
extruded samples, and between extruded and non-extruded samples. However, a

few bands which were present in the low molecular weight region of non-extruded

101

samples disappeared after extrusion. Similar observations were made in the
electrophoretic patterns of the samples containing 20 and 30% protein (data not
shown), even though these two contained more protein bands than the 9% protein
content sample in view of their mixture with vital gluten. Thus, without reduction of
disulfide bonds, extractability of low molecular weight proteins in sodium dodecyl
sulfate (SDS) was markedly decreased after extrusion, consistent with previous
studies (Koh et al 1996). Dexter and Matsuo (1977) reported a decrease in protein
extractabilities of four different flours after extrusion. Wen et al (1990) have also
reported previously that the size distribution of dimethyl sulfoxide-soluble proteins
of corn meal extrudates was unchanged after extrusion even though significant
amounts of protein became insolubilized.

Previous studies have demonstrated reductions in solubility as a function of
temperature for soy protein (Hager 1984) and corn meal protein (Racicot et al
1981). The disappearance of the bands at the lower molecular weight region after
extrusion may suggest that extrusion might have depolymerized the lower molecular
weight proteins into even much smaller units which then eluted off the gel into the
running buffer; or the proteins might have polymerized into larger aggregates
making those proteins too big to enter the running gel. However, gels run under
non-reduced conditions in which running was terminated when the tracking dye just
reached the bottom of the running gel, showed similar patterns. This suggests that
polymerization was more likely to have occurred during the extrusion process.
Ummadi et al (1995) observed the aggregation of polypeptides of albumins,
globulins, and glutenins of semolina under non-reduced conditions after extrusion.

Figure 4.3 shows the electrophoretic patterns of reduced 9% protein content

102

samples. After reduction of disulfide bonds with 2-mercaptoethanol (2-ME), strong
and distinct bands appeared at the high molecular weight protein region (labeled i)
in both non-extruded and extruded samples. There were no differences in
intensities of bands in this region. The electrophoretic patterns of both 20 and 30%
protein samples were similar. However, comparing the bands of non-extruded
samples to those of the extruded samples, it was observed that a single band
(arrow) in the middle region labeled “ii” of the non-extruded sample lightened and/or
disappeared after extrusion. Simultaneously, there was an appearance of heavy
bands (white arrows) in the lower region (labeled iii) for extrudates. There was a
very light band at the same location in the non-extruded sample. It therefore
appears that proteins in the middle region depolymerized into lower molecular units
during the extrusion process, which then appeared in the extruded products in the
lower region. However, examination of Figure 4.3 shows that samples extruded at
the highest screw speeds did not show the “disappearance-appearance” pattern
described, which might indicate that a combination of high shearing forces and
temperatures might have provided alternate formation of new disulfide bonds
through interchange reactions or by additional cystine formation from cysteine.
Thus, depolymerization of proteins was followed by protein aggregation, especially
at higher shear forces.

The absence of any visible differences between extruded and non-extruded
samples, and among all extruded samples, in the high molecular weight protein
region (glutenins) suggests that the native intermolecular disulflde cross-links
remained unaltered by extrusion, and that extrusion even at the higher temperature

levels used in this study did not significantly affect disulfide bonds. Alternatively, the

103

 

proteins might have aggregated through inter- and intra-molecular disulfide cross-
Iinking after extrusion indicating that disulfide bonds still play a major role in the
protein structure after extrusion under the conditions studied.

The marked differences between SDS-PAGE patterns of reduced and non-
reduced samples and the decrease in protein disulfide content after extrusion are
in part inconsistent with thiol-disulfide interchange reactions. According to Koh et
al (1996) the characteristics of such interchange reactions include net increase in
protein SH, net reduction in cross-linking, and no increase or decrease in disulfide
bonds. Thus, reduction with 2-ME produces no change on SDS-PAGE patterns
under such conditions. The fact that net protein disulfide content decreased and the
SH content increased with extrusion in the present study indicates that extrusion
might have resulted in shear-mediated scission of disulfide bonds. This observation
together with changes in SDS-PAGE patterns suggest that thiol-disulfide
interchange had only a minor effect under the extrusion conditions used in this
study. Possible explanation of the absence of thiol-disulfide interchange reaction
may be the low water contents and very short reaction times of extrusion. Studies
in doughs have shown that the thiol-disulfide interchange reactions require longer
periods of mixing time and also require sufficient water (Stewart and Mauritzen

1966, Jones and Carnegie 1971).

104

 

4.5 SUMMARY

Extrusion processing of wheat flour resulted in the decrease in protein
disulfide content and an increase in the free sulfhyde content while total cysteine
content remained fairly unchanged. SDS-PAGE patterns of the total proteins
showed marked differences between reduced and non-reduced samples indicating
that there was very little or no thiol-disulfide interchange reaction during extrusion
processing under the conditions of this study, and that the intramolecular scission
of disulfide bonds seemed to be the predominant occurrence during the extrusion
process. lntramolecular fragmentation of proteins, as evidenced by SDS-PAGE
patterns, led to the formation of low molecular weight protein aggregates. This
information should be helpful in determining the relationship between disulfide and
free sulfl'tydryl contents of an extruded product and the textural characteristics of the

extrudates.

105

 

4.6 LITERATURE CITED

BREDIE, W.L.P., HASSELL, G.M., GUY, R.C.E., and MOTTRAM, D.S. 1997.
Aroma characteristics of extruded wheat flour and wheat starch containing
added cysteine and reducing sugars. J. Cereal Sci. 25:57.

BURGESS, LD. and STANLEY, D.W. 1976. Research Note - A possible
mechanism for thermal texturization of soybean protein. J. Inst. Can. Sci.
Technol. Aliment. 9:228

BUSHUK, W. and WRIGLEY, CW. 1974. Proteins: Composition, structure and
function. Pages 119-145. In Wheat: Production and Utilization. G.E. lnglett,
ed. AVI Pub. Co., Westport, CT.

CAMIRE, ME. 1991. Protein functionality modification by extrusion cooking. J. Am.
Oil Chem Soc. 68:200.

CAMIRE, M.E., CAMIRE, A., and KRUMHAR, K. 1990. Chemical and nutritional
changes in foods during extrusion. Crit. Review Food Sci. and Nutr. 29235.

CHAN, K.-Y. and WASSERMAN, B.P. 1993. Direct colorimetric assay of free thiol
groups and disulfide bonds in suspensions of solubilized and particulate
areal proteins. Cereal Chem. 70:22.

CUMMING, D.B., STANLEY, D.W., and deMAN, J.M. 1973. Fate of water soluble
soy protein during thermoplastic extrusion. J. Food Sci. 38:320.

DAHL, SR. and VILLOTA, R. 1991. Effect of thermal denaturation on the
texturization of soybean protein via twin-screw extrusion. Can. Inst. Food Sci.
Technol. J. 242143.

DEXTER, J.E. and MATSUO, RR. 1977. Changes in semolina proteins during
spaghetti processing. Cereal Chem. 54:882.

HAGER, D. F. 1984. Effects of extrusion upon soy concentrate solubility. J. Agric.
Food Chem. 32:293.

HARPER, J.M. 1986. Extrusion texturization of foods. Food Technol. 40:70.

JENKINS, S.L. 1970. Method for preparing a protein product. Ralston Purina Co.,
St. Louis, MO. US. Patent 3,496,858.

JEUNINK, J. and CHEFTEL, J.C. 1979. Chemical and physicochemical changes in

field bean and soybean proteins texturized by extrusion. J. Food Sci.
4421322.

