£1 IIHIHHIIU!llllHIHUI!!!"HIHIHIIHUIIUINIIHIINW I 193 10409 8839 “58:5 Date— 0-7 639 /_/ ‘1- .w LIBRARY Michigan State University , ‘ ‘ .— This is to certify that the thesis entitled FUNCTIONALITY OF LIQUID CYCLONE PROCESSED COTTONSEED FLOUR‘IN L BREAD SYSTEMS presented by Mona A. El—Minyawi has been accepted towards fulfillment of the requirements for Ph.D. degree in Food Science & Human Nutrition w fiajor profuzt 7/21 /yc/ ‘IV1ESI_} RETURNING MATERIALS: Place in book drop to LJBRARJES remove this checkout from ” your record. FINES wiH — , be charged if book is returned after the date stamped be10w. i - ‘ r WW9 if 1 .3‘ ,——J- , 3 i 3 3k-IO" {tbfl‘tr 9 / I ., 3m like? 1K) E. ,5 ~’ ;+ . , . 5/ fT / ; W) FUNCTIONALITY 0F LIQUID CYCLONE PROCESSED COTTONSEED FLOUR IN BREAD SYSTEMS By Mona A. Ei-Minyawi A DISSERTATION Submitted to Michigan State University in partiai fulfiiiment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1980 ABSTRACT FUNCTIONALITY OF LIQUID CYCLONE PROCESSED COTTONSEED FLOUR IN BREAD SYSTEMS By Mona A. El-Minyawi The effects of Liquid Cyclone Processed cottonseed flour (LCP) substitution were studied in wheat dough systems. Farinograph studies showed increase in the water absorption and arrival time as well as decrease in sta- bility as the level of cottonseed flour increased in dough systems. It was found that increasing the level of sub- stitution with starch decreased farinograph water absorp- tion, arrival time, and increased stability and resistance to mixing. LCP cottonseed flour breads were evaluated for acceptability; color was found to be the most objectionable character of the bread. The effect of salt, conditioner and oxidant were studied in 0, 8 and 16% LCP cottonseed flour substituted dough systems. Mixograph studies showed that each addi- tive added separately had strengthening effect on dough systems; this strength was much more noticeable in the substituted dough with 8 and 16% LCP cottonseed flour. Potassium bromate in combination with any other additive did not affect dough rheology; salt showed a strengthening effect in all the double and triple combinations of Mona A. El-Minyawi additives and it increased with increasing the salt level. Baking studies using 8% LCP cottonseed flour with single additives indicated that 1% salt produced high volume bread. The effect of double combinations of addie tives significantly increased crumb softness, and maximum softness was obtained with the combination of 1% salt, 30 ppm KBro3. Triple combinations of additives had better effect on bread characteristics with combination of 1% salt, 1.5% dough conditioners (Tween 20), and 50 ppm K‘Bro3 producing the softest crumb. The functionality of LCP cottonseed flour was studied in Egyptian "Baladi" bread system. Bread baked with O, 4, 8, 12 and 16% cottonseed flour showed a decrease in bread volume as the cottonseed level increased. -Sensory data indicated that bread was acceptable even at 12% level of substitution. Mixograph studies showed the maximum effect of four types of dough conditioners on peak time, peak height and stability to be 0.5% conditioner. Egyptian bread baked with these conditioners, retained its softness after 3 days of storage when Tween 20 and Tandem 552 were added at 0.5% level. The softness was retained after 6 days when Tween 20 was added. The protein content of substituted bread increased significantly, as well as the lysine content of the substituted bread. Mona A. El-Minyawi Control and substituted doughs were modified using different chemical reagents. Low levels of urea, sodium- dodecyl-sulfate and succinic anhydride, caused slight strengthening to both dough systems. High concentrations of reagents caused weakening of dough. The ratio of SS/SH that are involved in dough mixing is higher in stronger doubh than weaker ones. This ratio also indicated that there was more disulfides involved in resistance to mixing than thiols in both control and LCP substituted systems. Scanning electron microscopy of control and substi- tuted dough systems shows that LCP cottonseed flour incor- porated in dough gave thicker, less flexible gluten matrix than the control and some pockets were evident when dough conditioner (Tween 20) was added (0.5%), the protein matrix seemed more fluid, flexible and continuous. Fixation, dehydration and critical point drying as sample preparation procedures drastically altered the ultrastructure of both dough systems when compared to nitrogen freezing and freeze drying procedures. To My Mother, My Husband and My Children ii ACKNOWLEDGMENTS I wish to express my deepest appreciation to my major professor, Dr. Mary E. Zabik, for her patience, continu- ous support and encouragement during the course of this study, and for her assistance in the preparation of the thesis manuscript. Sincere appreciation is also extended to members of my committee, Dr. Wanda Chenoweth for her support and encouragement, Dr. J.R. Brunner and Dr. P. Markakis for their valuable advice and support, Dr. L. Minor of the Department of Hotel, Restaurant and Institu- tional Management for serving on the guidance committee and for his valuable advice. I also extend my deepest thanks to Dr. L.E. Dawson for attending my oral exam, and Dr. J. Gill of the Department of Dairy Science and Dr. C. Cress of the Department of Crop and Soil Science for their statistical advice and help during the preparation of this study. My deepest thanks go to Dr. N.T. Yamazaki of the Soft Wheat Lab, Wooster, Ohio for supplying the small farinograph bowl used in this study. A special acknowledgment of appreciation goes to my mother, my husband and my children who provided encourage- ment, motivation and support to make this achievement possible. TABLE OF CONTENTS LIST OF TABLES. LIST OF FIGURES . INTRODUCTION. REVIEW OF LITERATURE. Wheat Proteins Dough Formation. . Chemical Bonds Cottonseed Flour . Cottonseed Flour Processing. Cottonseed Use in Food . Nutritional Value. . . Supplemental Value of Plant Protein on Wheat Flour. Supplementary Value and Protein Quality of Oilseed Flour. . . . Effect of Processing on Protein Quality. Protein Modification and Functional Properties Salts. . . . . . . . . . . . . . . . Oxidizing Agents Surfactants. . . Scanning Electron Microscopy . Principle of Operation Application. . Sample Preparation EXPERIMENTAL DESIGN . MATERIALS AND METHODS . Dough Rheology Study . . Farinograph Testing. Mixograph Testing. . . Viscoamylograph Testing. Baking Study . . Baking Procedure for American Bread. iv Page vii NNc—lu—l—l NOU'IU'IOLDUT 01 pH of Dough. 50 Compressability. 51 Color. . . . 51 Sensory Evaluation . . . 51 Baking Procedure for Egyptian Bread. 52 Sensory Evaluation . . . . . 53 Tenderness . 53 Chemical Analyses. 54 Moisture . . 54 Kjeldahl Total Nitrogen. 55 Lipid. . . . 56 Ash. . . 58 Sulfhydryl Groups. . . 58 Total Sulfhydryl Groups. . 59 Estimation of Rheologically Active Thiol and Disulfide Groups in Dough. . . . 61 Evaluation of Bonding Systems in Dough . 62 Amino Acid Analyses. . . . . . . 62 Scanning Electron Microscopy Study . 63 RESULTS AND DISCUSSION. 65 Effect of LCP Cottonseed Flour on Dough Rheo- logy . . . . . . '55 Farinograph Study. . 55 Viscoamylograph Study. 74 Baking Study . . . 77 Mixograph Study of Single Additive Effect. 84 Salt . . . 86 Tween 20 . . . 88 Potassium Bromate. . 91 Mixograph Study of Double Additive Effect. 93 Mixograph Study of Triple Additive Effect. 97 Baking Study . . . . . . 100 Sensory Evaluation . . . 102 Egyptian Bread Substituted with LCP. Cottonseed Flour. . . . . ll7 Sensory Evaluation . . 120 Effect of Four Dough Conditioners on Dough Rheology . . . . . . 120 Mixograph Study. . . . 120 Baking Study (Egyptian Bread). . . 129 Supplemental Value of Cottonseed Flour on Egyptian Bread . . 135 Chemical Modification Studies. 139 Urea . . . . 140 Sodium- -Dodecy1- Sulfate . l4l Succinic Anhydride . . 14l Disulfide and Sulfhydryl Groups. 147 N-ethylmaleimide. Dithiothreitol. Scanning Electron Microscopy Studies: APPENDIX . American Bread Score Card . Egyptian Bread Score Card . Optimum Bread Scbre Card. SUMMARY AND CONCLUSIONS. PROPOSAL FOR FUTURE RESEARCH REFERENCES . vi Page . 148 . 148 . 154 . 164 . 164 . 165 . 166 . 167 . 173 . 175 Table 10 11 12 LIST OF TABLES Farinograph data and SD for HRW wheat flour dough substituted with LCP cottonseed flour. Farinograph data and SD for HRW wheat flour dough substituted with wheat starch. . Proximate analysis of bread prepared with dif- ferent levels of LCP cottonseed flour. Mean and standard deviation of objective measurement of wheat/cottonseed flour bread. Mean and standard deviation of sensory evalua- tion of wheat/cottonseed flour bread . . Mean squares and F statistics for mixograph parameters of HRW wheat/LCP cottonseed dough Effect of interaction of double combinations of additives on mixograph characters at 0% LCP cottonseed flour dough Effect of interaction of double combinations of additives on mixograph characters at 8% level of LCP cottonseed flour dough. The effect of the interaction between the three additives on the mixograph characters of a control wheat dough. . . . . The effect of the interaction between the three additives on the mixograph characters of 8% LCP cottonseed flour dough All the possible combinations of salt, condi- tioner and bromate each at 3 levels. Mean square of analysis of variance of 8% sub- stituted bread, and the effect of additives and their interaction on objectiVe measurements. Page 66 69 79 8'1 83 85 94 96 98 99 . 101 . 103 Table Page 13A The mean effect of single additives on bread volume and compressability. . . . . .3. . . . . . 104 138 The mean effect of double combinations of addi- tives on 8% LCP cottonseed flour bread volume and compressability . . . . . . . . . . . . . . . . . 105 13C The mean effect of triple combinations of addi- tives on 8% LCP cottonseed flour bread volume and compressability . . . . . . . . . . . . . . . . . 105 14 Mean squares and F statistics significance for sensory evaluation parameters of 8% LCP cotton- seed flour bread baked with additives . . . . . . 107 15 The effect of interactions between two additives on the sensory evaluation characters of 8% LCP cottonseed flour bread. . . . . . . . . . . . . . 109 16 The mean effect of interactions between three additives on the sensory evaluation characters of 8% LCP cottonseed flour bread. . . . . . . . . 111 17 Sensory evaluation data of optimum 8% LCP cotton- seed bread and untreated ones . . . . . . . . . . 116 18 Effect of LCP cottonseed flour on Egyptian bread volume and specific volume. . . . 119 19 Effect of LCP cottonseed flour on breadmaking properties. . . . . . . . . . . . . 121 20 Mean square of mixograph characters as affected by cottonseed level, conditioners and conditioner level . . . . . . . . . . . . . . . . . . . . . . 125 21 The average effect of dough conditioners on mixo- graph properties of HRW wheat/LCP cottonseed flour dough . . . . . . . . 126 22 Mean squares of analysis of variance of effect of dough conditioners on volume or specific volume of Egyptian bread . . . . . . . . . . . . . . . . 129 23 Effect of conditioners on loaf volume and specific volume of Egyptian bread made with 0,4, 8, 12 and 16% LCP cottonseed flour. . . . . . . . . 130 viii Table 24 25 26 27 28 Effect of conditioners on bread tenderness after 3 storage times. Proximate composition of bread prepared with different levels of cottonseed protein Protein and amino acid composition of wheat flour (WF), LCP cottonseed flour and their mixtures . . . . . . . Essential amino acid content of wheat/LCP cot- tonseed flour mixtures and FAD/WHO suggested pattern of amino acid requirement. Some chemical and physical properties of wheat flour and 8% substituted flours and doughs ix Page 131 136 137 138 153 Figure 4A 4B BA BB 8C LIST OF FIGURES Farinograph absorption of dough systems substi- tuted with cottonseed flour, and starch. Farinograph arrival time of wheat/cottonseed and wheat/starch doughs. . Farinograph peak time of wheat/cottonseed and wheat/starch doughs. . . Effect of cottonseed flour and starch on Farino- graph Mixing Tolerance Index . . . . Effect of cottonseed flour and starch on Farino- graph Twenty Minute Drop Viscoamylograph peak viscosity and viscosity at 60 min. for wheat flour substituted with LCP cottonseed flour . . . . . . Effect of cottonseed flour on pH of dough during fermentation . . . . . . . . Bread substituted with LCP cottonseed flour a) Control (untreated) b) 4% cottonseed flour c) 8% cottonseed flour d 12% cottonseed flour e 16% cottonseed flour. Effect of salt on mixograph properties of 0, 8, 16% dough substituted with cottonseed flour. Effect of conditioner "Tween 20" on mixograph parameters of O, 8, 16% doughs substituted with cottonseed flour . . . . . . . . . . . . . . Effect of potassium bromate on mixograph proper- ties of 0,8,16% dough substituted with cottonseed flour . . . . Page 68 71 72 73 75 76 78 82 87 89 92 Figure Page 9A Effect of additives on 8% LCP cottonseed bread Single Additives a) Control Untreated Bread b) Potassium Bromate 30 ppm c) Potassium Bromate 50 ppm d) Salt 1% e) Salt 2%. . . . . . . . . . . . . . . . . . . .112 98 Effect of additive interaction on 8% LCP cot- tonseed bread Double Combinations Control Untreated Conditioner 0.5% + KBro3 30 ppm Conditioner 1.5% + KBro 50 ppm Conditioner 1.5% + KBrog 30 ppm a) b) C) d) e) Conditioner 0.5% + KBro3 50 ppm f) Salt 1% + KBro 30 ppm 9) Salt 1% + Cond tioner 0.5% h) Salt 1% + KBro3 50 ppm i) Salt 1% + Cond1tioner 1.5% j) Salt 2% + KBr03 30 ppm k) Salt 2% + Cond1tioner 0.5% 1) Salt 2% + KBr03 50 ppm m) Salt 2% + Cond1tioner 1.5% . . . .-. . . . . .113 9C Effect of additive interaction on 8% LCP cot- tonseed bread Triple Combinations a) Control Untreated Bread b) Salt 1% + Conditioner 0.5% + KBr03 30 ppm c) Salt 1% + Conditioner 1.5% + KBr03 50 ppm d) Salt 1% + Conditioner 0.5% + KBr03 50 ppm e) Salt 1% + Conditioner 1.5% + KBr03 30 ppm f) Salt 2% + Conditioner 0.5% + KBr03 30 ppm 9) Salt 2% + Conditioner 1.5% + KBr03 50 ppm h) Salt 2% + Conditioner 0.5% + KBro3 50 ppm 1) Salt 2% + Conditioner 1.5% + KBr03 30 ppm. . .114 10A Effect of optimum levels of additives on LCP cottonseed bread a) Untreated 8% LCP cottonseed bread b) Salt 0.7% + Conditioner 0.4% + KBro3 37 ppm. .118 xi Figure 108 11A 11B 12A 12B 13 14A 14B 15 16 17 18 Comparison between optimum bread made of white flour, dark flour, and 8% LCP cottonseed flour a) Bread made of 85% extracted HRW wheat flour b) Bread made of 8% LCP cottonseed substituted flour c) Bread made of straight grade HRW wheat flour. . . . . . . . . . . . . . . . . A typical loaf of Egyptian bread. Effect of LCP cottonseed flour on Egyptian bread characters. 0, 4, 8, 12 and 16 repre- sent the levels of substitution with cottonseed flour percent . . . . . . . . . . . . . . Tenderness of Egyptian bread substituted with cottonseed flour after 0,3 and 6 days of storage . . . . . . . . Effect of dough conditioners on substituted bread after 0, 3 and 6 days of storage. Effect of urea on 0,8 and 16% substituted dough rheology. . . . . . . . . . Effect of $05 on 0% substituted dough rheology. Effect of $05 on 8,16% substituted dough rheology. . . . . . . . . . . . . . . . . Effect of succinic anhydride on 0,8 and 16% substituted dough rheology. . . . Effect of NEMI on loss of resistance in A. wheat dough B. 8% LCP cottonseed substituted dough systems Effect of dithiothreitol on the resistance of A - control dough and B - 8% LCP cottonseed substituted dough systems . Effect of dithiothreitol on development times of A - Control dough and B - 8% LCP cottonseed substituted dough systems . . . . . Page 118 122 123 132 134 142 143 144 146 149 151 152 Figure Page 19 Scanning Electron Micrograph of HRW wheat flour dough a) 1000X b) 3000X c) 5000X. . . . . . . . . . . . . . . . . . . . 156 20 Scanning Electron Micrograph of 8% LCP cotton- seed/HRW wheat flour dough a) 700X b) 1000X c) 3000X. . . . . . . . . . . . . . . . . . . . 157 21 Scanning Electron Micrographs of Dough and 0.5% Conditioner Tween 20 a) HRW wheat flour dough at 8000X b) HRW/cottonseed flour dough at 1600X c) HRW/cottonseed flour dough at sooox. . . . . 159 22 Scanning Electron Micrographs of HRW Wheat Dough Prepared with Different Methods IL. Fixed 1" glutaryldhyde, ethanol dehydra- ted and CPD a) 1600X b) 4000X B. Nitrogen frozen, freeze fractured, and freeze dried c) 1000X d) aooox. . . . . . . . . . . . . . . . . . 161 23 Scanning electron micrographs of HRW wheat dough substituted with 8% LCP cottonseed flour pre- pared with different methods A. Fixed in glutarldhyde, ethanol dehydrated and CPD a) 800x b) 1600X B. Nitrogen frozen, freeze fractured, and freeze dried c) 700x d) 1000X e) 3000X . . . . . . . . . . . . . . . . . 