106

JONES, I.K. and CARNEGIE, PR. 1971. Binding of oxidized gluthathione to dough
proteins and a new explanation, involving thiol-disulfide exchange, of the
physical properties of dough. J. Sci. Food Agric. 22:358.

KOH, B.K., KARWE, M.V., and SCHAICH, KM. 1996. Effects of cysteine on free
radical production and protein modification in extruded wheat flour. Cereal
Chem. 732115.

LEDWARD, DA. and MITCHELL, JR. 1988. Protein extrusion - More questions
than answers? In Foods Structure - Its Creation and Evaluation. J.M.
Blanshard and JR. Mitchell (Eds), p. 219. Buttenrvorths, London.

NEUMANN, P.E., JASBERG, B.K., WALL, J.S., and WALKER, CE. 1984. Uniquely
textured products obtained by co-extrusion of corn gluten meal and soy flour.
Cereal Chem. 61:438.

PHILLIPS, DR. 1989. Effects of extrusion cooking on the nutritional quality of plant
proteins. In Protein Quality and Effects of Processing. pp. 21-246 R.D.
Phillips and J.W. Finley, eds. Marcel Dekker, New York.

POGNA, N.E., AUTRAN, J.-C., MELLINI, F., LAFIANDRA, D., and FEILLET, P.
1990. Chromosome 1B—encoded gliadins and glutenin subunits in durum
wheat: genetics and relationship to gluten strength. J. Cereal Sci. 11:15.

RACICOT, W.F., SATTERLEE, L.D., and HANNA, MA. 1981. Interaction of lactose
and sucrose with corn meal proteins during extrusion. J. Food Sci. 4621500.

REDAELLI, R., MOREL, M.-H., AUTRAN, J.-C., and POGNA, NE. 1995. Genetic
analysis of low Mr glutenin subunits fractionated by two-dimensional
electrophoresis (A—PAGE x SDS-PAGE). J. Cereal Sci. 21 :5.

SCHOFIELD, J.D., BOTTOMLEY, R.C., TIMMS, M.F., AND BOOTH, MR. 1983.
The effect of heat on wheat gluten and the involvement of sulphydryl-
disulfide interchange reactions. J. Cereal Sci. 1:241.

STEWART, PR. and MAURITZEN, CM. 1966. The incorporation of 35S cysteine
into the proteins of dough by disulfide—sulfhydryl interchange. Aust. J. Biol
Sci. 19:1125.

STRECKER, T.D., CAVALIERI, R.P., ZOLLARS, R.L., and POMERANZ, Y. 1995.
Polymerization and mechanical degradation kinetics of gluten and glutenin
at extruder melt-section temperatures and shear rates. J. Food Sci.
602532.557.

SYNOWIECKI, J. and SHAHIDI, F. 1991. Heat-induced changes in sulfhydryl
groups of harp muscle proteins. J. Agric. Food Chem. 3922006.

107

THANNHAUSER, T.W., KONISHI, Y., and SCHERAGA, HA. 1987. Analysis for
disulfide bonds in peptides and proteins. Methods Enzymol. 1432115.

UMMADI, P., CHENOWETH, W.L., and NG, P.K.W. 1995. Changes in solubility
and distribution of semolina proteins due to extrusion processing. Cereal
Chem. 72:564.

WEEGELS, P.L., de GROOT, A.M.G., VERHOEK, J.A., and HAMER, R.J. 1994.
Effects on gluten of heating at different moisture contents. II. Changes in
physico-chemical properties and secondary structure. J. Cereal Sci. 19:39.

WEN, L.-F., RODIS, P., and WASSERMAN, B.P. 1990. Starch fragmentation and
protein insolubilizaticn during twin-screw extrusion of corn meal. Cereal
Chem. 67:268.

108

 

 

 

 

 

120
-0- Free Sulfhydryl
-l- Dlsulflde
+Total Cysteine
90 -t-
.E
*3
o.
a 60 -n
E
i
:t.
30 'r
\ fl.
0 : 2 t . 4.
9 20 30
Protein Content, %
Figure 4.1

Variation in free sulfhydryl, disulfide, and total cysteine
contents in non-extruded wheat flour samples of varying
protein content.

109

 

 

 

Figure 4.2

SDS-PAGE patterns of non-reduced total protein of extruded wheat flour
containing 9% protein.
N = non-extruded flour sample.
Screw Speeds: A = 240 rpm; B = 320 rpm; C = 400 rpm.
Die Temperatures: 1 = 120°C; 2 = 140°C; 3 = 160°C.

i, ii, iii: see text for explanation.

110

 

 

123123123

Figure 4.3

SDS-PAGE patterns of reduced total protein of extruded wheat flour containing
9% protein.

N = non-extruded flour sample.
Screw Speeds: A = 240 rpm; B = 320 rpm; C = 400 rpm.
Die Temperatures: 1 = 120°C; 2 = 140°C; 3 = 160°C.
i, ii, ill: see text for explanation.

111

 

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Table 4.2

Statistical F-Values for Analyses of Free Sulfhydryl, Disulfide and Total Cysteine
Contents in Wheat Flour Extrudates

 

 

 

 

F-Value

Source df Free Sulfhyde Disulfide Total Cysteine
Screw speed 2 63.34“" 29.79‘“ 663*"
Temperature (Temp) 2 2183.09*** 955.43*** 154.40***
Screw speed x Temp 4 20.27*** 4320*“ 3996*“
Protein (Prot) 2 6936.16*** 31980.65*** 47513.58***
Screw speed x Prot 4 2358*" 183.36” 202.98***
Temp x Prot 4 612.06“ 265.85” 55.16***
Screw speed x Prot x Temp 8 25.64*** 47.92*** 76.23***
R2 0.998 0.999 0.999

*** ps0.001

113

CHAPTER 5

EFFECTS OF TWIN-SCREW EXTRUSION PROCESSING ON STRUCTURAL
AND CHEMICAL PROPERTIES OF THE MAJOR WHEAT FLOUR
COMPONENTS AS MEASURED BY FLUORESCENCE SPECTROSCOPY
AND DIFFERENTIAL SCANNING CALORIMETRY

114

5.1 ABSTRACT

The effect of twin-screw extrusion processing on the structural and chemical
changes in the starch and protein components of wheat flour were studied using
differential scanning calorimetry (DSC) and fluorescence spectroscopy (FS). Much
higher temperatures for thermal transitions were observed for starch than are
normally associated with starch gelatinization in DSC. The formation of amylose-
lipid complex has been suggested for such levels of transition enthalpy changes.
DSC data further suggest that extrusion promoted the exothermic aggregation
process between amylose and lipid. Increase in protein content of raw materials
increased transition peak temperatures, Tm, and decreased transition thermal
energy, AH, in non-extruded samples. The addition of vital gluten therefore
increased the stability of the system and conferred hydrophobic properties on the
system, thus reducing the net favored AH. Fluorescence spectroscopy data
indicated changes in the conformational structure of wheat proteins consequent to
extrusion processing. All extrudates showed higher peak fluorescence emission
wavelengths, Amuthan non-extruded samples. Increasing extruder die temperatures
resulted in changes in the hydrophobic environment of proteins as measured by
fluorescence intensity (Fl) and changes in the maximum fluorescence emission

peak wavelengths.

115

5.2 INTRODUCTION

Cereal starches are generally transformed into melts through gelatinization.
However, Qu and Wang (1994) have reported that the transformation of starch can
also be achieved under conditions of high shear rate, high temperature and high
pressure such as are found during extrusion cooking. The phase transition
temperatures and energies associated with such transformation are known to be
affected by additives such as sodium chloride; thus information derived from
thermal studies can be used to determine processing conditions and type and
quantity of additives used in formulated-extruded food systems.

Unfortunately, studies conducted with the aim of elucidating information on
transition phases of cereals are mostly done with pure starches or extracted protein
fractions. Phase transitions in pure starches and fractionated proteins of several
cereals, as they undergo heat-induced thermal events, have been studied using
differential scanning calorimetry (DSC) following the pioneering work of Stevens and
Elton (1971). The DSC can provide both qualitative and quantitative information in
terms of transition temperatures and heat of transition, respectively, of the heating
process. Eliasson (1983) studied the effect of gluten on the gelatinization of wheat
starch using DSC. Biliaderis et al (1986a,b) have conducted DSC studies on the
multiple melting transitions of rice starch/monoglyceride systems (a), and the
thermal characterization of rice starches (b). Thermal properties of heat-moisture-
treated wheat and potato starches (Donovan et al 1983), cat globulin (Hanrvalker
and Ma 1987), thermal stability of wheat gluten (Eliasson and Hegg 1980), and the

phase transitions of amylose—Iipid complexes in wheat, maize, and potato starches

116

(Kugimiya et al 1980) have all been studied using DSC.

Information on more complex systems, however, such as wheat flour which
contains both starch and proteins in addition to other components, is limited.
Therefore, differential scanning calorimetry and fluorescence spectroscopy were
used in the present studies to investigate phase transitions and changes in surface
hydrophobicity, respectively, of wheat flour components due to extrusion

processing.

5.3 MATERIALS AND METHODS

5.3.1 Extrusion
Extrusion processing conditions and materials used were described in

Chapter 3 of this document.

5.3.2 Differential Scanning Calorimetry (DSC)

A Dupont 2920 DSC unit (Dupont, Wilmington, DE), calibrated with indium
for temperature and enthalpy, was used to analyze phase transition peak
temperatures (Tm) and enthalpy changes (AH) associated with the transition change
for extruded and non-extruded samples. Approximately 10 mg of non-extruded flour
or ground extrudate samples were weighed and sealed in an aluminum sample pan
(TA Instruments, Newcastle, DE) by an encapsulating press. The DSC cell was
flushed with nitrogen at 50 ml/min to maintain an inert environment during the
measurements. Thermograms (plots of differential heat flow as a function of

temperature) were obtained at a heating rate of 10°C/min, from 20 to 250°C. For

117

each therrnogram, T,,, and AH values were obtained using the General V4.1C data

acquisition software program which controlled the DSC unit.

5.3.3 Fluorescence Spectroscopy

The hydrophobicity of total proteins in extruded and non-extruded samples
was determined using steady-state fluorescence measurements of total proteins by
interacting the proteins with the extrinsic fluorescence probe 1-anilino-8-
naphthalene sulfonic acid (ANS). Flour from non-extruded and ground extruded
samples were extracted with 20 ml of acetic acid-urea buffer (3mM urea in 0.1M
acetic acid). Samples were varied to obtain equal protein concentrations of 10
mglml. The mixture was stirred for 1 hr and centrifuged at 13,000 x g for 20 min at
room temperature. Ten pl of ANS (8 mM in 0.1 M phosphate buffer, pH 6.9) were
then added to 2 ml of sample supernatant. Relative fluorescence intensities were
measured with a SLM 4800 spectrophotometer (Urbana Champaign, IL) connected
to a data acquisition and operating system from On-Line Instruments Systems
(Bogart, GA). Measurements were made in duplicate in 1 cm pathlength semi-micro
quartz fluorescence cuvettes held in a therrnostable block maintained at 22°C. The
emission spectra of supematants were measured between 400 and 600 nm at an
excitation wavelength of 390 nm (excitation wavelength of ANS). Slit width for both
excitation and emission was 4 nm.

The relative fluorescence intensity (Fl), measured as the height of the
emission maximum, and the shift in the wavelength of maximum emission (km),
were recorded and used as indices of change in hydrophobic or polar environments

(Strasburg and Ludescher 1995) of wheat total proteins.

118

5.4 RESULTS AND DISCUSSION

5.4.1 Thermal Transitions

The effect of protein content of raw extruder feed on the transition
temperatures, Tm, and energies associated with phase change, AH, in non-
extruded samples are shown in Figure 5.1. Increasing the protein content of raw
feed material increased the transition peak temperature while, conversely, the
energy associated with the phase change decreased. Increasing protein level
therefore has the effect of increasing the exothermic component of the overall
enthalpy. Protein aggregation is an exothennic reaction that contributes negatively
to the overall transition enthalpy, AH (Myers 1990). The addition of vital gluten
protein to wheat flour conferred more hydrophobic properties to the raw material
thus decreasing the net AH values and increasing overall stability, as evidenced by
the increases in Tm upon increasing the protein content of the raw material. Eliasson
(1983) found similar decreasing effect of gluten on wheat starch gelatinization
enthalpy and increasing effect on the gelatinization temperature. It was assumed
that the increase in Tm on addition of gluten may be due to a delay in diffusion of
water into the starch granules because of the presence of gluten on the surface of
the starch granules, since higher starch gelatinization temperature in wheat flour
compared to the isolated starch has been observed (Olkku and Rha 1978). Since
the Tm values reported in the present study (111-125°C) are much higher than the
gelatinization temperature range of starch (60-80°C), the increasing effect of protein
content on Tm an be assumed to be due to decreasing amount of water available

for the starch, as reported by Eliasson (1983).

119

Table 5.1 shows the Tm and AH values for extruded and non-extruded
samples, containing 9% protein, with some representative therrnograms shown in
Figures 5.2 and 5.3, respectively. The DSC therrnograms give information on both
the temperature and heat required for thermal transitions. The AH, at the molecular
level, involves the cleavage of existing hydrogen bonds between starch molecules,
and the formation of new ones with water molecules. The result is a less-ordered
structure with increased entropy, hence the overall process is endothermic (Stevens
and Elton 1971).

For the extruded samples with 9% protein content, transition endotherm
peaks were observed in the range 112.62-125.34°C. In general, increasing extruder
die temperatures decreased Tm values. However, the Tm value for the non-extruded
sample (111.21°C) was lower than the values for all extrudates containing 9%
protein. Such increase in Tm values, according to Harwalker and Ma (1987), would
indicate an increase in the heat stability of extruded samples relative to non-
extrudates, possibly due to strengthening of the amylose-lipid complex. The
extruded samples thus required relatively higher amounts of energy for thermal
transition as evidenced by the higher Tm values. However, within the extruded
samples, die temperature increases reduced the stability of samples and weakened
the lipid complex formation of the extruded amylose molecules as seen by
decreasing Tm values. It therefore appears that extrusion processing increased the
stability of extruded samples relative to non-extrudates, but stability among extruded
samples varied depending on the range of extrusion temperature used, with higher

temperatures tending to reduce stabilities.

120

Based on the assumption that the thermal transitions occurring in starch at
higher temperatures between 100-140°C are due to the presence and
disorganization of amylose-lipid complexes, as stated earlier, the data for the
present study would indicate that extmsion promoted the complexation of amylose
with lipid, but the stability of the formed complex was subsequently weakened by
increasing the exit die temperature in extrusion processing. Biliaderis (1990)
reported that the formation of the amylase-lipid complex is an intermolecular
aggregation process which is exothermic, which agrees with the interpretation of the
Tm data for extruded and non-extruded samples in the present study.