163 xiii Figure 24 25 26 Score card used for sensory evaluation of white bread. Score card used for sensory evaluation of Egyp- tian bread . Score card used for sensory evaluation of optimum bread. . . . . . . . Page 164 165 166 INTRODUCTION The fortification of bread with oilseed proteins is expanding rapidly. At first the attention was focused on soy flour, and soy protein substitutes for bread and similar bakery foods. The initial purpose was to substitute other proteins for the nonfat dry milk in bread formulas. The rising price of nonfat dry milk made it economically feasible to search for alternative protein substitutes for white bread. Another major reason of the growing interest in the use of various nonwheat flour in bakery products is the desire to improve the nutritive value of wheat flour bakery foods by increasing their protein content, in particular their lysine level. In many of the third world nations, not only is the level of food consumption inadequate by the American stan- dards, but the optimum utilization of indigenous crops which are often of the oilseed variety, is hindered by a lack of proper technology. With cereal foods constituting the prin- cipal staple in the diet of many peoples of those nations, any nutritional improvement by means of protein substitution would represent a major step toward a dietary improvement. It Should be remembered that any benefited results achieved in the area of nutrition improvement, would also apply to America's aged and poor pe0ple whose quality of nutrition may not meet the U.S. recommended dietary standard (Pyler, 1979). Cottonseed offers a potential solution to increase the human consumption of protein since its annual consumption was estimated as one hundred million tons of protein, and twice as much for feeding to livestock (Bressani, 1965; Lawhon et al., 1972). In 1970 approximately 24 million tons of cottonseed was readily available as a source of protein (Harden et al., 1975). The liquid cyclone processed (LCP) cottonseed, a relatively new processing procedure, yields a low gossypol cottonseed flour. A LCP plant has been installed in Texas, and the anticipated daily yield is 20- 25 tons (Ziemba, 1972). In this research, the rheological properties of wheat flour substituted with LCP cottonseed flour was studied. As a control the untreated wheat flour was substituted at 0, 4, 8, 12 and 16% levels of LCP cottonseed flour. Bread was baked to determine the effect of the different levels of LCP cottonseed flour on final product. Salt (0, 1.0, 2.0%), potassium bromate (0, 30, 50 ppm) and dough conditioner polysorbate (20) (0, 0.5, 1.5%), were added to HRW wheat/LCP cottonseed flour dough and the effect on dough rheology was studied using the mixograph. The effect of these additives and the potential of their interactions was studied in bread substituted with 8% cotton- seed flour. The main objective was to determine the level of each additive that would produce optimum bread volume, grain texture, and tenderness. Four dough conditions at three levels each were added to the HRW wheat/LCP cottonseed flour dough, and their rheological properties were studied using the mixograph. Egyptian bread was baked with 0, 4, 8, 12 and 16% LCP cotton- seed flour, and the effect of dough conditions on the Egyp- tian bread were evaluated objectively, and for taste, appearance and tenderness after 3 different periods of storage. The objective was to test the functionality of LCP cottonseed protein in Egyptian bread, and the effect of different conditioners in retaining its freshness after 3 and 6 days of storage. The effect of LCP cottonseed substitution on the amino acid content of wheat flour was calculated. Proteins in wheat/cottonseed flour systems as well as a control were modified using sodium dodecyl-sulfate, succinic anhydride, Urea, N-ethylmaleimide and dithi- othreitol on flour bonding during mixing. The effect of cottonseed flour on the ultrastructure of wheat dough was studied using a scanning electron microscope. Dough conditioner Tween 20 was added to the control and sub- stituted dough and the electron micrographs were evaluated. Sample preparation for electron microscopy involves fixation, dehydration and drying. Chobat (1979) reported the effect of some sample preparation methods on the image formed. The objective of this section was to compare two different sample preparation methods on the control and LCP cottonseed flour substituted doughs. REVIEW OF LITERATURE Wheat Proteins The protein content of wheat which is an important index of its quality for the manufacture of different food products is influenced by climate, weather, soil and the variety of grain. The quantity of protein is influenced to a large extent by environmental factors; the quality of protein is heritable. For many years it has been believed that differences in protein content among varieties of wheat grown under the same conditions were small as compared to the environmental differences. In recent years, it has been shown that the protein content of wheat can be increased greatly by selective breeding (Pomeranz, 1980). The wheat proteins include water—soluble proteins (albumins), salt-soluble proteins (globulins), alcohol- soluble proteins (prolamins or gliadins), and acid- and alkali-soluble proteins (glutelins). The wheat endosperm proteins (gliadin and glutenin) form a colloidal complex known as gluten, when water is added to them. The gluten complex.is responsible for the superior performance of wheat over other cereals for the manufacture of leavened products, because of its ability to retain carbon dioxide produced by yeast or chemical leaving agents (Pomeranz, 1980). In 1745 Becarri reported the first separation of gluten from the starch of flour by adding 60 to 65% water to a hard wheat flour, mixing and allowing the dough to rest for 30 minutes, then washing out the bulk of starch under steady stream of water. An elastic, rubberlike material holding, roughly, two thirds of its weight of water was obtained (Sullivan, 1954). The gluten protein can be about equally divided into two classes - gliadin which is soluble in 70% ethanol and glutenin which is insoluble in ethanol. The cohesive and elastic properties of gluten are a com- posite of these two fractions. The gliadin when hydrated is fluid, whereas glutenin is cohesive and elastic but much tougher than the gluten formed. Gluten proteins compose about 80-85 percent of the wheat flour proteins, while the water soluble albumins and salt-soluble globulins constitute the remaining 15-20 per- cent (Krull and Wall, 1969). For a single wheat variety, it is well known that loaf volume is directly proportional to the protein content, however, there is disagreement as to which wheat protein fraction is responsible for the variation in loaf volume among wheat varieties (Khan, 1978). Pomeranz (1965) published one of the first reports on the functionality of glutenin in bread making. He reported that there was an inverse relationship between the wheat proteins solubilized by 3M urea and baking quality, while loaf volume was directly related to the proteins insoluble in 3M urea. The baking studies on reconstituted flour (Shorgan et al., 1969) confirmed these findings. Conver- sely, Orth and Bushuk (1972) concluded that glutenin is responsible for the variations in loaf volume (at constant protein content), while Hoseney et a1. (1969) had concluded that gliadin proteins control the loaf-volume potential of a wheat flour. Thus, this apparent controversy remains unsolved. The key to the functional behavior of glutenin lies in its physiochemical properties, molecular size, shape, tendency to aggregate and its amino acid composition and sequence (Khan and BUshuk, 1978). The molecular weights range from 150,000 to 3 million (Jones et al., 1961; Nielsen et al., 1962; Taylor et al., 1973). The gliadin proteins have a relatively narrow molecular weight distri- bution from 20,000 to 50,000 (Jone et al., 1961). The work of Pence and Olcott (1952) on the effect of disulfide reducing agents on the viscosity of gluten led to the idea that glutenin molecules consists of polypeptide subunits. Later, Woychik et a1. (1964) showed by starch gel electro- phoresis that some of the subunits obtained by reducing glutenin resembled gliadin but glutenin also contained subunits that were different which indicate that glutenin is not a polymer of gliadin as was believed at that time. Bietz and Wall (1973) and Orth and Bushuk (1973) both demonstrated the uniqueness of glutenin proteins by applying sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SOS-PAGE). The technique separated a highly complex mix- ture of subunits ranging in molecular weight from 12,000 to 134,000. Gel filtration studies by Wall (1974) and Khan (1977) showed that glutenin is composed of three groups of subunits. Group I contained subunits of MW of 68,000 to 12,000 and therefore exhibited a strong tendency to aggre- gate, group II contained the largest glutenin subunits, while group III contained the two major gliadin proteins (MW 35,000 and 45,000). Each group may contribute to the functional properties of glutenin. ‘ The amino acid composition of gliadin and glutenin are very similar. Wheat proteins are composed of 21 different amino acids, and a single protein chain may contain more than 150 amino acids. Different proteins contain different proportions and sequences of the 21 amino acids, which result in variation in protein structure (Krull et al., 1969). Glutamine constitutes about 37 percent of all amino acids in single protein (Kasarda et al., 1976), glycine, alanine, valine, isoleucine, phenylalanine and proline compose 40 to 50 percent of the residues of wheat proteins. Proline, an amino acid which occurs in high levels in gluten, is of interest because of its cyclic side chain which interferes with helix formation by causing the poly- peptide chain to bend (Wall and Beckwith, 1969). It is well known that wheat proteins have an abundance of hydro- phobic (e.g. valine, leucine) and hydrophilic (e.g. gluta- mine) residues, and a scarce amounts of charged residues (aspartic acid and arginine). As a result of this amino acid composition, the wheat proteins are compactly folded to minimize hydrophobic residue exposure to aqueous solu- tions thuszivery small number of charged amino acid occur in the surface of the protein. In fact there are sufficient hydrophobic amino acids that all cannot be buried in the interior of the molecule and some of them must be exposed to the solvent at the surface of the protein. The low charge density on the surface of the proteins combined with hydrophobic areas and an abundance of resi- dues capable of forming hydrogen bonds, result in a high sensitivity to salt concentration, even at low concentra- tion (0.005 M NaCl) (Berardin, 1970). As a class, cereal proteins are not as high in biologi- cal value as are certain legumes, nuts, or animal proteins. The limiting amino acid in wheat endosperm is lysine (Pomeranz, 1980). Dough Formation The amino acid studies of different wheat varieties failed to explain the difference in their breadmaking char- acteristics (Pomeranz et al., 1970). The viscous and elastic properties of dough are primarily due to the 10 properties of its continuous phase or gluten phase. It has been reported that the viscoelasticity of gluten and dough is considered to be due to a network of protein mole- cules, while the rheological properties of such a network greatly depend on the number amjstrength of the cross-links between the protein molecule (Pomeranz, 1978). Kneading, of wheat proteins with water causes hydration of the poly- peptides folded in the aleurone granules, permits their partial'uncoiling (particularly, the glutenins), and facili- tates extensive intra- and intermolecular association of these polypeptides via several types of chemical and physical forces which together influence size, shape and subunits of wheat proteins hence their functionality (Krull and Wall, 1969; Kinsella, 1976). Chemical bonds The chemical bonds are divided into two major types: covalent and noncovalent forces. The only covalent bonds that are known to be significant in dough structures are the disulfide linkage between proteins, which have an energy of 49 Kcal per mole and are not broken at room temperature except by chemical reaction (Wehrli and Pomeranz, 1969). The amino acidscystine or cysteine form about 1.4% of the amino acids of gluten protein and supply the disulfides or sulfhydryls, respectively. ‘In glutenins the disulfide bonds-bind protein subunits of MW 20,000 together to form a giant structure with molecular weight of some millions, 11 while the disulfides in gliadin are intramolecular bonds. As early as 1936, Balls and Hale suggested that the stif- fening of a dough made by oxidative flour improve is due to the formation of cross-linked ($5) from thiol groups (SH), corresponding the conversion of the amino acid cysteine into cystine. 2 Pr-CHz-SHfzzsPr-CHZ-SS-CHZ-Pr+2 H Cysteine Cystine Total disulfide and thiol contents of wheat flour are of the order of 10 and 1 umole per g of flour respectively (Mecham, 1968), but only 1-2% of the disulfides of gluten ~protein are crucial to the rheological properties (Mauret- zen, 1967). Goldstein (1957) was the first to suggest that the thiol-disulfide interchange reactions can explain the opening of the rigid cross-linked structure to give the viscous flow which characterizes the bread dough. The SS-SH interchange reaction can also explain the effect of oxidizing agents on the rheological properties of dough. Bloksma (1968) explained that effect as first the breaking disulfide crosslinks, and secondly their concurrent refor- mation by an exchange reaction with a free sulfhydryl during which the velocity of the reaction would be expected to be proportional to the combined number of sulfhydryl and 12 disulfide groups. The baking quality of wheat is governed by protein content and SS:SH ratio; optimum breadmaking results were obtained if the ratio SS:SH is around 15 (Belderok, 1967). This SS:SH ratio increases during flour storage. The ratio of reactive to total SS groups increase with decreasing mixing strength, and the ratio of reactive to total SH groups also increases with decreasing flour strength. Thus mixing strength appears inversely related to reactive SH and SS contents (Tsen and Bushuk, 1968). The thiol and disulfide groups in dough can be affected by some compounds other than oxidants. The effects of cysteine, glutathione, mercaptoacetic acid, gammaglutamyl cysteine, thioctic acid and dithiothreitol, as thiol com- pounds on dough consistency were reported by Pomeranz (1978). The decrease in dough consistency, an increase in extensibility and acceleration of stress and structural relaxations were explained by the increase in the rate of SS-SH interchange reactions brought about by these addi- tions. Thiol blocking compounds such as N-Ethylmalermide were reported to hinder the interchange reaction, which results in a higher resistance to extension. Three types of non-covalent chemical bonds (such as ionic, hydrogen and VanderWaals) also occur in the dough system. The importance of ionic bonds is demonstrated by several effects of adding salt to the dough. Ions in dough may complex with some groups of lipids (Fullington, 1967), 13 proteins (Bennet, 1965), and pentosans (Neukom et al., 1967). Low salt concentrations (e.g. 0.005M NaCl) can effectively mask the repulsion of one charged protein mole- cule for another of like charge which allows a sufficiently close approach of one molecule to another to allow hydro- phobic and hydrophilic interactions to form. Therefore by altering the salt concentration, the protein-protein inter- actions may be increased or decreased. The effect of salt concentration on protein aggregation is straight forward and it follows the ionic strength. Trivalent ions would be expected to be more effective than divalent ions which are more effective than monovalent ions (Bernardin, 1978). Hydrogen bonds result from the affinity of hydrogens of hydroxyl, amide, or carboxyl groups for the oxygen of carbonyl or carboxyl groups. The energy of this bond, however, is low (about 8 Kcal per mole) but it is compen- sated for by their abundance, which is due to the high amide content of the gluten proteins (Beckwith and Wall, 1963). Hydrogen bonds cause the insolubility of gluten proteins due to the intermolecular bonds (Wehrli & Pomeranz, 1969). Reagents like urea, acetamide and sodium salicylate can salinate the hydrogen bonding capacity of a single chain and by competition with other peptide chains, prevent the formation of intermolecular hydrogen bonds. AS a conse- quence, these reagents readily dissolve gluten (Bloksma, 1978). Hydrogen bonds are among the factors that determine 14 the rheological properties of dough in spite of their low energy. This importance was clearly demonstrated by repla- cing the water (hydrogen oxide) by deuterium oxide (higher bond energy than hydrogen bonds), resulting in a strengthened gluten as was shown by farinogram and extensigram studies (Tkachuk, 1968). Van der Waals bonds and dipole-induced-dipole interac- tions provide very weak bonds (up to 0.5 Kcal per mole). They are not significant in the presence of stronger bonds, and at distances longer than 5 Angstroms. They may play a role in attraction between nonpolar amino acid residues or fatty acid side chain in systems with limited water where hydrophobic bonds are impossible. They play a role in stabilizing starch-glyceride complex which has been postu- lated to affect baking and bread characters. Hydrophobic bonds may contribute to both plasticity and elasticity. The bond energies are low enough to allow rapid interchange at room temperature and to contribute to dough plasticity. 