The AH values generally increased with increasing die temperature at
constant screw speed, while varying screw speed at constant die temperatures did
not show any specific pattern of AH values. However, in contrast to Tm, the AH
values for the non-extruded sample (9% protein) were higher than that of all
extrudates containing 9% protein. This suggests that in converting the raw materials
into an extrusion-cooked product, there was an increase in the exothermic
(negative) component of the enthalpies of reaction, at least in the samples
containing the lowest amount of protein, since AH is a combination of endothermic
reactions, such as hydrogen bond disruption, and exothermic reactions including
aggregation (hydrogen bond formation) and the breakup of hydrophobic interactions
(Privalov and Khechinashvili 1974, Evans et al 1979). This also further supports the
Tm data to the effect that the lowering of the AH in extrudates corresponds to the
formation of amylase-lipid complexes. Biliaderis (1990) reported lower AH values
for high lipid content starches which reflected an exothermic amylose-Iipid

complexation. Camire et al (1990) noted that lipid-complexed starch has a more

121

narrow gelatinization temperature than untreated starch, and has a lower enthalpy
of gelatinization that may give it valuable functional qualities.

The data for extruded samples containing 20 and 30% protein showed some
inconsistencies for both AH and Tm values (data not shown), perhaps due to high
gluten protein, and therefore were inconclusive within the extrusion parameters
studied. As reported in the literature, gluten proteins do not give good AH or Tm
values. Nevertheless, the enthalpy value for the non-extruded 30% protein sample
was higher than those of the extrudates, which is similar to the observation for the

9% protein sample.

5.4.2 Fluorescence Spectroscopy

Emission peak wavelengths (Am) and relative steady-state fluorescence
intensities (Fl) of extruded and non-extruded samples are given in Table 5.2. All
extruded samples showed higher wavelengths of maximum emission than non-
extruded samples. For the 9% protein content sample, peak wavelengths varied
between 493.0 and 511.5 nm for the extrudates compared to 482.5 nm for the non-
extruded sample. The 20% protein content sample showed a maximum emission
variation between 490.5 and 500.0 nm for extrudates and of 489.5 for non-extruded
samples, and similarly, peak emission wavelengths for the 30% protein content non-
extruded samples shifted from 482.0 nm to between 491.5 and 502.0 nm after
extrusion.

Data for fluorescence intensities showed opposite trend to the emission
wavelength data. The relative fluorescence intensities for the non-extruded samples

were all several magnitudes higher than those of extruded samples. The shift in the

122

emission wavelengths to higher values (i.e., red shift) and the decrease in relative
fluorescence intensities in extruded samples reflect a change in the conformation
of the proteins after extrusion. Yeboah et al (1994) reported an increase in
wavelength of maximum emission for denatured, and reduced/alkylated wheat
gliadin proteins relative to the native proteins.

The position of the maximum in the emission spectrum (km) and the
fluorescence intensity are sensitive to the polarity of the environment. An increase
in fluorescence intensity occurs only when the ANS interacts with a nonpolar group
on the surface of the protein (Javor et al 1991). Increases in peak wavelengths and
decreases in fluorescence intensities are indicators of polar (hydrophilic)
environment, and conversely, decreases in emission wavelengths and increases
in fluorescence intensities reflect non-polar (hydrophobic) environment (Javor et al
1991, Lakkis and thlota 1992, Yeboah et al 1994). Thus, the present data indicate
that extruded samples were in a comparatively more polar environment than the
non-extruded samples. One possible explanation for this observation is that
extrusion probably led to aggregation of proteins which buried the hitherto exposed
hydrophobic sites on the protein surface thereby making them inaccessible to the
ANS. The observed decreases in enthalpies (AH) of extrudates relative to non-
extruded samples support the aggregation theory since aggregation is an
exothermic process which contributes negative value to the overall AH of a reaction.

As shown in Figure 5.4, increasing extruder die temperature at constant
screw speeds shifted the peak emission to lower wavelength (la, blue shift) for all
extruded 9% protein content samples. At the same time, there appeared to be slight

increases in the relative fluorescence intensities. Increases in fluorescence

123

 

 

intensities and blue shifts in maximum emission wavelengths are indicators of
decreases in polarity (i.e., increased hydrophobicity) of the environment (Strasburg
and Ludescher 1995). This suggests that within the extruded samples, temperature
increases favored the unfolding of proteins thus exposing hydrophobic sites in the
proteins to ANS binding. Such a change in the conformation of the proteins
probably leads to an increased number of hydrophobic sites on the surface of the
protein which also results in aggregation. Javor et al (1991) observed that if
aggregation occurs after ANS binding and holds the ANS in a hydrophobic
interaction environment, the nonpolar environment produces an extra increase in
fluorescence intensity, as was reflected in the present data. In general, the surface
hydrophobicities of proteins are increased by heating due to increased exposure of
hydrophobic groups at the outer surface of the molecule (T anford 1980, Matsudomi

et al 1982, Voutsinas et al 1983).

5.5 SUMMARY

The effect of temperature, screw speed and protein content on the
maximum emission wavelengths and relative fluorescence intensities for extruded
samples were difficult to discern due to absence of any clear and definite pattern
among the 20 and 30% protein content samples. Nevertheless, for the 9% protein
content sample, exit die temperature had a decreasing effect on the peak emission
wavelengths at all screw speed levels, while increasing screw speed at each
temperature level appeared to increase the emission maximum.

The DSC and FS data reported in this study indicate that there were some

124

conformational changes in the starch and proteins of wheat flour as a result of twin-
screw extrusion processing. DSC data suggest the formation of a complex between
amylose and lipid in the wheat. This is an exothermic aggregation process that
leads to a decrease in the overall thermal energy of the process. Furthermore,
fluorescence data suggest that proteins in the extruded products may be in a more
polar environment than those in the non-extruded products. Protein aggregation
(hydrogen bond formation) prevents the exposure of hydrophobic sites on the
surface of the protein and thus prevents their contact with ANS. However, for all
extruded products in this study, higher temperatures seemed to unfold the proteins
and expose the hydrophobic core of the proteins to ANS binding.

It seems probable that the formation of amylose-Iipid complex and protein
aggregation, under the present extrusion conditions, are responsible for the initial
decreased access of ANS to hydrophobic sites in the protein in extruded samples.
However, hydrophobicities increased (decreasing AM) as die exit temperature was
increased for all extruded products containing 9% protein. Even though the same
observation cannot be made for samples at higher protein levels, it appears,
nevertheless, that higher extruder exit die temperatures possibly lead to the
weakening of the amylose-lipid complex and disruption of hydrogen bonds which
permit the unfolding of the proteins with a resultant exposure of more hydrophobic
sites to ANS binding.

The information obtained from this study suggests that DSC and FS may be
useful techniques for studying conformational changes in the major components of
wheat after extrusion processing of wheat flour. DSC is capable of providing both

qualitative and quantitative information, though limited to flour samples of

125

comparatively high starch and protein concentrations. Such information may include
the aggregation of the proteins as well as the interaction of starch components with

other minor components of the flour.

126

 

5.6 LITERATURE CITED

BILIADERIS, CG. 1990. Thermal analysis of food carbohydrates. In Thermal
Analysis of Foods pp. 168-220. V.R. Harwalker and C.-Y. Ma, eds. Elsevier
Applied Sci., New York.

BILIADERIS, C.G., PAGE, CM, and MAURICE, T.J. 1986a. On the multiple
melting transitions of starch/monoglycerides systems. Food Chem. 22:279.

BILIADERIS, C.G., PAGE, C.M., MAURICE, T.J., and JULIANO, B.O. 1986b.
Thermal characterization of rice starches: a polymeric approach to phase
transitions of granular starch. J. Agric. Food Chem. 3426.

CAMIRE, M.E., CAMIRE, A., and KRUMHAR, K. 1990. Chemical and nutritional
changes in foods during extrusion. Crit. Rev. Food Sci. Nutr. 29235.

DONOVAN, J.W., LORENZ, K. and KULP, K. 1983. Differential scanning
calorimetry of heat-moisture treated wheat and potato starches. Cereal
Chem. 60:381.

ELIASSON, A.-C. 1983. Differential scanning calorimetry studies on wheat starch-
gluten mixtures. I. Effect of gluten on the gelatinization of wheat starch. J.
Cereal Sci. 12199.

ELIASSON, A.-C., and HEGG, P.-O. 1980. Thermal stability of wheat gluten. Cereal
Chem. 572436.

EVANS, M.T.A., PHILLIPS, MC, and JONES, MN. 1979. The conformation and
aggregation of bovine B-casein A. ll. Thermodynamics of thermal association
and the effects of changes in polar and apolar interactions on micellization.
Biopolymers 1821123.

HARWALKAR, V.R. and MA, C.-Y. 1987. Study of thermal properties of oat globulin
by differential scanning calorimetry. J. Food Sci. 522394.

HOSENEY, RC. 1994. Gluten Proteins. In Principles of Cereal Science and
Technology, 2"“ ed. pp. 197-21 1. Am. Assoc. Cereal Chem. St. Paul, MN.

JAVOR, G.T., SOOD, S.M., CHANG, P., and SLATTERY, CW. 1991. Interactions
of triply phosphorylated human B-casein: fluorescence spectroscopy and
light scattering studies of conformation and self-association. Arch. Bioch.
Bioph. 289239.

KUGIMIYA, M., DONOVAN, J.W. and WONG, R.Y. 1980. Phase transitions of
amylose-Iipid complexes in starches: a calorimetric study. Starke 322265.

127

LAKKIS, J. and VILLOTA, R. 1992. Effect of acylation on substructural properties
of proteins: a study using fluorescence and circular dichroism. J. Agric. Food
Chem. 402553.

MATSUDOMI, N., KATO, A., and KOBAYASHI, K. 1982. Conformation and surface
properties of deamidated gluten. Agric. Biol. Chem. 4621583.

MYERS, CD. 1990. Study of thermodynamics and kinetics of protein stability by
thermal analysis. In Thermal Analysis of Foods. pp.16-50. V.R. Hanrvalker
and C.-Y. Ma (eds). Elsevier Applied Science, New York.

OLKKU, J. and RHA, CK. 1978. Gelatinisation of starch and wheat flour starch - a
review. Food Chem. 32293.

PRIVALOV, PL. and KHECHINASHVILI, 1974. A thermodynamic approach to the
problem of stabilization of globular protein structure: a calorimetric study. J.
Mol. Biol. 86:665.

QU, D. and WANG, SS. 1994. Kinetics of the formations of gelatinized and melted
starch at extrusion cooking conditions. Starch 462225.

STEVENS, DJ. and ELTON, G.A.H. 1971. Thermal properties of the starch/water
system. Part I. Measurement of heat of gelatinisation by differential scanning
calorimetry. Starke 2328.

STRASBURG, GM. and LUDESCHER, RD. 1995. Theory and applications of
fluorescence spectroscopy in food research. Trends Food Sci. Technol. 6269.

TAN FORD, C. 1980. The Hydrophobic Effect: Formation of Mice/[es and Biological
Membranes. Wiley-lnterscience, J. Wiley & Sons, New York.

VOUTSINAS, L.P., NAKAI, S., and HARWALKER, V.R. 1983. Relationships
between protein hydrophobicity and thermal functional properties of food
proteins. Can. Inst. Food Sci. Technol. J. 16:185.

YEBOAH, N.A., FREEDMAN, R.B., POPINEAU, Y., SHEWRY, PR, and TATHAM,
AS. 1994. Fluorescence studies of two y-gliadin fractions from bread wheat.
J. Cereal Sci. 19:141.

128

 

 

 

 

 

350 125
+Transition Energy
-e— Transition Temperature
300 1%
-- 120
2, 250 -- é’-
7. t-5
I ..
at
Z 200 -- 5
a 8
g -- 115 g.
L“ at
.5150" '2
E '2
'- 100 - - E
- - 1 10
5o ..
0 : : 4 ‘r : 105
9 20 30

Protein Content, %

Figure 5.1

Endothermic thermal transition energy and temperature changes of non-
extruded wheat flour as a function of protein content of flour.

129

 

Figure 5.2

Representative differential scanning thermograms of non-extruded wheat flour.
(a) = 9% protein content; (b) = 20% protein content; (c) = 30% protein content.

130

Endotherm ic Heat Flow

9 55.os°c

 

 

 

 

233.5Jrg ”——
a
1 1 1.21 °c
63.07%
210.7Jlg
b
11 c.3o°c
ee.2s°c
2727.113
c
122.56°C

131

 

Figure 5.3

Representative differential scanning thermograms of wheat flour extruded at die
temperature of 120°C and screw speed of 400 rpm.

(a) 9% protein content; (b) 20% protein content; (c) 30% protein content.

 

132

Endotherm ic Heat Flow

 

 

79.04%
265.8.1/9

89.70°C
215.9Jlg

 

110.92%

 

 

122.34%
100.37%
248.2Jlg
c
125.80°C

133

 

 

515
Screw Speed
+240 rpm
510-.
+320 rpm
+400 rpm
E
‘2. 505 4»
E
.2
3%
3 500 -—
x
8
c
E
5
.§ 495 «L
:5
::
4901~
485 -_
480 5 : t t :

 

 

 

120 140 160
Die Temperature, °C

Figure 5.4
Effects of extruder die temperature on the shifts in fluorescence

emission peak wavelengths of extruded wheat flour containing 9%
protein.

134

Table 5.1

Endothermic Reaction Temperatures and Enthalpies of Extruded and Non-
Extruded Wheat Flour Containing 9% Protein‘

 

Protein Content
9%

 

 

 

 

Exit Die Temperature °C Screw Speed (rpm) Tm (°C) AH (Jlg_)__
Non-Extruded 111.21 288.5
120 125.34 187.2
140 240 122.64 215.0
160 119.85 260.5
120 122.49 267.5
140 320 118.66 279.8
160 115.02 284.9
120 118.92 265.8
140 400 113.98 271.7
160 112.62 278.5

 

1Data are mean of two replicates.

135

 

 

 

 

 

 

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CHAPTER 6

GENERAL CONCLUSIONS

137

Extrusion processing has become one of the important new technologies that
is assuming an increasing role in the food industry in an effort to provide an
unprecedented array of novel products to satisfy consumer preferences, especially
in the ready-to-eat cereal market. In spite of the apparent success of the process,
fundamental understanding of the behavior of ingredients in the extrusion raw
materials is lacking, and therefore needs research attention. This research project
assessed the impact of extrusion processing on the functionality and physical
characteristics of the major components of wheat flour in an attempt to gain some
fundamental information and understanding on the physical and biochemical
processes that occur during extrusion processing of cereals.