0n the other hand they might stae bilize conformations with small surfaces. Hydrophobic bonds could also be important in early stages of baking. Hydrophobic bond formation, in contrast to all the other chemical bond formations, is an endothermic process favored by increasing temperature up to 60°C (Wehrli and Pomeranz, 1969). 15 Cottonseed Flour Cottonseeds, in contrast to cereal seeds, are composed primarily of embryo tissue with a very thin layer of resi- dual endosperm. These seeds store oil as the major energy source. The cotyledons of the cottonseed embryo typically contain three major classes of cells: epidermal, palisade and spongy mesophyll (Wall, 1965). In addition to these cell types, glanded cottonseed varieties contain intercel- lular structures called pigment glands which are the depo- sition sites of gossypol pigments. These glands are distributed throughout the colyledons and.the periphery of the axial tissue. They vary in diameter from 50-400 u, with the majority being 80-120 0 (Boatner, 1948). Moore and Rollins (1961) showed that the center of the pigment glands consists of a complex net-like structure holding the globules of gossypol which were found to be less than 1 u in diameter. The intracellular structure of the cottonseed .is highly organized, where lipid, protein and phosphorus are neatly packed and embedded in the cytoplasm, in addition to the other basic cell structures (Engleman, 1966; Yatsu, 1965). This natural organization of the cell is extremely important to the processing of edible cottonseed products. Cottonseed flour_processing Very early in the processing history of cottonseed it was recognized that cottonseed could be a multiple source of edible products - both protein and oil (Richardson, 16 1917). However the presence of the chemically reactive, antinutritional factor - gossypol (Berardi and Goldblatt, 1969) affected any rapid development of edible protein products, therefore cottonseed processing became a compro- mise between oil quality, gossypol inhibition, and protein quality, where the economically important oil and poten- tially toxic gossypol were given major emphasis. As a result, the full potential of the quality and functionality of the protein of cottonseed was seldom evident in commer- cially produced meals (Altschul et al., 1958; Bailey, 1948). The two major developments that renewed the interest in the potential of edible cottonseed protein products were: the elimination of gossypol through breeding (McMichael, 1959; Miravall, 1959) and the development of the liquid Cyclone method (Gastrock et al., 1969) for processing glanded cottonseed. Cottonseed flour as defined by Martinez et a1. (1970) is the finely ground material produced from dehulled and defatted seed. The heat, moisture, and pressure used in the process of oil removal also affect the protein constitu- ents of the seed, as well as the product's color (Martinez, 1969; Vix, 1968). Further concentration of the proteins of the cottonseed and the removal of gossypol can be accom- plished by either wet or dry processing of the defatted flour. Ninety percent ethanol was used for the optimum extraction of lipid and sugar with a minimum loss of 17 nitrogen (Berardi et al., 1968). Aqueous extraction at neutral pH (6.3-6.8) is another wet procedure for prepara- tion of cottonseed protein concentrate (Martinez, 1969), where water or a very dilute divalent cationic salt solu- tion (0.008M CaClz) can be used to extract the defatted flour. The outer structure of the pigment glands is fairly rigid, thus allowing properly conditioned kernels to be processed without appreciable damage and subsequent ejec- tion of gossypol. The differences in density, size, and shape of pigment glands and of other extraglandular seed material, particularly in the solvent media, have been employed in a number of procedures to obtain a gland free, low gossypol protein product. However there are two major difficulties associated with wet operations: the processing of the extract, the byproduct in an economically feasible manner, and the drying of the major product. The first difficulty involves low-yield product recovery, solvent recovery, and pollution problems. The second difficulty involves denaturation of the major product during drying, which is more severe when alcohol is part of the extracting solvents. These two problems hinder the applicability of wet operations commercially (Kadan et al., 1979). The dry procedure of air classification has neither of these difficulties (Martinez, 1969; Martinez et al., 1967). It is based on the mechanical separation of particles, clusters of unruptured cells, cell wall fragments with 18 adhering residual cytoplasm, and residual sphersome mem- branes are separated from free, intact protein bodies. It is also used for the removal of pigment gland (Meinke & Reiser, 1962, 1964; Vix et al., 1972). The free gossypol of the air-classified product was not low enough to meet the standards of Food & Drug Administration (1974). Graza (1959) and Schaller and Lapple (1971) showed that air classification of solids results in a fine fraction and a coarse fraction that have overlapping particle sizes. Successive air classifications for the removal of intact and partially broken pigment were suggested by Kadan (1979) to produce an edible cottonseed protein product, without affecting its nutritional value. The non-aqueous, liquid classification procedure of Liquid Cyclone Procedure (LCP) was developed primarily for the removal of the pigment glands (Gastrock et al., 1969). Appropriately defatted flakes are ground in hexane to remove the cellular tissue from the pigment gland, which was then separated from the flour particles in a liquid centrifuge called a Liquid Cyclone. Cottonseed flour contains about 0.8% free-gossypol which is responsible for the toxicity associated with the protein as a food or feed for non-rumi- nants. According to the Protein Advisory Group of the United Nations Systems (FAO-PAG, 1975), free gossypol con- tent of edible-grade cottonseed flour should not exceed 0.06%. 19 El-Tinay et al. (1980) studied the effect of pH, salts and the combined effect of pH and salts, on the extracta- bility of protein and free-gossypol from fully gossypolised cottonseed flour. These researchers reported that a high salt concentration (0.25 M) was needed to bring about reasonably high protein extractability, and alkaline extrac- tion at pH 10 is the best method to obtain a protein isolate totally free from free—gossypol with a high protein content. Several workers (Jonassen, 1955; Smith, 1970; Smith and Clawson, 1970) have shown that the addition of ferrous salts to nonruminant animal rations containing cottonseed meal reduces the toxicity of gossypol to varying degrees. Bressani et a1. (1964), Jarquin et a1. (1966), Braham et a1. (1967) have reported the addition of iron in the presence of calcium hydroxide reduces the free gossypol in meals containing cottonseed flour, to low levels. Mayorga et a1. (1975) found that the addition of 1% Ca(0H)2 or 0.1% FeSO4 reduced free gossypol in prepress solvent cottonseed meal slightly. When both were added together, the free gossypol was reduced to a greater extent. The combined treatment of calcium hydroxide and ferrous salts plus heating the meal up to 130°C for 90 min was the most effective in reducing 60% of the initial free gossypol, with no reduc- tion in the available lysine. 20 Cottonseed flour use in foods The edible use of cottonseed proteins is influenced by its functionality, color, flavor and nutritive value. The full range of cottonseed products from flour to isolates have been tested by the American Institute of Baking in both sponge, dough and continuous bread formulations. With slightly reduced mixing times and the addition of 30 ppm bromate, the flour and air-classified concentrate at 3% and 2.6% levels substitution respectively, produced bread generally comparable in all characteristics to the control containing 3% nonfat dry milk. The bread substituted with the air-classified concentrate had reduced break and Shred, and firmer texture. Neither the flour nor the concentrate performed satisfactorily in continuous bread formulation. Volume, bread characteristics, and shock resistance were all lacking (Martinez et al., 1970). There are two cottonseed protein isolates obtained by the two-step procedure of isolation. The major isolate of qlandlesscottonseed proteins has a low moderate flavor profile and its acid solubility suggests its use in citric acid based beverages. The minor isolate has an interesting whippability at acid pH and produces foam volumes as much as nine times that produced by sodium caseinate at pH 4, and 75% of that produced by sodium caseinate at pH 7 (Ber- ardi et al., 19691. 21 Matthews et a1. (1970) studied the effect of both glandless and glanded cottonseed flour, peanut, safflower and full fat soy at 25% level of replacement of wheat flour on bread characteristics. They reported the need for more research to determine the maximum amount of the oilseed flour that can be used to meet consumer acceptance stan- dards. When liquid Cyclone Processed cottonseed flour was used substituting 13% of cake flour, it gave cakes and doughnuts with a very desirable yellow color comparable to that of an egg rich product. In devils food cake, a substitution of 10 percent LCP flour for cake flour pro- duced an excellent product with good color, flavor and texture. The LCP imparted an undesirable greenish color to waffles and pancakes and a grayish cast to white.cake. Cottonseed flour was also used at levels up to 8% in beef patties and up to 6% in sausage. Research on extruded products containing LCP flour at Grain Processing Corpora- tion and Texas A&M University showed that cottonseed flour materials have promise for use in meat products and cereals (Olsen, 1973; Gardner et al., 1973). Cottonseed proteins were used for replacing 10% and 30% of the meat in frank- furters (Terrell et al., 1979). Texturized cottonseed products with good nutritional and functional properties were obtained by an extrusion cooking process using cotton- seed flour (Cabrera et al., 1979). 22 Cottonseed products are influenced by pigments present in seeds, or the glands contained in the seed (Hedin et al., 1976).' The basic pigment groups that affect the quality of cottonseed flour or proteins are: Terpenoids: of which gossypol has been the compound of the greatest interest, and is concentrated in discrete glands. . Flavonoids: The need for a color-free cottonseed pro- tein led to the study of pigments other than gossypol in cottonseeds. Blouin et a1. (1978) reported that there are six major flavonoids in both glanded and glandless seeds which were present in the surrounding embryo meats in both varieties. The flavonoids added to biscuit gave yellow- brown color (Blouin and Cherry, 1978). Flavor is another important factor in the acceptance of a vegetable protein for human food applications. Cottonseed flour is notably bland compared to other vegetable sources, but there are several free phenolic acid fractions which have been identified and might contribute to taste in cottonseed flour (Jones, 1979). Nutritional Valgg The nutritional value of cottonseed proteins is influ- enced by the reaction of free gossypol with available epsilon amino group of lysine, and in this state is much less physiologically active in the animal gut (Conkerton, 1959). The gossypol that is thus reacted is called "bound 23 gossypol" and that gossypol that is still not bound to protein is designated as "free gossypol". The main concern of nutritionists is the amount of free gossypol consumed by animal, which has some toxic effects on different animal species (van Sumere et al., 1975; Sabir et al., 1974; Sosulski et al., 1969). Bressani et a1. (1969) conducted long term feeding studies with rats to determine the effect of constant intake of gossypol in cottonseed flour. He found that rats were able to detoxify the ingested gossy- pol, since the amounts consumed daily were relatively small (0.011-0.028%), however they do not resist the toxic effects of higher levels of pigment as reported by Bressani and Elias (1968). Hale and Lyman (1957) and Sharma et a1. (1966) have reported the effect of high protein levels of diet on counteracting gossypol toxicity. The phenol compounds in seeds when oxidized by atmospheric or enzyme- catalyzed oxidation, results in quinoidal production and the formation of hydrogen peroxides, which are both des— tructive to labile amino acids, denature proteins, and inhibit enzymes such as indole acetic acid oxidase (Rabin and Kein, 1957), trypsin and lipase (Milic et al., 1968) and arginase (Muszynska and Reifer, 1970). Cinnamic acids and their esters are of particular significance in oilseeds because they are a preferred substrate for phenol oxidase. Caffeic and chlorogenic acids are oxidized to o-quinones by a copper containing enzyme which occur in plants. Once 24 oequinones are formed they react nonenzymatically to poly- merize, and are reduced or bound covalently to amino thiol and methylene groups. The epsilon-amino group of lysine and the thioether group of methionine are commonly attacked to render them nutritionally unavailable to monogastric digestive system (Sosulski, 1979). Lysine is considered to be the first limiting amino acid in cottonseed, there- fore, the air-classified concentrates and the liquid cyclone processed flour were lower in lysine than the parent flours and their PERs were 2.3 and 2.6, respectively, as compared to 2.5 for the standard sodium caseinate (Martinez et al., 1970; Ridlehuber and Gardner, 1974). Supplemental Value of Plant Proteins on on Wheat Flour Because of the universal acceptance of bread and the potential of its fortification with proteins, amino acids, vitamins, and minerals, the measurement of the effect of added proteins on dough formation and bread's physical and nutritional quality have been extensively studied. Effect of_plant¥proteins on the functionality of wheat proteins in dough and bread The dough forming capacity of protein mixtures and the properties of doughs are routinely determined by the farinograph and mixograph which measure development time, dough strength, consistency and stability. Soy flour has 25 traditionally been added to wheat flour as a source of lipoxygenase, which aids maturation and bleaching. In -addition small amounts of soy proteins are being increasingly added to bakery products for several claimed functions (Wolf, 1970). Replacement of wheat flour by oilseed flour up to 5-10% has been successful (Bacigalupo et al., 1967). However at higher levels of replacement, loaf volume is severaly decreased along with serious deterioration of crumb color, and grain texture (Matthews et al., 1970; Sidwell and Hammerle, 1970). The maximum level of replace- ment depends on the type of nonwheat flour, the strength of wheat flour, the baking procedure, and the dough sta— bilizing compounds used (Dendy et al., 1970; Pringle et al., 1969). .Tsen and co-workers (1971) used sodium and calcium stearyl-Z-lactylate to produce sponge dough breads with 12% soy flour. The dough conditioners increased loaf volume, and the organoleptic properties of soy bread were comparable to bread with 100% wheat flour. Rooney et a1. (1972) compared the functional bread-making pr0perties of heat-treated and non-heat-treated flour from cottonseed, peanut, sesame and sunflower flours. The oilseed flour replaced wheat flour to produce blends at two protein levels, 17.5 and 20.5 percent. He reported that the farino- graph water absorption increase with the increased levels of replacement of wheat flour with oilseed flour at both protein levels. Moreover, the heat treated flours had 26 higher water absorption than the noneheatetreated ones. The type of oil seed did not significantly influence the water absorption of the various flour/oilseed blends. The sunflower substitution drastically weakened the dough struc- ture while cottonseed destroyed the dough stability. Peanut flour incorporation at the high protein blend (20.5%) caused noticeable weakening of dough structure, whereas sesame showed only a slight decrease in mixing strength. The heat treatment of oilseed flours improved mixing strength and stability. The heat-treated cottonseed flour was more compatible with the proteins of wheat which accounts for the dramatic increase in loaf volume. The functional properties of sunflower were improved by heating while sesame and peanut flours responded negatively to the heat-treatment and the bread baked had poor internal pro- perties. Khan et a1. (1976) studied the baking properties of cottonseed protein concentrate (CSPC) spray dried at dif- ferent pH levels, and compared the influence of Ca++ and Na+ on its baking properties, in an attempt to optimize the CSPC processing method. They reported that acidic pH (4.5) adversily affected the baking properties of Spray- dried concentrate. pH adjustment with Ca(0H2) significantly reduced the loaf volume and bread crumb grain score. Acceptable loaves were obtained with spray-dried concen- trates at near neutrol pH adjusted with NaOH, and they 27 were similar in quality to breads baked with parent gland- less cottonseed flour or commercial soy flour. Lawhon et a1. (1972) demonstrated that solvent- extracted cottonseed proteins prepared from glandless cottonseed flour and spray-dried at pH 4.5 had signifi- cantly poorer baking properties than a comparable concen- trate spray-dried near neutral pH (6.8). Recent studies on flours from faba beans (McConnell et al., 1974) showedthat the addition of faba bean flour to hard red spring (HRS) wheat flour at the rate of 10, 20, 30, and 40% resulted in a progressive decrease in loaf volume and a deterioration in crumb grain, even in the presence of the conditioner SSL. 