In Chapter 3, the role of increased protein level in extrusion raw material and
how this protein augmentation, together with other extrusion variables, influenced
the physical characteristics of the extruded product was investigated. The level of
protein content in the feed material was found to have negative correlation with
expansion ratio and water absorption index, but positive correlation with bulk
density. The expansion ratio of extrudates was also found to correlate negatively
with bulk density but positively with water absorption index. Similarly, bulk density
correlated negatively with water absorption index. These results suggest that the
protein content of raw material, water absorption index, and bulk density are all
related to the expansion characteristics of extruded products.

The changes in total protein patterns as well as the possible interchanges
between the disulfide and sulfhyde contents of wheat flour proteins as a result of
extrusion were examined in Chapter 4. Extrusion processing of wheat flour resulted

in the reduction of protein disulfide bonds to form free sulfhyde groups while total

138

cysteine content remained fairly unchanged. Marked differences between SDS-
PAGE patterns of reduced and non-reduced total protein samples were observed.
Observed data suggested that there was very little or no sulfhydryl-disulfide
interchange reaction during extrusion processing, and that the intra molecular
scission of disulflde bonds seemed to be the predominant occurrence during the
extrusion process. This information should be helpful in determining the relationship
between disulfide and free sulfhydryl contents of an extruded product and the
textural characteristics of the extrudates.

Data from differential scanning calorimetry and fluorescence spectroscopy
studies in Chapter 5 indicate that there were some oonfonnational changes in the
starch and proteins of wheat flour as a result of twin-screw extrusion processing.
Differential scanning calorimetry data suggest the formation of a complex between
the amylose and lipid contents in the wheat flour. This reaction is an exothermic
aggregation process that leads to a decrease in the overall thermal energy of the
process. Furthermore, fluorescence data suggest that extruded products may be
in a more polar environment than non-extruded products.

Like many other studies in extrusion processing, data obtained from this
study have established that extrusion is a complex process which involves several
variables, each of which contributes differently under different sets of extrusion
conditions to the overall quality characteristics of the extrudates. The interactions
among variables are complex, but have significant impact on quality attributes of
extruded products, and make it challenging to differentiate among the individual
variables in terms of changes in characteristics of the final product. Nevertheless,

the results of this study add to the pool of available knowledge on the subject of

139

extrusion processing of cereals and further provide evidence that, continuous

research in this area will be needed to fully understand the phenomena of extrusion

processing as they apply to cereals, and how fundamental knowledge can be
transformed into improvement of current products and the design of new ones.
The significant findings of this research can be summarized as follows:

0 The protein content in extrusion raw materials has significant effects on the
water absorption and bulk density of extrudates, which in turn, are all related
to the expansion characteristics of the extrudates.

0 High protein content in wheat flour reduces expansion capability of wheat
flour extrudates and their ability to absorb water. Thus, in extrusion of wheat
flour, supplementation of the protein content of the flour may not be
beneficial to improving the quality characteristics of the extruded products.

0 Extrusion of wheat flour results in the reduction of disulfide bonds to form
free sulfhydryl groups. The total cysteine content is not changed during
extrusion processing, and intra-molecular scission of disulfide bonds appears
to be the predominant occurrence. This information can be helpful in
determining the relationship between and among the total cysteine, disulfide
and sulfhydryl contents of wheat flour and the textural characteristics of the
extrudates.

e Extrusion processing results in protein aggregation which increases the
stability of the proteins in addition to the apparent increase in hydrophilic

(polar) environment of proteins after extrusion.

140

CHAPTER 7

FUTURE RESEARCH

141

Extrusion technology has many present and possible future applications in
the food industry for cooking, forming, and expanding cereal grains, as well as for
texturizing proteins. There is, however, still much to understand with extrusion
processing and its impact on the components of the starting raw ingredients, and
their effects on the quality of the extruded products. Certainly, extrusion processing
will play a much broader role in both traditional and non-traditional products in the
future. Understanding the mechanisms of ingredient interactions will be the key in
this direction. Thus, future work should aim at gaining more understanding of
ingredient interactions. Research will be needed to give accurate estimation of
gelatinization and melting during extrusion processing.

The role of protein in extrusion technology will need much more focus in view
of the current controversy about the involvement of disulfide bonds in extrusion
processing. Furthermore, there is the need for more understanding of the role
disulfide bonds play with respect to the texture and quality of extruded cereal
products. More specifically, research will need to determine the exact relationship
between disulfide and sulfhydryl contents of cereal proteins and the exact
mechanism by which they are involved in the texturization process.

In view of the success of extrusion of soy protein, and considering the
functional properties of vital gluten, it should be feasible to subject vital gluten to
extrusion processing. Future research should therefore aim at gaining more
understanding of the mechanism of vital gluten interactions with starch components
and possibly the chemical and physical modifications of gluten.

Having established in the present research that extrusion operating variables

have a direct impact on the quality of extrudates, future research work will need to

142

 

establish quantitatively the relationship between changes in molecular weights of
proteins and starches and time, shear, and extrusion variables. Studies on modeling
of extrusion parameters using response surface methodologies will be necessary
to establish much more reliable relationships between dependent and independent
variables. Such studies will also be useful in the upscaling of extrusion research

from the laboratory into industrial processing conditions.

143

 

APPENDICES

144

APPENDIX 1

Composition and Farinogram Data1 of Soft Wheat Flour (cv. Harus), Vital Gluten
and Flour-Gluten Mixture Samples Used in Experiments

 

F lour-Gluten Samples

 

 

Component (%) cv. Harus Vital Gluten 20% Protein 30% Protein
Moisture 10.3 9.2 - -
Protein (N x 5.7) 9.2 69.8 - -
Ash 0.5 0.4 - -
Fat 1.6 2.1 - -
Carbohydrates2 78.4 18.5 - -
Eatinogram
Absorption (%) 52.2 nd 63.0 74.6
Arrival time (min) 0.5 nd 2.0 6.5
Peak time (min) 1.0 nd 7.75 9.0
Peak height (mm) 25 nd 48 69
Stability (min) 1.5 nd 29.0 > 30

 

‘Constant flour weight method.
2Determined by difference.
nd = not determined.

145

APPENDIX 2

Raw Data1 for Extrusion Experiments

 

 

8qu Water Abs. Sulfhydryl Disulfide Total

Expansion Density Index Content Content Cysteine Content

Ratio (91ml) (9/9) (uMImg Prat) (uMImg Prat) (uM/mg Prat)