'D'Appolonia (1977) 'studied the physical dough properties and baking potential of five legumes including: mung bean, faba bean, navy bean, pinto bean and lentil, substituting HRS wheat at 5, 10, and 20% levels. Results indicated that mung bean, and lentil had the least water holding capacity while pinto and navy bean flours retained the water better than the other legume flours. Farinograph water absorption also increased with increasing the level of pinto and navy beans in flour blends, while the dough development times for all bean flour combinations were less than the all wheat flour and stability decreased. . Fleming and Sosulski (1977), using 15 percent faba bean and field pea, found it necessary to add 2 percent vital 28 gluten, and about 1 g of dough conditioner per 100 g flour to produce acceptable bread quality. Yellow peas (raw and cooked) have been studied as a bread fortifier by Jeffers et al. (1978). D'Appolonia (1977) reported that as the level of legume flour in the blend was increased, the crust color of the bread became increasingly darker; the 20% blend level of the mung bean bread produced the darkest 'crust color. The whitest crumb color was noted with the 5 and 10% legume flour breads and these were whiter than the control bread. The 5 and 10% navy bean flour blends produced the best grain and internal appearance of the various legume flour-containing breads. They substituted raw pea, cooked pea and soy flour for 5, 10, 15 and 20% of wheat flour and they reported the significant difference between the cooked and raw yellow pea on physical dough and bread making where raw pea flour was better. At 5% level of substitution the functional baking performances of peaesubstituted flours were superior to soy-substituted flours. At the 15 percent level, yellow pea flours produced acceptable breads, and no dough con- ditioners were needed to produce acceptable bread with up to 15 percent yellow pea flour. The supplementary value and protein quality of oilseed flours. Great emphasis has been placed on the use of composite flour in bread-baking in the last 10 to 15 years. Composite flour refers to flour containing blends of wheat flour with 29 nonwheat flours (D'Appolonia, 1977). The nutritional value or protein quality of a food protein depends not only on its content of amino acids, but on thehrphysiological availability. This availability varies with the protein source, processing method, and interaction with other diet components. The availability also depends on the condi- tion of the consuming animal (Boloorforooshan, 1977). The biological value of a protein is one way of expressing its nutritional value. The biological value is based on the amino acid content of a protein food as well as the essential amino acid balance, that is the relative propor- tions of the amino acids present in a protein. Thus, any factor that affects the protein food and causes the amino acid balance to change, would also alter the biological value of the protein (Boloorforooshan, 1977). The storage proteins of cereals, oilseeds and legumes are variable in their amino acid composition in contrast to the similar amino acid distribution of proteins in all the metabolitically active tissues whether they are animal or plant or microorganism (Bressani, 1968). Plant storage proteins are deficient in one or more essential amino acids. Furthermore, the ratio of essential to total amino acids is smaller in plant storage protein in comparison to that of animal protein, which causes lower utilization of some plant proteins, even after the essential amino acid pattern is corrected by substitution (Harper and DeMuelenaere, 30 1963). Evans and Bandemer (1967) studied the nutritive values of peanuts, safflower, sesame, soybean and sunflower seeds and found that methionine and lysine are the most limiting amino acids in the seeds studied. Only soybean proteins had high nutritive value, but these were deficient in the sulfur amino acids, while sesame seed proteins have an abundance of the sulfur amino acids (Evans et al., 1967) but are deficient in lysine. Effect of Processing on the Protein Quality The loss in nutritional value of a protein due to 'thermal processing is measured by determining the available lysine loss (Lea and Hannan, 1950; Carpenter, 1973). The available lysine destruction rates for pure proteins stored at aw = 0.68 and temperature = 35°C, and included in model food systems containing 10% gluCose varied with the type of protein. Albumin was the most stable protein while soy protein was the least (Schnickels et al., 1976). The effect of heat on the nutritive value of proteins was observed as early as 1917 by Osborne and Mendel in their attempts to destroy the toxic pigment gossypol in cotton- seed by steaming the seeds for a long period of time. These researchers reported heats effect on reducing the nutritive value of seeds. Hopkins (1967) determined the PER of cottonseed flour and found it equal in nutritive value to casein control. Lysine is considered to be the 31 first limiting amino acid in cottonseed flour. Therefore, the air-classified concentrates were slightly lower in lysine than the parent flour. However, the residues from the air classification process were equal in PER to the parent flours (Martinez et al., 1970). The available lysine in the wet-processed cottonseed concentrate was lower than in its parent flour (Lawhon et al., 1972). Solvent extrac- tion of glandless cottonseed was recommended by Vix et a1. (1968) as the defatting method because of the mineral effects on protein quality and versatility of use with different oilseeds. Desolventization is the most impor- tant step for the protein, because high residual solvent will cause off-flavors. However the use of heat and added moisture in the absence of fat resulted in disruption of cellular integrity and produced rapid browning, coagulation of water-soluble proteins, and reduction in lysine availa- bility (Martinez et al., 1970). The potential use of edible cottonseed protein pro- ducts could be extensive. The inherent differences between cottonseed and soy in protein products should complement rather than compete to fulfill the spectrum of needs in the food industry (Martinez, 1970). Bressani et a1. (1966), and Squibb et a1. (1959) have supplemented corn, sesame flour and sorghum with cottonseed flour in formulating a vegetable mixture to upgrade their protein content. Jones and Divine (1944) substituted white wheat flour with cotton- 32 seed flour, and found that the addition of 5 parts of cotton- seed flour to 95 parts of wheat flour produced mixtures containing 16 to 19% more protein than the wheat flour alone, and a protein combination that was definitely superior in its growth promoting value in rats when compared to feeding the same amount of wheat flour. The more recent liquid cyclone process technique (Gas- trock et al., 1969) yields a flour superior in quality and higher in protein. The liquid cyclone processed cottonseed flour had PER value (2.3) very close to casein (PER = 2.5). Castro et a1. (1976) investigated the substitution effect of LCP cottonseed flour on the protein quality of soybean concentrates and isolates, triticale, wheat, and rye. They found a significant improvement in the protein quality of all the grains investigated which suggests that LCP cotton- seed flour is a valuable supplement to these proteins and possibly to other grain products as well. Breads made with 18.8% LCP cottonseed flours substituted for wheat flour were tested for their nutritional value by amino acid analyses, and biological tests (Harden and Yang, 1975). Amino acid analyses of breads indicated that lysine content was higher than the comparable wheat breads. Protein Modification and Functional Properties Protein modification usually refers to the intentional alteration of the protein structure by physical, enzymatic, or 33 chemical agents, to improve functional properties. Thus, modification of food protein may involve alterations in structure or conformation at all levels of organization (primary, secondary, and tertiary structures). It may include disruption and reformation of covalent bonds and secondary forces using physical (thermal and pressure), chemical, or enzymatic treatments. Dough formation is one example of protein modification (Kinsella, 1976). Modifi- cation procedure may also result in improvements of flavor, color, elimination of off-flavors, and destruction of undesirable enzymes, antinutritive factors, hemagglutinins, and allergens. Salts. In breadmaking, salt (sodium chloride) forms a part of the dough ingredients (Bloksma, 1978). Altering the salt content in a protein solution causes the breadkown of one of the physical bonds (electrostatic bond) between protein molecules in a food system. /The role of salt can be explained by the action of salt ions on reducing the repul- sion of the alike chargescniprotein molecules, which causes protein to aggregate and then to precipitate (Krull and Wall, 1969). The effect of salt ions on the protein aggregation have also been described by Bernardin, 1978. He reported that the close approach of one protein molecule to another resulting from the masking effect of salt ions on the repulsion of the similarly charged protein molecule 34 allows hydr0phobic and hydrophilic interactions to form, causing protein to aggregate. This effect of salt ions was also found at very low concentrations (0.005 M NaCl). The effect of salt concentration on protein aggregation is straightforward, and follows directly the ionic strength. Trivalent will be more effective than di and mono valent ion in reducing the charge-charge repulsion of similarly charged protein molecules. The concentration of salt in most dough systems is 2% (Miller et al., 1947). The addition of sodium-chloride to dough makes it stiff and less sticky, as confirmed by the measurements with the extensigraph (Fisher et al., 1949; Grogg et al., 1967; Calvel, 1969; Margulis and Campagne, 1955). They reported that the curves with added salt Show a higher resistance and increase extensibility. Farinograph studies usually fail to show this trend, showing only de- crease in consistency (viscosity) (Bennett et al., 1953; Hlynka, 1962; Tanaka et al., 1967). This observed decrease in consistency has been explained as being due to the de- crease in stickiness rather than in stiffness (Bennett et al., 1953). Mixographs were also used to Show the increase of stiffiness with salt addition (Bennett et al., 1953). The specific effects brought about by ions, particularly organic acids and lipid molecules, which bind to the protein surface and bear a charge and which can reduce or increase the net charge on the proteins have been studied by 35 Bernardin (1978). Bernardin reported their effect on the aggregation of A-gliadin. The use of salts of L- ascorbyl G-palmitate (AP) and D-isoascorbyl 6-palmitate (IAP) in breadmaking has been reported by Ofelt (1958) to be as effective as monoglyerides on bread-crumb softening. Nevertheless it was found that at levels of 0.4% based on flour, the compounds darkened bread crumb. Hoseney et al. (1977) used the salts of 6-acyl esters of L-ascorbic acid and D-isoascorbic acids in breadmaking. These researchers found that both salts behaved similarly in breadmaking because of their closely related structure. They reported that 2-acy1 esters of L-ascorbic acid did not give a desirable dough-conditioning effect because of their effect on tightening the dough structure. OxidizingiAgents. Oxidants are used to modify and control dough consis- tency and strength (Kinsella, 1976). The effect of oxidizing agents is twofold: flour improvement which refers to changes in rheological properties of the dough and flour bleaching, which is due to destruction of its yellow pigments; and results in whiter flour and bread (Bloskma, 1978). It is generally believed that the agents function by controlling disulfide bond rupture and the extent of disulfide inter- change reactions (Ewart, 1972; Wall, 1964, 1971). Bromate and iodate improve dough pr0perties without bleaching, while nitrogen dioxide and benzoyl peroxide bleach only effects 36 are exerted. Chlorine dioxide and acetone peroxides both improve flour and bleach. The reaction of bromate has been explained by Bloksma (1978) as the formation of reactive groups on the protein chains that form cross-links only during structural activation. He said that it is possible that the reactive groups are too far removed from one another during dough resting and they only come together by the work of rounding and shaping. The exact nature of the assumed reactive groups is still unknown; there is little doubt, however, that bromate and iodate act via the oxidation of thiol groups. The reaction of bromate is slow except at elevated temperatures (Dempster et al., 1956). Surfactants. Surfactants have been used in commercial bakeries for ' many years. The first ones to be used were monoglycerides, introduced in the 1930's, and lecithin. Research during the 1950's and 1960's resulted in more sophisticated and effective products. The need for these sophisticated sur- factants was caused by the use of high speed mixing equip- ments and large scale production in the baking industry (Tenney, 1978). Surfactants are sometimes referred to as dough conditioners which have a number of distinct actions on dough and bread. Dough conditioners have been reported to improve the handling of properties of dough, to increase the loaf volume, to increase the water absorption of the dough, to 37 replace shortening in the formula, to counteract the dele- terious effect of foreign proteins, and to retard the rate of firming of bread. Basically, surfactants are esters composed of polyhydric alcohols and longechain fatty acids. Thus they are characterized by both hydrophilic and lipo- philic groups. Their performance in breadmaking requires a proper balance between both groups, which is affected by the presence of ionic charges on the hydrophilic groups and even by the counter-ion associated with that charge (Hoseney et al., 1976). The use of softeners and conditioners in breadmaking is controlled by government regulation. The FDA approved bread softeners included: mono- and diglycerides, diacyl tartaric acid esters of mono- and diglycerided and propylene glycol mono- and diesters of fat forming fatty acids. The regulations state that the total weight of the individual softeners may not exceed 20 percent of the combined weight of the softeners and the shortening in the bread. The dough conditioners that were approved by the FDA regulations included: polysorbate 60, calcium and sodium stearoy1-2- lactylate, lactylic stearate, sodium stearyl fumerate and succinylated mono- and diglycerides. The regulation further stated that conditioners may be used alone or in combination of both a conditioner and a softener at the level not to exceed 0.5 percent based on flour weight for each additive separately (Newbold, 1976). 