lamp SS Prot 1 2 1 2 1 2 1 2 1 2 1 2
120 240 9 3.15 3.20 0.132 0.130 6.16 6.14 7.00 6.90 38.28 38.43 83.55 83.76
140 240 9 2.76 2.90 0.125 0.122 6.24 6.16 9.55 10.00 36.91 36.76 83.37 83.51
160 240 9 1.80 1.95 0.163 0.164 6.91 6.70 10.95 11.00 35.39 35.46 81.72 81.92
120 320 9 3.16 3.05 0.124 0.120 5.88 5.86 7.85 7.25 41.04 40.99 89.92 89.22
140 320 9 2.66 2.55 0.129 0.132 5.72 5.82 8.60 8.95 37.40 37.57 83.40 84.09
160 320 9 1.83 1.80 0.159 0.153 8.32 6.16 10.45 10.75 35.28 34.97 81.01 80.68
120 400 9 2.55 2.63 0.116 0.119 5.45 5.55 8.15 7.80 40.24 40.21 88.62 88.21
140 400 9 2.16 2.05 0.134 0.130 5.79 5.83 9.10 9.06 37.32 37.67 83.73 84.40
160 400 9 1.80 1.75 0.132 0.129 6.07 5.93 13.10 13.47 37.90 37.55 88.89 88.56
120 240 20 1.98 2.07 0.339 0.331 5.50 5.41 3.65 3.50 36.18 36.52 76.00 76.53
140 240 20 2.50 2.52 0.164 0.165 5.57 5.65 4.90 5.10 32.89 33.02 70.67 71.14
160 240 20 2.93 2.92 0.090 0.090 5.46 5.51 9.45 9.40 31.89 31.92 73.23 73.24
120 320 20 2.33 2.42 0.242 0.237 5.92 5.43 3.95 4.10 33.47 33.34 70.89 70.77
140 320 20 2.48 2.53 0.143 0.145 5.50 5.32 4.90 4.80 32.86 32.43 70.62 69.66
160 320 20 2.89 2.98 0.074 0.074 5.28 5.07 10.80 10.50 29.30 29.68 69.39 69.85
120 400 20 2.66 2.76 0.190 0.189 5.43 5.49 3.95 4.20 33.56 33.26 71.06 70.72
140 400 20 2.40 2.37 0.149 0.147 5.31 5.11 5.10 5.30 32.73 33.05 70.55 71.40
160 400 20 2.71 2.71 0.097 0.094 5.08 4.97 9.85 10.00 28.47 28.35 66.79 66.69
120 240 30 2.15 2.30 0.184 0.181 4.23 4.32 2.78 2.80 22.66 22.73 48.09 48.26
140 240 30 2.10 2.15 0.209 0.209 4.40 4.45 2.98 2.90 22.07 21.86 47.11 46.61
160 240 30 2.15 2.20 0.218 0.214 4.50 4.39 2.95 2.91 22.43 22.31 47.80 47.52
120 320 30 2.20 2.15 0.233 0.235 4.48 4.55 2.78 2.79 22.59 22.95 47.96 48.69
140 320 30 2.15 2.25 0.183 0.183 4.44 4.52 3.18 3.17 22.87 22.82 48.92 48.81
160 320 30 2.20 2.10 0.194 0.194 4.59 4.55 3.15 3.10 21.92 21.55 46.98 46.19
120 400 30 2.10 2.13 0.202 0.204 4.76 4.63 3.46 3.29 20.41 20.90 44.27 45.08
140 400 30 2.10 2.11 0.218 0.218 4.58 4.76 3.32 3.28 21.89 21.60 47.10 46.48
1 .1 .1 .174 4. 4. . 0 3.41 1 21.7 435 4&87

146

 

Appendix 2, cont’d

 

 

Specific
Mechanical Fluorescence Peak Transition Transition
Energy Intensity Wavelength Temperature Energy
(W hlkg) (A.U-) (nm) (°C) (Jig)

1991p 88 Prot 1 2 1 2 1 2 1 2 1 2
120 240 9 144.00 143.80 0.05 0.05 502.00 500.00 125.34125.32187.20 188.90
140 240 9 153.60 150.00 0.05 0.05 495.50 497.00 1226412160 215.00 214.20
160 240 9 122.40 123.80 0.06 0.05 493.50 493.50 1198511899 306.50 307.10
120 320 9 208.00 209.00 0.05 0.05 507.00 510.00 1224912400 267.50 267.00
140 320 9 208.00 210.20 0.05 0.06 503.00 502.50 1186611845 289.80 288.40
160 320 9 179.20 180.40 0.05 0.05 496.50 496.00 115.02114.70 284.90 284.50
120 400 9 268.00 266.00 0.05 0.04 511.50 510.00 118.92117.82 265.80 265.20
140 400 9 260.00 263.20 0.05 0.05 495.50 494.50 113.98113.01 288.70 289.00
160 400 9 240.00 239.00 0.08 0.08 493.00 493.50 1126211244 278.50 277.40
120 240 20 144.00 143.00 0.06 0.06 495.00 496.50 121.63120.99181.70 182.20
140 240 20 139.20 139.00 0.05 0.05 500.00 502.00 1092010921 212.10 213.00
160 240 20 132.00 135.00 0.06 0.05 485.50 484.50 1245912388 222.80 221.00
120 320 20 195.20 195.00 0.05 0.05 494.00 493.00 116.62116.32157.00 157.50
140 320 20 192.00 192.20 0.05 0.06 498.50 498.00 115.20115.43 207.30 207.30
160 320 20 176.00 177.80 0.07 0.07 492.50 490.50 101.59100.47 205.40 205.60
120 400 20 240.00 239.00 0.05 0.05 495.50 494.50 1223412300 215.90 215.30
140 400 20 220.00 222.20 0.06 0.06 499.00 498.50 111.68110.98 204.10 204.90
160 400 20 240.00 239.00 0.09 0.09 490.50 490.00 1257712465 257.20 258.20
120 240 30 144.00 145.80 0.06 0.06 491.50 490.00 126.18126.77 218.50 217.90
140 240 30 139.20 139.00 0.05 0.05 496.50 495.50 1279012743 222.90 223.10
160 240 30 127.20 129.00 0.05 0.05 498.50 498.00 1244812504 214.40 213.80
120 320 30 192.00 192.20 0.05 0.05 495.00 494.00 140.21 141.21 201.80 200.60
140 320 30 176.00 174.00 0.05 0.04 502.00 500.00 121.77120.94 242.20 243.10
160 320 30 176.00 177.00 0.05 0.05 495.50 495.00 1246712500 239.40 239.10
120 400 30 220.00 219.20 0.05 0.05 494.50 496.00 1258012532 248.20 249.00
140 400 30 220.00 222.00 0.04 0.04 499.00 498.50 1293312967 224.20 223.60
1 4 212. 212.2 . 0.06 494.00 499.99 1296612699 233.29 293m

 

‘Abbreviations and symbols: A.U., arbitrary units; Temp, exit die temperature (°C);
SS, screw speed (rpm); Prot, protein content (%); 1,2, replicates.

147

Analyses of Variance Tables for Experimental Data1

APPENDIX 3

I. Expansion Ratio

Sum of Mean
Source DF Squares Square F Value Pr > F
SS 2 0.20979259 0.10489630 27.56 0.0001
TEMP 2 0.42789259 0.21394630 56.22 0.0001
SS‘TEMP 4 0.13680741 0.03420185 8.99 0.0001
PROT 2 1.51922593 0.75961296 199.61 0.0001
SS‘PROT 4 0.56877407 0.14219352 37.36 0.0001
TEMP’PROT 4 4.29790741 1.07447685 282.34 0.0001
SS'TEMP'PROT 8 0.63099259 0.07887407 20.73 0.0001
Error 27 0.10275000 0.00380556
Total 53 789414259

R-Square: 0.986984 C.V.: 2.586147
ll. Bulk Density

Sum of Mean
Source DF Squares Square F Value Pr > F
SS 2 0.00471693 0.00235846 343.28 0.0001
TEMP 2 0.02381404 0.01190702 173.10 0.0001
SS‘TEMP 4 0.00395230 0.00098807 143.82 0.0001
PROT 2 0.04184293 0.02092146 3045.17 0.0001
SS’PROT 4 0.00529074 0.00132269 192.52 0.0001
TEMP‘PROT 4 0.06506430 0.01626607 2367.57 0.0001
SS'TEMP‘PROT 8 0.01570137 0.00196267 285.67 0.0001
Error 27 0.00018550 0.00000687
Total 53 0.16056809