38 Scanning_Electron Microscope The direct examination of a tissue structure was limited to the images obtained in the light microscope, and at the ultrastructural level in the transmission electron microscope which are both two-dimentional images. Only the low-power stereascopic light microscope reproduced the object in a manner corresponding to man's eye. The standard light microscope is limited by its low resolving power (2000 A) because of the wavelength of the visible light. The transmission election microscope (TEM) also is limited by the power of the electron beam which requires the use of complex and time consuming specimen preparation and sectioning techniques, at the same time only extremely small areas of the specimen can be viewed. The scanning electron microscope (SEM) was commercially produced in the 1960's. The instrument combines the advantages of the stereoscopic light microscope for produ- cing a three-dimensional image with a better resolution (200 A), and the transmission electron microscope of pro- ducing images with very high magnification. The SEM has an additional advantage over both types of microscOpes, that is the possibility of viewing a larger specimen area (1 cm2) than the TEM. Since the SEM is a surface examining method, the observed image is limited by the details pre- served in the viewed structure (Aranyi and Hawrylewicz, 1969). 39 Principle of Operation. In the scanning electron microscope (SEM) the sample surface is scanned by an electron beam, and the emitted secondary electrons create an image of the structure viewed. As the scanning beam of electrons moves across the specimen, another electron beam of a standard cathode ray tube is driven in synchrony with it. The brightness of the cathode ray tube is modulated by a signal produced from the secondary electrons emitted from the specimen surface and amplified by a scintillator-photomultiplier system. The magnifica- tion is determined by the ratio of the two synchronized electron scans. The electron micrographs of the viewed images can be taken instantly with a Polaroid camera (Hayes et al., 1966; McDonald et al., 1967; Pease, 1968; Pease et al., 1968). Application. The goal of the food scientist is to understand struc- tural features of a material that are important in its functional role in a food system. Despite the rapid advances in instrumental design of scanning electron microscopes, there are still limits to the image resolution set by the biological specimen preparation problems. Food science samples are biological materials that differ in nature, hardness, form, moistness, and size, so the viewing of their structure is limited by the two major problems that limit the biological sample resolution in SEM. These 40 are: the removing of water from the specimen without destroying the structural relationships, and making the specimen conductive; by coating with metal layer deposited by high-vacuum—evaporation. The applications of SEM in the food science area has proved to be useful in some areas more than others (Pomeranz, 1976). Aranyi et a1. (1968) found scanning electron microscopes were well suited for the study of wheat flours and doughs, because of the absence of fixation, embedding, and thin-sectioning techniques, which permit the sample to be viewed in their natural state. The fine structure of developing and mature wheat endosperm has been investigated by Buttrose (1963), Seckinger et a1. (1967), and Simmonds (1972). The structural relationships of protein and starch in aigood quality bread flour at various dough stages and in bread crumb have been studied by Khoo et a1. (1975) using scanning and transmission microscopy. All microscopic studies on wheat kernel struc-. ture, flour components, and dough formation showed that only wheat flour produces the type of loaf that is now expected for bread. Both gluten forming proteins and starch granule size and morphology are important, since other starch or proteins drastically alters crumb structure. An understanding of the precise functional relationships between all the components in a loaf of bread is important not only from a need for basic knowledge of foods, but also aiding attempts at adding other nutrients such as single 41 cell proteins (Evans et al., 1977) or fiber (Pomeranz et al., 1977). Sample preparation for SEM; The routine techniques of fixation, dehydration and drying have been critically discussed by Chabot, 1979. He concluded from a study of fixation conditions on bread morphology that it produced profound changes in structure. These occurred precisely in the area of the interaction between protein and starch which have attracted the most attention. Dehydration in alcohol resulted in non-uniform shrinkage of starch protein connections. Varriano-Marston (1977) compared different dough preparation procedures for scanning electron microscopy and reported a definite effect of fixatives on the dough structure. He reported that the method of drying had affected the structure where air- drying caused more distortions to the dough structure than freeze drying. EXPERIMENTAL DESIGN The purpose of this research was to study the influ- ence of liquid cyclone processed cottonseed flour on dough rheology and bread baking performance in American and Egyptian breads. Bread formulas were optimized with dough conditioners. Specific chemical and physical effects were followed through measurement of chemical bonds and scanning electron microscopy. Thus the study was divided into five sections. 3 The first section included an overall physical and rheological characterization of hard red winter (HRW) wheat flour substituted with liquid cyclone processed (LCP) cottonseed flour and with unmodified wheat starch at 0, 4, 8. 12 and 16% of flour weight. Testing in this section included an evaluation of dough rheology using farinograph and Viscoamylograph. Baking studies including sensory evaluations were also conducted. Proximate analyses on flour and bread were performed. The second section was a study of the effects of additives on the rheology of dough substitued with 0, 8, and 16% cottonseed flour. Sodium chloride 0, l and 2%, potassium bromate 0, 30 and 50 ppm and conditioner 42 43 polysorbate (20) (Tween 20) 0, 0.5, 1.5% of flour weight were added and the effect of each additive at their three levels were investigated. I The potential for all the possible interactions among the three additives used and their three levels were con- ducted on doughs prepared with 0, 8. 16% cottonseed SUb- stitutions and breads prepared with the 8% level of LCP cottonseed substitution. The mixograph was used to measure dough rheology for these additive studied. Bread was baked with single, double and triple combinations of additives according to a 33 factorial design. Baking studies were repeated twice, and breads were evaluated by 32 panelists for the sensory characters of interest. These data were subjected to Multiple Regression analyses, and the optimum level of each additive to produce an 8% cottonseed flour-substituted bread with an optimum volume and texture was calculated. Optimum bread was baked and evaluated for sensory characteristics and objectively for texture. The third section of testing was a study of the functionality of 0, 4, 8, 12 and 16% LCP cottonseed flour substitution for wheat flour in Egyptian bread. Bread was evaluated for the characteristics of interest by 6 middle- eastern panelists, and the data were subjected to analysis of variance. Proximate analyses were performed on these breads. 44 A 43 factorial experiment was designed to test the effects of four dough conditioners at three levels each on the Egyptian bread character. The effect of conditioners on bread softness was tested after 0, 3 and 6 days of storage. The fourth section was a mixograph and farinograph investigation of the effects of sodium dodecyl-sulfate,_ succinic anhydride, Urea,N-ethylmaleimide and dithiothrei- tol on the flour bonding during dough mixing. The final section was a scanning electron microscope study of the untreated and 8% substituted doughs. For the first part of this microscopic study, the effects of 0.5% conditioner on both dough systems were investigated. For the second part, samples were prepared by two different methods to evaluate the effect of preparation procedures on SEM electron micrographs. Materials Straight grade, and 85% extraction hard red winter (HRW) untreated wheat flour was purchased from Department of Grain Science, Manhattan, Kansas. Liquid cyclone processed (LCP) cottonseed flour was supplied by the Southern Regional Research Center, New Orleans, Louisiana, sample no. 85400-A. Red Star Active Dry Yeast was purchased from Universal Foods, Milwaukee, WI. Sugar was supplied by Michigan Food Com- pany, Saginaw, MI. Analytical reagents potassium bromate 45 and sodium chloride were purchased from Mallinckrodt Chemi. cal Works, St. Louis, MO. Dough conditioners Polysorbate 20 (Tween 20), Polyoxyethylene Sorbitan Mono-Stearate (Tandem 552) were supplied by ICI United States Inc. Specialty Chemical Division, Wilmington, DE. Polysorbitan, Mono and Diglycerides Polysorbate 60 (36%) and Polyoxyethy- lene 10 stearyl ether were purchased from Sigma Chemical Company, St. L0uis, M0. Dough Rheology Studies Farinograph Testing:. A C.W. Brabender Instruments, Inc. Farinograph was used, and it was equippped with a Type Pl-2H Dynamometer and a Type 3-S-300 Measuring Head. Temperature of this instrument was kept constant at 30 t 0.l°C by a Heat-Transfer Circulator Type T-60-B. The AACC Constant Dough Weight Procedure 54-21 B (1962) was followed using a 300 g bowl maintained at 30 i 0.106. 0n "as-is" moisture basis, 300 g flour as flour/cottonseed mixtures were weighed to i 0.5 9, then mixed in the Farino- graph bowl for l min. starting at the 9.0 minute mark on the chart paper. At zero minute, the water was added from a fast delivery burette (within 25 seconds), the bowl sides were scraped with plastic spatula and then covered with plastic plate to prevent evaporation. When the peak was centered on the 500 BU-line the Farinogram was run for 20 46 minutes. If the curve was not centered at the peak on the 500 BU-line, re-estimation of absorption was done according to the approximate relationship: 20 BU = 0.6 ml water. From Table 54-28 of the AACC handbook, the weight of flour and water which correspond to the estimated "as-is" absorption were used to run triplicate Farinograms for each level of supplementation with cottonseed flour. Farinograms produced were evaluated for the following parameters: Water Absorption: The amount of water necessary to center the peak on the 500 BU-line. It was corrected to 14% moisture basis as illustrated in Note No. 2 of Table 54-29 of the AACC Test procedure. Arrival Time: The difference between zero minute and the point where the top of the curve first interSects-the 500 BU-line. Peak Time: The interval from the first addition of water (zero time) to the top of the curve (peak). Stability: The difference between the departure time and the arrival time. Departure: The time from zero to the point where the top of the curve leaves the 500 BU-line. Mixing Tolerance Index (M.T.I.): The difference in BU from the top of the curve at the peak to the top of the curve measured at 5.0 minutes after the peak. Twenty Minutes Drop (T.M.D.): The difference in BU from the 500 BU-line to the center of the curve measured at 47 twenty minutes from the addition of the water. Mixograph Testing. Mixographic studies were performed on a National Mfg. C0- Mixograph equipped with 35 g bowl, and Gra-Lab timer for automatically stopping mixograms. The spring was set on position number A on the damping arm. The procedure outlined in AACC 54-40 (1955) was followed where 30 i 0.1 g flour on 14% moisture basis were placed in the mixograph bowl, mixer head was lowered to operation position, the pen was lowered to the chart about 0.25 to 0.5 minutes before it reached a vertical line. The mixer was turned on and the amount of water needed to give the required absorption was added when the pen reached a vertical line. The curve was run for 9 minutes, then was evaluated for the following characters: Peak Time: The time from the first addition of water to the highest point of the curve, in minutes. Peak Height: The distance in cm between the center of the peak to the base line. 3 Curve Height After 9 Minutes: The distance between the center of the curve at the 9-minute point and the base line. Area Under Curve: The area uhder the curve measured by a Compensating Polar Planimeter expressed in cmz. 48 Viscoamylograph Testing. Tests were performed on a Brabender Viscoamylograph type VA-VL equipped with a sensitivity cartridge 700 cm-g, 7 pin style stirrer, 8 pin style stainless steel bowl of 500 ml capacity, and a thermoregulator 20—9700 and a programmed heating rate l.5°C/min. The diastic activity of flour was determined according to AACC method 22—10 (1960), where 100 g flour was weighed on 14% moisture basis, and was put in 1000 ml flask to which 360 m1 diluted anhydrous sodium phosphate buffer (pH 5.3-5.35) were added and mixed well with flour by shaking. The flour slurry was put into the Viscoamylograph bowl and the flask was washed with 100 ml diluted phosphate buffer, which was added to the slurry. The starting tem- perature was adjusted by hand, then the heating stage started with the bowl in motion. The viscosity was recor- ded as temperature increased from 30°C to 95°C at l.5°C/min. to reach the maximum viscosity, after which a holding at ambient temperature for 15 minutes followed. A rapid cooling at l.5°C/min followed the holding period and was run for 30 minutes. The viscoamylograms were evaluated for peak viscosity in BU and the viscosity after 60 minutes in BU. 49 Baking Studies A Hobart Kitchenaid K5-A mixer which was equipped with a dough hook was used for mixing dough; fermentation was performed in Cres-Cor Model 210-1828 fermentation cabinet. A National Manufacturing Compahy Sheeter and Holder was used for white bread shaping, after which the breads were baked in 9x5.3x5.8 cm pans. All breads were baked in Reel Type Test Baking Oven, National MFG Company, Lincoln, Nebraska. Bread volume was measured by a National Mfg. Company loaf volumeter; texture was evaluated by Food Technology Cor- poration Texture Test System, Rockville, MD., which was equipped with TP-2 Texturpress and FT-100 force transducer, TC-l compression test cell and TR-3 Texturecorder with thermal writing chart paper. Bread tenderness was measured on an Instron Universal Testing Instrument Standard-low speed-Metric Floor Model TT-B, Instron Corporation, Canton, Massachusetts. Cross head speed was 5 units. The single blade test cell of Food Technology Corporation Texture Test System CA-l was used for tenderness. The color of baked loaves was evaluated on a 025-2L Hunter Lab equipped with automatic reading for the color difference and a Tri scale simultaneous display. It was standardized with a yellow tile. Bakinq Procedure for American Bread. The formula for baking bread was 100 g flour, 2 g dry yeast, 2 9 sugar and 2 9 salt. The amount of water added was 50 the Farinograph water absorption for each mix. The baking procedure was optimized for the 0% formula according to the AACC method 10-10 (1961). The yeast was hydrated in water for 5 minutes in the mixing bowl, then the dry ingredients were added and mixed at speed 2 for 2 minutes, after which the bowl sides were scraped with a rubber spatula. Mixing was completed at speed 6 (standar- dized to 144 rpm) for 5 minutes; after which the dough was transferred to a floured board where it was shaped into a ball by folding in half for six times. The dough balls were transferred to slightly greased stainless steel bowls and fermented for 90 minutes at 88°F e 2 and 90% relative humidity. The fermented dough was scaled to 150 9, then degassed with a dough Sheeter set at 7/32 in, molded into loaves and panned. After it was given a 10 minute bench rest at room temperature, loaves were proofed for 60470 minutes at 88 : 2°F and 90% relative humidity. The bread was then baked at 425°F reel oven for 20 minutes. Bread was cooled to room temperature before being weighed and wrapped in plastic wrap. Volume was measured by rapeseed displace— ment. Bread was then stored in a freezer until objective and sensory evaluations were done. Testing Procedure for American Bread. pH of Dough. pH was measured at 0, 45, 90 minutes during fermentation by weighing 3 g of dough and dispensing it into 10 ml distilled water. 51 Compressability. To measure the bread texture, a 3 cm high slice of each loaf was cut with a round cutter of 5.0 cm diameter, and was compressed to 30% of its original height, using a Food Technology Corporation Texture Test System. The compressability was expressed as lbs force. 9213:. Hunter Lab color meter was standardized with a yellow tile for L=lightness (Black & white), a=redness and greenness, b=yellowness and blueness. A round slice of 5 cm diameter bread was read for the 3 scales. Sensory Evaluation. Bread SUDStltUtEd with cottonseed flour was judged for color, crust and crumb characters, grain texture and for overall acceptability by six judges. A linear scale was applied, where the score card was designed so that a line 10 cm long represents each charac- ter t0 be tested. Minimum and maximum descriptions were printed on the 0 and 10 cm mark on the line. Judges were asked to put a (X) mark along the horizontal line indicating their evaluation for each character. The sensory evaluation tests were repeated five times before the data were subjec- ted to statistical analyses. The same bread score card was used for evaluation of bread baked with different levels of additives and for the interaction between the additives. The data were used for applying Multiple-Regression analyses to optimize the levels of additives in bread substituted with 8% cottonseed flour. 52 A slice of this optimized bread was presented to each of 16 panelists, and scored for color, grain texture, tenderness, flavor and overall acceptability. These tests were duplicated. Samples of score cards appear in the Appendix. Bakinq Procedure for Egyptian Bread. Bread was substituted with cottonseed flour at 0, 4, 8, 12 and 16% of the wheat flour weight. The effect of four conditioners; Polysorbate 20 (Tween 20), Polyoxyethy- lene Sorbitan Mono Stearate (Tween 60), Mono and Diglyceride Polysorbate 60 (Tandem 552) and Polyoxyethylene 10 Stearyl Ether, each at 0, 0.5 and 1.5% of flour weight, on bread quality was tested. The effect of these conditioners on tenderness was tested after different storage periods. The bread formula was 200 g of 85% HRW wheat flour, 3 9 salt, 9 g dry yeast, and distilled water, 20% more than the Farinograph absorption. Dry yeast was hydrated for 5 minutes in the mixer bowl, then the dry ingredients were added and mixed at speed 2 for l min. The bowl sides were scraped down with a rubber spatula, after which the mixing was continued on speed 6 for 7 minutes. The dough was then transferred to a slightly greased stainless steel bowl and fermented at 88 i 2°F for 4 hrs. The fermented dough was scaled into two 150 g loaves, which were put on a cookie sheet previously dusted with shorts. The loaves were shaped by padding by hand to a round loaf of 18 cm in 53 diameter and 1 cm high. Loaves were proofed for 30 minutes at 90°F and 88% relative humidity. Bread was baked at 550°F in a reel-oven for 5 minutes, cooled to room temperature, then weighed, wrapped in plastic wrap and volume was measured with rapeseed displacement. Testing Procedure for the Egyptian Bread: Sensory Evaluation. A score card was developed for judging bread substituted with cottonseed flour where crust color, crumb color, flavor, texture, aroma, and general accepta- bility were evaluated on a scale of 1-7 where l is very poor to 7 excellent for flavor, texture, aroma and general acceptability. Crust and crumb color, descriptions were given for each of the seven numbers. A sample score card appears in the Appendix. The bread was presented to six middle eastern panelists for evaluation 1 hour after baking. The test was repeated five times. The data were subjected to statistical analy- ses. Tenderness. Bread tenderness was tested with an Instrom equipped with single blade tenderness test cell after 0, 3 and 6 days of storing bread at 10°C. Bread was left at room temperature for 1 hour before tenderness was measured each day. A round slice of bread 5 cm diameter was sheared with the single blade. Tenderness was expressed as 9 force/ 5 cm of sample diameter. 54 Chemical Analysis All chemicals used were reagent grade, and deionized water was used for all the chemical analyses. Mercuric sulfate, boric acid, potassium sulfate, sodium borohy- dride and sodium lauryl sulfate were all supplied by Fisher Chemical Company, New Jersey, Di Na-EDTA, dithiothreitol, 5,5'dithiobis-(Z-nitrobenzoic acid) and succinic anhydride were purchased from Sigma Chemical Company, St. Louis, MO. N'ethylmaleimide was supplied by Aldrich Chemical Company, Milwaukee, Wisconsin. Moisture The AACC method 44-40 (1961) was followed for moisture determination in flour. A Well mixed sample of 2 g weighed to the nearest 0.001 g was weighed into a predried and weighed aluminum dish. Samples were dried at 90°C under vacuum equivalent to 25-30 mm Hg in a Hotpack #633 vacuum drying oven to a constant weight. Samples were cooled in a desiccator to room temperature then were reweighed. The percentage loss in weight was reported as percent moisture. Bread moisture was determined according to the AACC method 62-05 (1961) where a representative loaf of bread was weighed to :0.2 g, placed on a waxed paper and sliced to 2-3 mm thick slices. Cut slices were left at room temperature for 18 hours, then were weighed and the percent loss of weight was reported as moisture at air drying. 55 Dried bread was ground to pass 20-mesh sieve and moisture was determined according to the AACC method 44-40. Percentage weight loss in the two drying procedures was expressed as percent moisture. Kjeldahl Total Nitrogen (Micro-Method). The method of McKenzie (1970) was followed. Reagents prepared for this micro Kjeldahl method included: Mercuric Sulfate Solution: In a total volume of 100 ml of 2 M sulfuric acid, 13.7 g mercuric sulfate were dis- solved. Sodium Hydroxide-Sodium Thiosulfate Solution: In 400 ml deionized water, 200 g sodium hydroxide and 12.5 g sodium thiosulfate were dissolved. Boric Acid Indicator Solution: Twenty grams boric acid were dissolved in 800 m1 deionized water, 6.67 mg methylen blue in 50 m1 deionized water and 13.3 gm methyl red in 10 ml ethyl alcohol, were all combined and brought up to 1 liter with deionized water. Hydrochloric Acid Standard Solution 0.02 N: This solu- tion was prepared diluting 1.65 ml of 37% hydrochloric acid to 1000 ml. A dry sample of approximately 30 mg, 1.5 g powdered potassium sulfate, 2 ml sulfuric acid and 0.5 ml mercuric sulfate, were added to a narrow mouthed, 100 ml non-transfer 56 micro Kjeldahl flask, and digested on a Lab con-co di- gestion rack #21621 for micro Kjeldahl digestion. Flasks were cooled after digestion and the digest was diluted with about 15 ml deionized water before distillation. A scientific Glass Associates micro-Kjeldahl distilla- tion apparatus was used, where flask mouth was greased and transferred to the apparatus. When the boiling water started to distill over, 10 m1 of sodium hydroxide-sodium thiosulfate solution was added. The steam distilled mixture was collected in 50 ml beaker containing 5 m1 of the boric acid indicator solution. Distillation was continued until 45 ml of sample were collected, then the beaker was lowered and the tip of condenser was rinsed with deionized water and 5 more ml of distillable were collected, and titrated with 0.02 N hydrochloric acid solution to a grey-lilac end point. A Nitrogen recoveries were determined with dl—tryptophan that had been dried in a dessicator. A blank was run to correct for nitrogen contamination. The percentage protein was calculated by multiplying the percentage nitrogen by 5.70 for wheat flour and 6.25 for cottonseed flour. LLLIQ- Lipid was determined in flour and bread as crude fat according to the AACC method 30-10. The following reagents were used for crude fat analyses: 57 Ethyl alcohol 95%. Ethyl ether, free from residue on evaporation. Hydrochloric acid solution 25 + 11 (v/v) Petroleum ether b.p. below 60°C. A 2 g flour sample was weighed in a 50 m1 beaker and moistened with 2 ml alcohol and 6 ml hydrochloric acid solution were added and mixed well. The beakers were held in a water bath at 70-80°C for 30-40 minutes, stirring frequently during the incubation, after which they were cooled and 10 ml alcohol were added to each mixture. Samples were transferred to Mojonnier fat extraction flasks by washing each beaker with 25 ml ethyl ether divided into 3 portions, and the flasks were shaken vigorously for 1 minute after which 25 ml redistilled petroleum ether were added and again the flasks were shaken for 1 minute. Flasks were let stand at room temperature for 1 hour until the upper fat-ether layer was clear. The fat layer was drown off and filtered through glasswool packed on a filter paper in a funnel, into a dried preweighed 125 ml flask. The liquid remaining in the fat flask was reextracted twice with 10 ml of each ether. The upper layer was fil- tered into the same flask, then the funnel and the tip of it's stem were washed with a mixture of equal parts of the two ethers. The ether was evaporated slowly on a steam bath, then the fat was dried in a Labline, Inc. fat oven at 70°C for 2.5 hours. Flasks were cooled for 30 minutes 58 in a dessicator, then reweighed. The weight was corrected by running a blank determination on the reagents used and the fat was expressed as percentage fat by acid hydrolysis. Ash. The method 08-01 of the AACC (1961) was followed, where approximately 2 i 0.01 9 well mixed samples were weighed into dried preweighed porcelain ashing dishes. The dishes were put in a Temco muffle furnace equipped with Barber- Colman Thermostat, and the oven temperature was increased gradually to 525°C. The samples were incarcerated until a constant weight was obtained. Ashing dishes were cooled in a dessicator to room temperature, and were weighed. Ash was expressed as percentage of sample weight. Sulfhydryl Groups. The procedure developed by Ellman (1959) and modified by Volpe (1976), was followed for the determination of sulfhydryl groups in the two flour samples. The reagent used for these analyses were: Sodium phosphate buffer 0.01 M pH 8.0; containing 1.1% sodium lauryl sulfate and 0.4% di Na. EDTA. 5,5'dithiobis-(2-nitrobenzoic acid) solution (DTNB): Fourty mg DTNB were dissolved in 10 ml 0.1 M sodium phos- phate pH 7.0. To determine sulfhydryls, 5 ml of 0.01 M sodium phos- phate buffer pH 8.0 were added to 10 mg of dry sample. The mixture was boiled for 30 minutes, then cooled, and 0.2 ml 59 DTNB solution were added. The color was allowed to develop for 45 minutes after which the samples were centrifuged for 10 minutes at 1000 rpm in Sorvall Glc-l centrifuge with type GSA rotor. The absorbancy was read with Beckman DB-G Grating Spectr0photometer equipped with visible and ultra- violet light sources, and 1 cm pathlength quartz cuvettes, using the wavelength 412 nm and 600 nm to correct for the turbidity in the presence of starch. The extinction coeffi- cient of 13,600 was used for determining the umoles of sH/g of sample. The correction was made for the cottonseed flour color, and a blank was run along with each determina- tion. Total Splfhydryl Groups. A method based on the reduction of disulfides to sulf- hydryls, then the determination of sulfhydryls in the pro- tein sample was developed by Cavallini et a1. (1966), and modified by Volpe (1976) was followed. The following read gents were used for this test. Sodium Phosphate buffer 0.05 M and pH 7.4 was prepared and 10 ml of 0.02 M di Na EDTA were added to the buffer at the ratio of 10 ml di Na EDTA/200 ml buffer. Urea-Sodium Borohydride Solution: Ten grams urea and 0.25 g sodium borohydride were dissolved in 10 ml deionized water. 60 Potassium Phosphate-Hydrochloric Acid Solution: It was prepared by dissolving 13.6 g mono potassium phosphate and 1.66 ml of 37% hydrochloric acid in 100 ml deionized water. DTNB Solution: 40 mg of 5,5'dithiobis-(Z-nitrogen- zoic acid) were dissolved in 10 ml 0.1 M sodium phosphate buffer pH 7.0. Anti-Foaming Agent: l-Octanol Three mg dry sample were dispersed in 1 ml sodium phosphate buffer pH 7.4, then 2 ml l-octanol and 1 ml urea- sodium borohydride solutions were added. This mixture was shaken and incubated in 40°C water bath for 30 minutes. The mixtures were cooled, and 0.5 ml potassium phosphate- hydrochloric acid solution was introduced carefully to wet the walls of the test tube in order to destroy the traces of sodium borohydride. Five minutes were allowed for the destruction of sodium borohydride after which 1 ml acetone was added and mixed to complete the destruction. The determination of sulfhydryl groups was completed according to Ellman's procedure (1959) where 0.2 ml of DTNB solution was added to the mixture and the reaction was allowed to develop for 45 minutes after which the absorbancy was read at 412 and 600 nm and the umoles SH/g sample were calculated using 13,600 as an extiction coeffi- cient. A blank was run throughout all determinations. 61 Estimation of Rheologically Active Thiol and Disulfide Groups in Dough. The quantitative method developed by Jones et al. (1974) for distinguishing the rheologically-important thiol and disulfide groups from those that are unimportant was followed. All experiments were carried out in the 50 g bowl of the Farinograph, at 30°C. A solution of l g sodium chloride in a volume of water necessary to center the peak of the Farinograph curve on the 500 BU-line was added, and was kept constant throughout all the testing. Mixing was continued for 30 minutes after which the curves were evaluated for maximum resistance and resistance to mixing after 30 minutes. i I The dithiothreitol for the determination of disulfide groups in 0% and 8% cottonseed substituted doughs, was dis- °solved 1" 0-1 ml 6t03001 and added with the salt solution to the flour at zero time. Solid DTT was added (5 to 200 umoles) in 4-5 minute intervals after the dough reached the maximum develOpment for serial addition. Determination of the reactive sulfhydryl groups in 0 and 8% cottonseed substituted dough was performed by adding N-ethylmaleimide (0 to 60 umoles) to the salt solution added to the flour at zero time in 0.1 ml ethanol. 62 Evaluation of Bonding Systems in Dough. The mixograph was used for this study where 30 i 0.01 g on 14% m.b. of 0, 8 and 16% cottonseed substituted flour mixes were weighed and were titrated with water necessary to produce a dough of the consistency determined by a Farinograph curve peak centered on the 500 BU line. The absorption was kept constant throughout testing and all the solid reagents were added dry to the flour, while the liquid reagents were added at zero time with the addition of water, and on a substitution basis of the water volume. The reagents used for this study were: Urea: It was added at 0.02, 0.04, 0.06, 0.08, 0.1 and 1 M based on the total volume of both flour and water. Sodium-Dodecyl Sulfate: SDS was added as a solid reagent to the flour at the following amounts: 0.125, 0.25, 0.5 and l g. Succinic Anhydride: It was added as a dry powder to the flour and the quantities were: 0.02, 0.1, 0.2, 1.0 and 2.0 9. Amino Acid Analysis. Amino acid analysis of straight grade HRW wheat flour, 85% extraction HRS wheat flour and liquid cyclone processed cottonseed flour were performed on 9 hydrolysates of the 63 proteins of the three flours. A Beckman Model 120 C Amino Acid Analyzer was used where the amino acids were separated by column chromatography, and the intensity of the color resulted from their reactions with ninhydrin was automati- cally recorded according to the procedures described in the literature by Moore and Stein, 1948, 1951, 1954; Moore et al., 1958; Spackman et al., 1958. Samples were prepared according to the method described in the Beckman manual for amino acid analysis (Toeffer, 1965). A standard amino acid calibration mixture was used for comparing the.chromatograms obtained. Sulfur Containing Amino Acids. Methionine and cysteine are instable during acid hydrolysis according to Schram et a1. (1953). Preliminary oxidation of performic acid was per- formed to oxidize cysteine to cysteic acid and methionine to methionine sulfone. Samples were then hydrolyzed and amino acids were determined as previously described. Tryptophan. Tryptophan is labile during hydrolysis so it was not determined but values for tryptophan was supplied from the literature for each flour sample. Scanning Electron Microscopy Studies The effects of cottonseed substitution of wheat flour and emulsifier polysorbate 20 were studied on 0 and 8% substituted dough systems. The mixograph was used to mix 0 and 8% substituted dough, with and without 0.5% emulsi- fier (Tween 20) to optimum development. Immediately after 64 the mixing, thin strips of dough were cut with a pair of scissors from a smooth freshly exposed surface (Hooper and Volpe). Dough strips were quickly immersed in liquid nitrogen then fractured to pieces 2-3 cm long, then were freeze dried. Some dried samples were fractured. The effect of sample preparation on the electro- micrographs was studied, where 0, 8% substituted doughs were prepared by mixing dough to the maximum development in a mixograph with and without 0.5% emulsifier Tween 20. Thin strips of dough were cut with a pair of scissors from a smooth freshly exposed surface. Dough strips were then divided into small segments about 2-3 mm long then fixed for 24 hours at 10°C in 0.1 M phosphate buffered glutaralde- hyde (5%), adjusted to pH 6.0. After fixation, samples were dehydrated in graded ethanol series 10 to 100%, after which they were critical point dried (CPD) with C02 as the ambient liquid. Some CPD samples were fractured. All specimens were mounted on aluminum stubs using television tube coat, then were coated with gold, and viewed at 20 KEV in ISI Super Mini scanning electron microscope. All sample preparations were duplicated and the representative areas were carefully examined. RESULTS AND DISCUSSION Farinograph Studies of HRW Wheat Flour Supple- mented with LCP Cottonseed Flour and Unmodified Starch. Absorption is the amount of water that flour requires to form a dough of optimum consistency for bread-making. It is usually determined to be the amount of water required to center the Farinograph curve on the 500 BU line. The absorption is actually dependent upon by the amount of water required by the various flour components, particularly by starch and protein (Bushuk and Hlynka, 1964). The increased absorptions of wheat flours substituted with different oilseed proteins have been reported by many researchers. Cottonseed protein concentrates prepared by wet-extraction and spray or freeze dried at different pH wawashown to have different water absorptions (Khan et al., 1976). In the LCP cottonseed flour system, the water absorption increased as the level of cottonseed flour increased in the system (Table 1); however, the maximum absorption seems to be obtained at the 8% level of substitution, after which there was no significant increase in absorption. 