R-Square: 0.998845 C.V.: 1.570764
III. Water Abeorptlon Index

Sum of Mean
Source DF Squares Square F Value Pr > F
SS 2 0.37993704 0.18996852 17.31 0.0001
TEMP 2 0.09601481 0.04800741 4.37 0.0226
SS'TEMP 4 0.21068519 0.05267130 4.80 0.0047
PROT 2 20.75099259 10.37549630 945.30 0.0001
SS'PROT 4 1.31444074 0.32861019 29.94 0.0001
TEMP‘PROT 4 1.07902963 0.26975741 24.58 0.0001
SS‘TEMP‘PROT 8 0.15873704 0.01984213 1.81 0.1194
Error 27 0.29635000 0.01097593
Total 53 24.28618704

R-Square: 0.987798 C.V.: 1.970178

148

IV. Speclflc Mechanlcal Energy

Sum of Mean
Source DF Squares Square F Value Pr > F
SS 2 84921 .96000 42460.9800 31018.57 0.0001
TEMP 2 2458.404444 1229.20222 897.96 0.0001
SS‘TEMP 4 284.742222 71.185556 52.00 0.0001
PROT 2 3476.03111 1 1738.015556 1269.65 0.0001
SS’PROT 4 2008.34222 502.085556 366.78 0.0001
TEMP'PROT 4 1144.724444 286.181111 209.06 0.0001
SS‘TEMP‘PROT 8 636.488889 79.561 1 1 1 1 58.12 0.0001
Error 27 36.9600000 1.368889
Total 53 94967.65333
R-Square: 0.99961 1 C.V.: 0.622706
V. Free Sulfhydryl Content
Sum of Mean
Source DF Squares Square F Value Pr > F
SS 2 3.31895926 1.65947963 63.34 0.0001
TEMP 2 114.38589259 57.19294630 2183.09 0.0001
SS'TEMP 4 2.12449630 0.531 12407 20.27 0.0001
PROT 2 363.42934815 181 .71467407 6936.16 0.0001
SS‘PROT 4 2.47087407 0.61771852 23.58 0.0001
TEMP'PROT 4 64.13894074 16.03473519 612.06 0.0001
SS‘TEMP'PROT 8 5.37357037 0.67169630 25.64 0.0001
Error 27 0.70735000 0.02619815
Total 53 555.94943148
R-Square: 0.998728 C.V.: 2.578808
VI. Disulflde Content
Sum of Mean
Source DF Squares Square F Value Pr > F
SS 2 2.1254370 1.0627185 29.79 0.0001
TEMP 2 68.1644926 34.0822463 955.43 0.0001
SS‘TEMP 4 6.1648296 1.541207 43.20 0.0001
PROT 2 2281 .6417593 1140.82087 31980.65 0.0001
SS'PROT 4 26.1638963 6.5409741 183.36 0.0001
TEMP‘PROT 4 37.9341074 9.4835269 265.85 0.0001
SS'TEMP'PROT 8 13.6748704 1.7093588 47.92 0.0001
Error 27 0.9631500 0.0356722
Total 53 2436.8325426
R-Square: 0.999605 C.V.: 0.614507

149

Source

0
11

SS

TEMP

SS‘TEMP

PROT

SS'PROT
TEMP'PROT
SS'I’EMP‘PROT
Error

Total

SO‘nbNdeN

0|
0

R-Square: 0.999722

0
1‘

Source

SS

TEMP

SS'TEMP

PROT

SS'PROT
TEMP'PROT
SS'TEMP‘PROT
Error

Total

agar-Swen»

R-Square: 0.985545

Source DF

VII. Total Cystelne Content

SS

TEMP
SS‘TEMP
PROT
SS‘PROT

TEMP‘PROT
SS'TEMP'PROT

Error
Total

O-b-hNbNN

27
53

R-Square: 0.985117

Sum of Mean
Squares Square F Value
1.825378 0.912689 6.63
42.498433 21.249217 154.40
21 .997656 5.499414 39.96
13077.673900 6538.836950 47513.58
111.738489 27.934622 02.98
30.365067 7.591267 55.16
83.926278 10.490785 76.23
3.715750 0.137620
1 3373.740950
C.V.: 0.547628

VIII. Fluorescence Intenslty
Sum of Mean
Squares Square F Value
0.00045633 0.00022817 67.70
0.00175433 0.00087717 260.26
0.00165933 0.00041483 123.08
0.00072678 0.00036339 107.82
0.00042389 0.00010597 31.44
0.00068256 0.00017064 50.63
0.000501 11 0.00006264 18.59
0.00009100 0.00000337
0.00629533

C.V.: 3.413783

IX. Peak Wavelength

Sum of Mean
Squares Square F Value
6423148148 32.11574074 38.75
362.00925926 181.00462963 218.42
52.04629630 13.01157407 15.70
272.39814815 13619907407 164.35
4207407407 10.51851852 12.69
531 .29629630 13282407407 160.28
15698148148 19.62268519 23.68
223750000 0.8287037
1503.4120370

C.V.: 0.183292

150

Pr>F

0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001

Pr>F

0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001

 

X. Transition Temperature

Sum of Mean
Source DF Squares Square F Value Pr > F
SS 2 665415593 33.2707796 124.90 0.0001
TEMP 2 361 .9847259 180.9923630 679.45 0.0001
SS‘TEMP 4 301 .0947852 75.2736963 282.58 0.0001
PROT 2 1245.4396259 622.7198130 2337.70 0.0001
SS‘PROT 4 390.8122185 977030546 366.78 0.0001
TEMP‘PROT 4 89.8678852 22.4669713 84.34 0.0001
SS‘TEMP'PROT 8 612.0284704 765035588 287.20 0.0001
Error 27 7.1923000 0.2663815
Total 53 3074.9615704
R-Square: 0.997661 C.V.: 0.427215
XI. Transltlon Energy
Sum of Mean
Source DF Squares Square F Value Pr > F
SS 2 6115.278148 3057639074 8853.22 0.0001
TEMP 2 9857080370 4928.540185 14270.30 0.0001
SS'TEMP 4 4445.049630 1 11 1.262407 3217.60 0.0001
PROT 2 30814.044815 15407022407 44610.15 0.0001
SS'PROT 4 5845.781852 1461 .445463 4231.53 0.0001
TEMP'PROT 4 3360.422963 840.105741 2432.48 0.0001
SS'TEMP‘PROT 8 9898.167037 1237270880 3582.45 0.0001
Error 27 9.325000 0.345370
Total 53 70345.149815
R-Square: 0.999867 C.V.: 0.252222

1SS = Screw Speed; Temp = Exit Die Temperature; Prot = Protein Content.

151

APPENDIX 4
Electrophoregrams of Non-Reduced Total Proteins in Extruded Wheat Flour
Screw Speeds: A = 240 rpm; B = 320 rpm; C = 400 rpm

Exit Die Temperatures: 1 = 120°C; 2 = 140°C; 3 = 160°C
Gels: a = 20%; b = 30%

 

 

         

.14 5;

123123123 123123123

(a) (b)

152

APPENDIX 5
Electrophoregrams of Reduced Total Proteins in Extruded Wheat Flour
Screw Speeds: A = 240 rpm; B = 320 rpm; C = 400 rpm

Exit Die Temperatures: 1 = 120°C; 2 = 140°C; 3 = 160°C
Gels: a = 20%; b = 30%

 

 

 

(a) (b)

 

153

 

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