65 66 Table l. Farinograph data1and SD for HRW wheat flour dough substituted with LCP2 cottonseed flour Cottonseed 3 4 5 Flour Absorption Peak Time M.T.I. T.M.D. % % min B.U. B.U. 0.0 58.67 3.57 33.0 87.0 :0.5 $0.1 $4.8 $4.7 4.0 60.94 3.23 117.0 187.0 $0.6 $0.2 $4.9 $4.8 8.0 62.32 3.60 82.0 110.0 $0.6 $0.3 $10.6 $0.0 12.0 62.97 3.47 107.0 160.0 $0.6 $0.1 $2.8 $0.0 16.0 62.90 3.70 135.0 217.0 $0.7 $0.1 $10.8 $4.7 1) Average of three replications. 2) Liquid Cyclone Processed cottonseed flour. 3) Absorption is expressed on 14% M.B. 4) 5) Mixing Tolerance Index. Twenty Minute Drop. 67 Wheat flour was substituted with wheat starch at 0, 4, 8, 12, 16% of flour wheat in order to study the effect of dilution per se as compared to LCP cottonseed effects. The water absorption in the wheat flour/wheat starch system followed an opposite trend from that of wheat flour/ cottonseed system, as can be seen from Figure 1. Data presented in Table 2 shows a decrease in water absorption with increasing the level of wheat starch in the system, with a mimimum absorption obtained at the 8% level of substitution. Starch absorbs somewhat less than its own weight of water (27-35%), while gluten, on the other hand, absorbs more than its weight of water (109-215%) (Bushuk et al., 1964). This can explain the difference observed in absorp- tion between the two systems. Hagenmaier (1972) studied the water binding of some oilseed isolates and reported that there was an increase of water binding with larger values of hydrophilic groups. This could be another reason for the higher Farinograph water absorption values with higher levels of cottonseed flour. Cottonseed flour has high glutamic and aspartic acids content. Results suggest that there must be a minimum amount of gluten present in the system in order to get the maximum absorption, which can be seen from Tables 1 and 2. The dilution of wheat gluten with either plant protein or starch with up to 8% showed clearly that effect. Figure l. Farinograph absorption of dough systems substi- tuted with cottonseed flour, and starch Water Absorpt ion /% 68 Q.....Q......O LCP Cottonseed Flour D—D—OVIhou Starch 1 L: 9 Water Absorption /'/. .3 57.5 l 1 k l T p 0 4 O 12 16 Substitution Level (Der100g) 69 Tabie 2. Farinograph Data1 and SD for HRW wheat fiour dough substituted with wheat starch Starch Absorption3 Peak Time M.T.I. T.M.D.5 % min B.U. B.U. 0.0 58.55 3.67 42.0 37.0 :0.9 :6.2 :2.4 4.0 58.24 3.37 35.0 50.0 :0.2 $4.] :4.1 8.0 57.62 3.07 33.0 42.0 :0.1 $6.2 :2.4 12.0 57.40 3.45 47.0 55.0 :0.2 :4.7 14.1 16.0 57.08 3.22 48.0 68.0 :0.1 26.2 12.4 Average of three repiications. Unmodified wheat starch. Absorption is expressed on 14% M.B. Mixing Toierance Index. Twenty Minute Drop. 70 Arrival time is a measure of the rate of hydration of flour constituents. Bushuk et al. (1964) reported that there is a decrease in the rate of hydration with the increase in protein content. The substitution with cottonseed flour (Table l) caused an increase in the arrival time with increasing the level of cottonseed flour in the system, while wheat starch (Table 2) caused a decrease in the arrival time, as can be seen from Figure 2. That could be resulting from diluting the protein with wheat starch. The peak time is the time the dough takes to reach maximum consistency or minimum mobility. The results of peak time presented in Tables 1 and 2 are consistent with the results obtained on the rate of hydration (Figure 3); wheat/cottonseed systems show an increase in the peak time with increasing the level of cottonseed flour, with an unusual drop at the peak time at the 4% level of substitu- tion. The wheat/starch system show a different trend with a drop in the peak time at 8% level of substitution, which indicate that the peak time is actually related to physical development of gluten, rather than to the protein content. Mixing Tolerance Index (M.T.I.), which is the difference in BU from the top of the curve at the peak to the top of the curve measured at 5.0 minutes after the peak; and the Twenty Minute Drop (T.M.D.), are both measures of dough breakdown. Table l and Figure 4A show the increase in the dough breakdown with the high levels of cottonseed flour Figure 2. Farinograph arrival time of wheat/cottonseed ahd wheat/starch doughs Arrival Time/min 71 0......0 ...... Q LCP CottonaaOd Flour 4 r- U—U—D Whaat Starch ,,.o 3 " O ............... O". .0" (3 0—0 0 k 4 8 12 16 Substitution Level (per 1009) Figure 3. Farinograph peak time of wheat/cottonseed and wheat/starch doughs . 72 m .. .m F d e eh am mu - as 0 Ct u an LW 0.” O I . _ _ . 7 e 4 2 o. 3 3 3 3 3 EE\mE:. xmom 16 12 Substitution Level (periOOQ) Figure 4A. Effect of cottonseed flour and starch on Farinograph Mixing Tolerance Index 73 O......O......O LCP Cottonseed Flour H——CI Wheat Starch :m\xouc_ 8:338. @552 m 5 '-3o P 140 120 _ w w m 100 _ 3m \xmo: _ 8:928 9: 5.2 20- J L.T T 16 12 Substitution Level (per100g) 74 (l2 and 16%) as can be seen from the mixing tolerance index which indicates that the Farinograph curves drop as much as l35 BU. There is also an increase in the dough breakdown with the addition of starch, up to 47-48 BU at l2 and 16% levels. T.M.D. (Figure 4B) whows the same thing. The data suggest that starch contribute, to the sta- bility of wheat flour dough substituted with cottonseed flour, and that a certain balance between protein and starch must be obtained in order to get the Optimum Farinograph properties, which are used as an indication of flour per- formance in bread-making. Viscoamylograph Study. Viscoamylograph was used to determine the effect of alpha-amylase on the viscosity of flour as a function of temperature. The high viscosity of the starch gel is counteracted by the action of alpha-amylase, which liquifies starch granules during heating of slurry. The amylograph value provides information on the possible effect of alpha- amylase or on the starch gelatinization during baking process (AACC, l960). The potential of wheat/cottonseed flour blends to pro- duce good loaf volume was studied. The peak viscosity and the viscosity after 60 minutes are represented in Figure 5; a slight decrease in both viscosities with the higher levels of cottonseed flour in the blends is apparent. These results suggest that there is a good potential, Figure 48. Effect of cottonseed flour and starch on Farinograph Twenty Minute Drop 75 O.....O.....O LCP Cottonseed Flour Wheat Starch 7O 7 . Dm\ao.o 23:22 3cm): 0 5 -40 3m \aoco 33:22 3:93. 16 Substitution Level (per1009) Figure 5. Viscoamylograph peak viscosity and viscosity at 50 min for wheat flour substituted with LCP cottonseed flour 76 @0489 1033 modem mod? .ozcoo b_moom_> xmon. ho com 1 Gem doom 3m .55 co u< >~_moow_> oow com 3m com cow 1 com mEbEmczmmm—z 585038835 77 even at the high level of cottonseed flour (16%), to give- enough fermentable carbohydrate in the flour blend, there- fore, a good loaf volume. BakingAStudy, Bread was baked using 100 g flour, 2 g dry yeast, 2 9 active dry yeast, 2 9 sugar, 2 9 salt, and the amount of water added was exactly the Farinograph water absorption, previously determined for each flour blend. The pH of dough prepared with different levels of cottonseed flour increased as the level of cottonseed increased in the system (Figure 6). During fermentation, there was a drop in pH at all levels of substitution with cottonseed but the pH of the substituted dough was much higher than the control dough, as can be seen in Figure 6. Mathason (1978) reported that the optimum pH value of white bread and rolls is 5.2 when bread's PH 15 higher than 5.8, it would contribute to dark crust, an open, crumbly grain, lack of flavor and sharp corners on pan loaves. The protein content of bread made with cotton seed increased from 7.82% in the control to 12.68% at the 16% level of substitution, which is equal to 62.15% increase in the protein content (Table 3). Although the higher levels of cottonseed flour in bread did not produce bread that can be defined as high protein bread (15% protein), it increased the protein significantly with each addition of cottonseed flour. Breads made with LCP cottonseed flour Figure 6. Effect of cottonseed flour on pH of dough during fermentation 6-4 6.2 6-0 5.4 78 c—a—co .——e——e4 e—e——e8 96ch CSF e ........ .. ...... .012 e—o—-—016 \‘ \~ \.\~ \‘ --- ..... e \m. e- ————————— 3: ’ 4s 90 Time/min 79 Table 3. Proximate analysis1 of bread prepared with different levels of LCP cottonseed flour Substitution Moisture Protein Fat Ash level % % % % % 0 42.34 7.82 1.26 1.30 4 38.14 9.35 1.86 1.70 8 37.95 10.92 1.58 1.74 12 37.95 12.38 1.34 1.79 16 38.26 12.68 1.97 2.06 1Values are average of three replications and are expressed on as is moisture basis. 80 actually met the Federal standard level of moisture in bread of 38%. Ash content also increased with an increas- ing level of cottonseed flour in bread. White bread made from untreated HRN wheat flour and bread substituted with LCP cottonseed flour were baked, and were evaluated objectively for volume, specific volume, compressability and color. Breads were evaluated for crust color and character, crumb color, grain texture and for general acceptability by taste panelists. These results (Table 4) show that there is no significant effects of cottonseed flour substitution on loaf volume, specific volume or compressability. In fact there was a slight increase in loaf volume and specific volume with the substi- tution of wheat flour with 4, 8, and 12% LCP cottonseed flour. The addition of LCP cottonseed flour significantly influenced bread color. The lightness and the yellowness of bread substituted with cottonseed flour decreased signi- ficantly with an increasing level of substitution indi- cating a darker,more greenish color bread (Figure 7). Sensory evaluation data (Table 5) indicated the sig- nificant effect of cottonseed flour on bread crust and crumb color (p<0.0005) which indicate a darker product with higher levels of cottonseed flour; it is consistent with objective color measurement. Grain texture scores increased significantly (p<0.0005) with the addition of cottonseed, 81 Table 4. Mean and standard deviation of objective measure- ment of wheat/cottonseed flour bread \Substi- Loaf Color tution Comp level vol sv lb/cm L*** a*** b* % cc cc/g 0 474a 3.50a 37.35a 42.53c -1.23a 10.83c :23 $0.3 $4.1 $1.2 $0.2 $0.5 4 484a 3.68a 48.12a 36.73b -o.43b 10.30ab $49 $0.4 $11.9 $0.6 $0.2 $0.6 8 483a 3.68a 39.50a 31.90a -o.35b 9.90a $42 $0.4 $8.2 $0.2 $0.2 $0.4 12 481a 3.56a 46.08a 31.08a 0.70bc 10.05ab $17 $0.1 $10.5 $0.6 $0.2 $0.4 l6 468a 3.42a 44.83a 29.733, 0.83c 9.82a $12 $0.1 $5.0 $0.7 $0.1 $0.2 Means followed wuuaammg mooo.owa ecu moo.owa we mpo>mp mocmuwmwcmwm on ccoammggou ««e .ee .mm—mom opuo we cowuczmpaxm Low F xwucmaa< mom AN .eeeeee_em_m eoz A. ON.OH Fm.OH om.ow FN.OH mm.oH was 0 cum 0 vmm m mmm m cue a my m¢.ow mN.OH mo.cm me.ow N¢.OH men n cum 9 comp m capo 0 0mm m up om.ow m¢.ow ¢¢.on Fo.ow 00.0“ mam B one m up“ n. gnome m 0mm n m mm.ow mm.0m Fo.ow mc.ow ¢¢.OH own 5 new e new m com 5 nmo m w o¢.NH Nm.ow ¢O.PH em.PH mm.ow mom.~ nv¢.© moo.~ unn¢.n mmm.m o opio opio opio opio Nopio N xSPFanuamoo< mgauxm» Lo—ou gmaomgmzu gopoo Fm>m— Fpmgwcmw ##kcwmgw crcnssgu «rumzsu keeungu uwwmcouuou omega Lzope ummmcouuoo\umm;3 co cowgmzpm>m Acomcmm we :o_umw>mc ugmucmum uco :mm: .m mpnwh 84 indicating a much more Open grain with the high levels of cottonseed in bread, however the 8% level of substitution had the coarsest grain of all the levels. Similar changes in crumb grain have been reported by Fleming and Sosulski (1978) on bread containing soy flour, sunflower, faba bean and field pea. Rooney et a1. (1972) reported a similar effect of solvent extracted cottonseed flour, on bread texture. Brean was generally scored as hence slightly unaccep- table (between 6-8 on the sensory scale). Mixograph Studies of Effect of Sodium Chloride, Potassium Bromate, and Polysorbate (20) on HRH Wheat/LCP Cottonseed Flour Dough One approach to improve bread characters is_to use different additives. The effect of additives on mixograms was evaluated. Each additive was evaluated separately and in combination with the others. All the possible interac- tions, at all levels of additives were studied. Mixograms were evaluated for peak time, peak height, curve height at 9 minute point, and the area under the curve. Single Additive Effects From Table 6 it can be seen that cottonseed, salt and conditioners significantly affected all the mixograph characters studied (p<0.0005), potassium bromate's effect was less significant on peak height (p<0.05) and height of 85 mo.ova mz mooo.ova¥¥¥ moo.ovn «r mo.ova k ea.mp No.o m¢.o «teoo.o Pm Logcm Fmacpmmm ertmm.pm ¥¥«PP.O rekmp.o «eemm.o mp mmeosmrow cmmzhxupmmkumwmcouuou «oe.~¢ *««op.o «txm~.o eexmm.o m mumsocmtom cmmzheupmm ««¢~.mm mzmo.o mzmo.o «tepm.o m mumeocmtom :mmzhtummmcopuoo «teem.~m ttto~.o tet-.o ette~.o w momeogmeupmmxummmcoupoo xxtmm.Fm «mo.o mzmo.o xttmm.o w om cmozhtumetummmcoauou sxx-.-~ «mo.o mzmo.o «ttn¢.o e mumsogmeummmcouuou mz~m.om «emp.o te-.o tttun.~ e mumsogmtupmm «ex—m.mp_ «to—.o mzmo.o «xtoo.P S cm cwmzpecmmmcopuoo www.mm «««mm.o tem~.o xtto~.o e wumaocmtom cmmzh mzo~.o¢ «eepm.o tmp.o «xxmm.o v om :mmzhtapmm etemm.om~ «tt¢m.o mzp.o tetsm.~F v upmmtummmcoppoo ¥*¥0¢.oom «rwp.o km~.o *«rmh.np N oumeogm txeoo.mom «stem.o «««oo._ «etmo.op N om cmmzh ettmo.m~m_ «ttm¢.p~ e««¢m.o ttt¢~.F~P N upmm «tkmm.-mm *«rmm.mm *«emo.w~ #*«NP.PN N ummmcoupou m>cso ucwon cps agave: we?» soummgu mocmwgm> Love: mmL< m an a: Jew; xmmm yo mmmcmmo mo muczom \ummsz 3m: eo mgmumEmgma :amgmost Low mocmuwcwcmmm momumwumpm m can mmgmzcm cam: gmzoc ummmcouuou no; .m m_nmp 86 curve at 9 min point (p<0.0005). Figures 8A through C represent those effects. Salt. Adding salt to the flour markedly affected the physical characteristics of the dough, especially when 2% salt was added. Figure 8A shows that increasing the salt level from 1 to 2% increased the peak time, and this effect was noticable with the higher levels of cottonseed flour in dough. Salt increased the peak height especially at 1% level, and the curve height at 9 min point. The area under the curve decreased with the addition of salt. This effect was very pronounced for the 8% level of substitution with LCP cottonseed flour, and 2% level of salt. The effect of salt is thought to be primarily due to the changes in gluten hydration, a phenomenon Bushuk and Hlynka (1964) explained as free and bound water. The presence of salt in dough system increased the amount of mobile or free water in dough by occupying the sites once occupied by bound water, thus altering the gluten structure, which results in longer peak time. The increase in gluten strength is represented by the increase in peak height as well as the increase in the curve height at 9 min point (Figure 8A). The effect of salt on strengthening or tough- ening wheat flour dough has been reported in the literature (Galal et al., 1978). Figure 8A. Effect of salt on mixograph properties of 0, 8, 16% dough substituted with cottonseed flour 87 *—‘—* a./O I) N- 10F .d—-.16°/0 : ES .. 7 .. E e s E 3 \. .s i :6 .. .36 L C- . z / fl. 4 e 5 _. i t O 1 2 8 _ 56- /° 90 _ \ it I «a £4- / i 70. . 1' < 2 50 t t 0 1 2 5011(9/1009) Sa|t(g “009) 88 Gluten proteins precipitate at ionic strength above 0.04 corresponding to a level of addition of 7-8 mmoles of sodium chloride. Since gluten proteins are insoluble in the presence of salt, they will favor compact forms minimizing protein-solvent contacts. The salt-soluble proteins, on the other hand, favor elongated configuration giving a maximum number of protein-solvent contacts, thus enabling maximum inter-chain bonding. As the gluten protein content in dough increases, the ratio of salt-soluble to salt-insoluble proteins will decrease thus the strengthening effect of salt is more noticable. Substituting 8 and 16% of LCP cottonseed flour for wheat flour increased the protein content of dough, but decreased its gluten content. According to the effect of salt on protein explained before (Bennett et al., 1965), the dough strength would decrease with increasing the level of cotton- seed flour. This effect can be seen clearly (Figure 8A); all the doughswith cottonseed showed weaker gluten strength represented by lower peak height, lower height at 9 min. point and smaller area than the control flour. Polyoxyethylene Sorbitan (20) Mono Laureate (Tween 20). Tween 20 significantly affected all mixograph proper- ties (p<0.0005 (Table 6). As a dough conditioner, Tween 20 strengthened the dough as can be seen from Figure 8B, by the increase in peak time, peak height, height at 9 min point and the area under the curve. The conditioner was Figure BB. Effect of conditioner "Tween 20" on mixograph parameters of 0, 8, 16% doughs substituted with cottonseed flour Peal! "me/m"! “L9 NHL/min q 89 J- T . . 4 0 0.5 1's 6 l" O‘gxfi s - . __.———e_v e_em «Ne._~ ~o~.m~ ame._m ap_.m mmo.m om~.m aomm.o asm.o amm.o umm.m npm.m apm.m o.om m am—.- nsm.om nmm.om ampo.m amm.m op~.m u~—.o amm.o amm.o owe.o nm~.m omm.m o.om sag ocmx amm._m umm.~m uamp.om mec.m o-.m mmm.e n~¢.o a-.o amm.e nm¢.m om_.m mmm.e o.o m.p m.c c.c m._ m.o c.o m.~ m.c o.o m._ m.o o.o an pw>mpv om cmmxp com.mo mum.m~ www.mn omm.o ome.o omm.m ouc.o oo~.o umm.m um—.~ omm.e oom.o o.~ omm.e~ am~.~m aom.oh noo.m omp.o awo.o oom.e om~.o umm.m uum.m n_~.m um_.o o._ a Spam amo._m am~.om um~.mo a~—.m am~.m am~.m ama.m nomm.o uom.m um~.e mm¢.¢ a-.e o.o m._ m.o o.o m._ m.o c.o m._ m.o o.o m.. m.c o.o an po>mpv om cmwzh so So So =_E -ll .meé Ii} 1:... a a: .222. .2... 1...: 3.2.... sauce cao—w umomcouuoo qua we as» an mcwuomcugo :aucmox.e :o mo>.u.uua co mcopuu:_asou o_a=ou co :ovuoocoucp as» we uuocmu .5 m_no» 95 peak time significantly p30.0005 from 4.89 minutes in the control system to 6.42 minutes in the double additives system. Peak height was significantly affected by the inter- action between double combinations of additives (Table 6). The maximum peak height was obtained at the 0.5% level of conditioner (Tween 20) with 1% salt both the 0 and 8% and 30 ppm KBrg, for 8% LCP systems. Peak height generally decreased when potassium bromate and salt were added ’together; this effect can be seen from Tables 7 and 8. The peak height increased as salt and bromate were added at all levels of combinations, with maximum effect (6.63 cm) obtained at the combination of 30 ppm KBrg with 0.5% con- ditioner. The salt increased the stability of both 0 and 8% LCP dough systems through its interaction with conditioner and potassium bromate as can be seen from curve height at 9 minute point. Table 7 shows that the maximum stability was reached with the combination levels of 2% salt, 0.5% con- ditioner and 50 ppm KBrg, 2.0% salt. The 8% LCP dough system stability also increased when the double combinations that included salt were added (Table 8). The maximum stability was obtained when salt was added at 2% level with 30 ppm KBrg (5.98 cm) and 2% salt with 0.5% conditioner (5.95 cm). The interaction between potassium bromate and conditioner in both dough systems (Tables 7 and 8) indicated .umo» «scam o—a—u—az m.:ao==a mo.cwa um acaceccpc xpa:Nu—u¥cm.m ac: ecu couump «sum ogu an nozop—oe memo: p 96 o~o.eo nom.a~ a~o.ms ovm.m amo.m amo.m mom.~ umm.m ~m~.¢ o.om m www.mm www.ms a-.o~ uma.m aho.m aso.m owe.“ oem.m n~¢.¢ o.om Egg ocmx amm.me can em amo ms umo.m nmm e u~m.m cm~.o n¢~.e mmm.m o.o o.~ o.— o.c o.~ o.p o.o o.~ o._ o.o Am pe>eev e_em oom.o~ a-.o~ usa.p~ nmo.m nso.m amp.m uo~.o amm.m umm.m c.0m m amp mu awn us use as amo.m amm m «me o umo m up. a u-.m o.om Ema ocmx nmm Nu amp mm aco us puma e nap m omo 0 665 m can m mom e o o m._ m.o c.o m.p m.c o.o m._ m.o o.o pu>opv cw coax» am~.~m umm.o~ www.mo umm.m umm.m umc.m mam.“ vom.u wen.“ o.~ -~.mm am¢.mm am¢.m~ nsm.e am_.m um~.m umo.m uoe.m nm~.¢ o._ x u_om omm.es mmm.pm www.ms am~.e com.v moc.o amm.e noe.e oom.m o.o m.p m.c o.o m.— m.c o.c m._ m.o o.o Au po>mpv om cough so Eu ewe quc< Fave m u: Fugm.m: xooa Poewh goo; canoe :. Lao—c vmmmcouuoo mu; co pm>mp am on» an mo.um_cmuoacoco gaacmoxgs co mw>.a_uua mo mcopua:_nsoo mpnzou mo cowuomcmuzp wzu co uumwmu .m epoch 97 that the interaction between 30 ppm KBro3 and 0.5% condi- tioner had the maximum effect on stability (5.33, 5.35 cm) in the 0 and 8% LCP cottonseed dough systems, respectively. The area under the curve decreased significantly (p<0.05) when all levels of combinations of KBro3 and con- ditioner were added to the dough. The combination including salt as a factor did not have any significant effect on reducing the area. Triple Combinations Effects The effects of the three additive combinations were tested on the mixograph characters of 0 and 8% LCP dough systems. Table 6 shows that the interaction between the three additives significantly (p<0.0005) increased the mixograph peak time. In the 0% LCP dough, the interaction between the additives salt (2%), KBro3 (50 ppm), and condi- tioner (1.5%) produced the longest peak time of all inter- actions (Table 9). In the 16% LCP dough system the combi- nation of additives that increased the peak time to the maximum were conditioner (1.5%), KBro3 (30_ppm) and salt (2%) (Table 10). Peak height was significantly reduced (p<0.005) when the three additives were added together. The triple combi- nations that contained salt had slightly higher peak height as can be seen from Tables 9 and 10. The combination of 1% salt, 30 ppm Kqugand 0.5% conditioner in 0% LCP dough 98 .umew enema epewupez m.eeee=e newm: mo.owe we «emcewwwe x—ueeowwwemwm we: wee eeuuep mama me» he eczeF—ew meme:F ee.ee ee~.ee eee._e m¢.e eem.e e3.... em~.e emm.e eee.e em~.~ meme.“ emN.e m._ eem.e~ eem.me ee.me eem.e eme.e aS... eee.e ee~.e eee.e eeee.~ coca.“ e_~.m m.e wow eeezw eem.e~ eae.m~ em.ee eme.e eee.e ee~.e ee~.e emm.e em~.e eaee.e eeNe.e em~.m e.e e Auue Pe>e_ upem em.m~ e_.m~ eem.e~ eneme.e eeee_.e neme.m eee.e eee.e eem.e eeem~.e eee.eeee_e.m m._ eeee.eeeaem.ee a.ee eeeee.e emm.e eeee_.e eem.e eee.e eee.e eeme.e ease.m em~.m m.e eeN eeezw ea eee_.ew ee.m~ e_.~e eemP.e eem_.e em~.m eem.e eee.e eme.e eemm.e em~.e eeem.m e.e Aupe _e>ep e_em e~.ee e~.ee eee.ee enme.m eee.e uee.m eeee_.e eme.m eeee_.e eme.e eee.e eem.e m.. em.ee aes.me eh.ee. emm.e eeee.m eem.m eeee.e eeeem.e eeh.e e_e.e eem.e eee.e m.e gem eeezw em.pa ee.ea em.em eemh.m eee.m eemm.m eeeme.e eeeem.e_ eumm.e em~.e aee.e em_.e e.e any .esep epem em on e em on e on on e om em e Aseee..e>e. meme so Eu Eu ewe .mee< Few: a a: .8: xeee .eeww some smeee gees: —ecueee e we mceuoeceee geeemest we» ee me>wuweee ewes» we» eeezuee eewueeemuew ecu we ueawwe mew .m epeew 99 .umew emeem epewupez m.eeeeee eewme pe>e~ mo.owe we ueocewwwe zpueeewwweewm no: use eeuue— «see one an eezeppew meme: p ep.po em.eo e¢.po emm.m eeemw.m eoemo.m eme.m eemm.m eemo.o eeomw.w eewm.w weesw.w m._ new we eem om ea ms eeem m eeeec 9 com: o eemo o emu m emm m eemm w eeeee w ewe m m 9 wow eeezw ep.mo eem.oc eem.we eo~.m euop.o emm.e eom.m gee—.o ecu.m e~m.m me—.m eom.m o.o Aumv —e>e_ “Fem e~.~w em.m~ ee—.mm eeop.m eeom.e eeecc.m eeow.e emm.e eeo~.o unw.m eee~.m eemm.m m.P nee.ms eew.mm ep.mm eeemm.¢ em~.m em~.m eoo.o eec~.m eom.m eew~.m eeow.m em—.m m.o wow eeezw ee~.~m em.w~ ee¢.om ee~.m eemp.m eow.¢ eemm.m eemm.m eemp.m eemm.m ee~¢.m eon.m o.o Aa—v pe>e_ «Fem eew.~w eeew.mw ew.ow emm.e e¢.e eoo.¢ eoeoe.m eoeo—.m eme.m e¢—.m emm.¢ eeo—.e m.p ens pm one we eem ~m oeme e eom e eemm e eemm o eeem e eeco m eem e em— m eeeoo e m o Rom eeezw ew.oh ew.wm oem.~m eeme.v eeme.e emm.e eeeom.m eeeom.m eo¢.o eeom.m oeeem.m eoe.m e.o Anov po>e_ upem em on o em on o cm on e em on o Azeev Fe>ep memx so So So ewe pmec< pews m a: we: gee; pesww xeee .smeee ceepw eeemeeuuee eue am we meeueeceee :eecmexwe use so me>wuweee ewes» use emexuee eewueeeeuew an» we weewwe mew .o_ epeew 100 system produced the highest peak (6.90 cm) vs (5.75 cm) peak height when the combination 0% salt, 30 ppm KBrg and 1.5% conditioner (Table 9). Similar effects were observed in the 8% LCP dough systems as shown in Table 10. Dough stability as measured by the mixograph curve at the 9-minute point followed the same trend as did the data for area. These results show clearly the effect of salt on strengthening the dough structure. The toughening effect of salt increased as the level of salt increased in both the double and triple additive combinations, and it was measured by increasing the peak time and decreasing peak height, 9 min ht and area under the curve. These decreases resulted from a very high resistance of dough to the mixer. When bromate was combined with salt at different levels, the bromate did not show a strong effect. This could be due to the fact that potassium bromate is a slow acting oxidant, and does not exert all its oxidative reaction during dough developing. When bromate was used at its highest level the action of bromate as an oxidant was clear, since it also toughened the dough in the presence of salt. Baking Study. Bread substituted with 8% LCP cottonseed flour was baked with all the levels of combinations according to a 33 factorial design (Table 11). .Eeew eweewp eeewo cw eumeeee eeez eepweLem Aomv weepaeeeXxezweem .e>wuweee seem cow Fe>e~ eeeN me» me emeee mew: meeeee e>wuweee ezF 101 meeme see Om .Opmm em meeme eOO Om .O_em mm ON eeezw em.. .e_em mm ON eeezw em.O .e_mm em moeme eOO Om .Om eeezw em.P .epem em meeme eOO Om .O_em e_ meeme gee Om .Om eeezw em.O .e_mm em meeme EOO Om .Opmm e_ em.. meeme see Om .ON eeezw em.F .Opmm em ON eeezw em._ .e_em e, em.O ON eeezw meeme gee Om .ON eeezw em.O .ewem em ON eeeze em.O .O_em e. see om meeme gee Om .ON eeezw em.~ .Opmm e_ meeme see Om .Om eeezw em.~ eOe Om meme meeme see Om .ON Oeezw em.O .e_mm e. _ meeme see Om .Om eeezw em.. mecme see Om .Om Oeezw mm.P .e.em e_ meeme eOO Om .ON eeezw em.O mm meeme eOO Om .Om eeezw em.O .e_em e_ moeme see Om .Om eeeze em.O e. e_em OOmOmOPOeOO eemememeeee e>wemeee epewcw epeeeo ewmewm mwe>e— m we seem mueseee eee eeeewuweeeo .HFem we meewpeeweeee epewmmee ecu _—< .ww epeew 102 The baked bread was subjected to factorial analysis (Table 12) where all the main effects as well as all the possible interactions were tested. Salt significantly (p50.005) affected the bread volume, 1% salt increased loaf volume while 2% decreased it (Table 13A). The very pro- nounced effect of additives was observed on the compressa- bility where all single additives as well as their inter- actions reduced the force needed to compress the bread sample to a constant height indicating these breads had a softer crumb. Results for volume and compressability are presented in Tables 13A, B and C. When single additives were added to bread, salt had a significant effect on volume (Table 13A) especially at 1% level (552 cc vs 512 cc untreated bread). The effect of salt on volume is obvious even when combina- tions of additives were added (Table 13A and B). The 2% salt in single or combined additives reduced loaf volume, and crumb softness (Table 13A). The double combinations of additives reduced bread volume and produced breadsthat have harder crumb than the triple combinations (Tables 13B, C). The highest volume (637 cc) and the softest crumb (17.8 g/cm) were obtained at the combination of 1% salt, 1.5% Tween 20, 50 ppm KBrg. Sensory Evaluation. Sensory evaluation data (Table 14) indicate the signi- ficant effect of additives and their interactions on grain texture. Salt and any additive combination that included 103 epeeeee Eewmmeueem AON :eezwv eeeewuweeeeN u—mmp mooo.o geek moo.o a we mo.o e w mm —euew Fm.opp Fo.o NN.o Np.eome NN eeecm —eeewmem «twmm.woop «mm.o m_.o mN.meom w mxuxm «wem.eme oo.o m_.o mm.emNN e mxu «meme.woeN we~o.o PN.o mN.Nmem e mxm «wwmm.mmmw weo.o N_.o mm.om_w e uxm wwm¢.pNoF «eo.o oo.o mm.mwmop N mm wwwmm.m~NP «em—.o N¢.o em.mmmm N No wewmm.momp *«me.o «hmm.N «wmw.wmmmm N Fm se\e e e\ee .ee seemeee eewuewem> xuwwweemmeceeeu : esewe> .em esewe> we meeemee we eeeeem mueeseeemmes m>weomnee ee eeweeeeeeew ewes» eee me>wuweee we eeewwe we» eee .eeeee eeeeuwumeem am we eeeewee> we mwmxweee we meeeem eee: .Nw epemw Table 13A. 104 The mean1 effect of single additives on bread volume and compressability Volume ***Com ressabilit Additives 1331113: figs pgégm y No additive 0.0 513:50.3 65.6$24.8a Salt % 1.0 **552$74.7 46.7$25.9a 2.0 460$62.2 64.6$25.Sa KBrg ppm 30 514$60.8 52.51223a 50 529:30.o 52.4$28.8a Tween 20% 0.5 505$63.0 63.4$29.8a 1.5 529$73.6 47.6$21.7a ** pSo.oos ***pso.ooos Means followed by the same letter are not significantly different p50.05 Duncan's Multipls Range Test. 105 Table 13B. The mean1 effect of double combinations of additives on 8% LCP cottonseed flour bread volume and compressability . . . . Volume ***Compressability Add1t1ve Comanations cc g/cm 0.5% Tween 20, 30 ppm KBrg 490 60.13a 0.5% Tween 20, 50 ppm KBrg 541 49.3a 1.5% Tween 20, 30 ppm KBrg 490 44.7a 1.5% Tween 20, 50 ppm KBrg 555 42.3a 1% salt, 0.5% Tween 20 541 57.1a 1% salt, 1.5% Tween 20 572 36.5a 1% salt, 30 ppm KBrg 556 35.4a '1% salt, 50 ppm KBrg 600 29.2b 2% salt, 0.5% Tween 20 464 59.26 2% salt, 1.5% Tween 20 468 43.8a 2% salt, 30 ppm KBrg 476 58.2a 2% salt, 50 ppm KBrg 467 57.6a ***p50.ooos 1Means followed by the same letter are not significantly different at p50.05 Duncan's Multiple Range Test. 106 m.emeeeo mo.owe we ueeeewwwe xpueeewwweewm pee wee meeueep esem wee xe eezeFFew meme: .pmew emcee epewepez mOOO.OwOwww me.~e mme mEme gee Om .Om Oeezw mm.F .epmm em mm.mm . mOO meeme see Om .ON eeezw mm.O .epem em m~.mm mme meeme Eee Om .ON eeezw em._ .epmm em mm.mm Owe meeme see Om .ON eeezw mm.O .emem mm ee.em mmm meeme see Om .ON emezw em.. .Oeem m. m..ON mOm meeme see Om .ON eeezw mm.O .SPOm e_ em.m~ mmm meeme gee Om .Om eeeze mm.F .epem m_ mm.mm Nmm meeme eOO Om .ON eeezw mm.O .O.mm mp Ee\a ee xuwpwewmmwgasouwww eEeFe> meewpeeweseo e>wuwee< eemmeeuuee eue am so me>wuweee we meewueeweEee mpeweu we poewwe muwpweemmeeesee eee eEeFe> eemee geepw weees mew .ump eweew 107 mooo.owawtw moo.owa we mo.owe « mm PQHOH mm.o mm.o mN.o mm.o KN LOLsu mo.N «hemm.¢ wwwpm.~ mN.o w ucmvwxo x smcowawvcou x upmm mm.o ww—o.P mo.o Nm.o w acmvwxo x meowuwvcou mo.o «wwNo.m «Nm.o m~.~ c ucwuwxo x “Pam om.o whemo.¢ ¢N.o «N.o e Lm:0wuwv:ou x upmm N~.p «mm.~ Nn.o mp.F N ucmtho wwwom.m weeNo.m 00.0 mn.o N meopuwvcou wwwmm.m «hemm.om «v.0 wttmm.© N ppmm auwwweeueeeo< eseuxew Leueeemeu eepeu Eeeeeee eeeewem> Pweee>o eweew umeeu emeeu we meeemee we eeeeem '1 meeeeseeee eewueepe>e aeemeem sew eeeeewwwemwm mewemwueum m use meeeeem new: me>memeem mew; Oeeee Omeee eOO.w Oeemeoeeee aee em we .ep mpeeh 108 salt significantly improved the grain texture (p<0.0005). However at the high levels of salt, some panelists indi- cated that grain texture was compact. The bread was gener- ally acceptable. Crust color and character were not signi- ficantly affected by the additives. The double combination results (Table 15) show that the grain texture tended to be more compact as the level of salt and conditioner increased. When 2% salt was added, the grain texture was scored 5.94 by the panelists on 0-10 scale, when 2% salt was combined with 0.5% conditioner the grain texture was more compact and scored 4.40, the combina- tion of 2% salt, 1.5% conditioner produced a very compact bread that was scored 3.92 by the panelists. The high level of salt (2%) had the same effect on grain texture when it was combined with potassium bromate (Table 15). As the level of KBro3increased in the combination, the bread grain tended to be more compact. The salt at the lower level (1%) acted differently. The grain texture was very acceptable when 1% salt was added with either conditions (0.5% level) or bromate (30 or 50 ppm levels). The interaction between conditioner and potassium bromate was somewhat different. The grain texture was scored more open as the level of the conditioner increased in the additive combination (Table 15). The best bread score for grain texture was obtained with 0.5% conditioner, 109 .umow enema o—ewH—ez uemewwwemwm eeemz epemeeeeemlepemeeeeemee eeeeluomQEeom e.ee-ee.eee eemeie—mem m.emoeee mc.cwe um aeeemwwwe >_uemewwwemwm we: eem eeaump esmm me» me eeze—pew meme:— mN.e pm.¢ co.e emu—.o emmo.N emmw.m mp.m om.m mN.m mo.m ee.m oe.m m.— eme :4 :6 ems...“ mNee emmse Nee eN.m —N.m mme mm.m com me uoN eeezw oN.m o—.m Np.m emNo.e em—.N emwo.w ec.m em.m eN.m Ne.m mN.m mm.e o.o em on o em em on o .om on o em on e Azeev mumseem Sewmmmuee me.m om.m F—.e mme.e emmm.o emoe.m Nm.m o_.m mw.e «N.e ee.e No.e e.N mo.m m¢.c ee.e eoNe.N eomm.N eeem—.o —e.e eo.m _e.m Nm.e Nm.m mp.e o._ a u—mm po.m ee.¢ we.e eeNc.e eeo.N eee¢.N NN.m mm.m Nm.m m_.m oN.e Nm.m e.o om an o em on e em on o em on o Azeev mumseem sewmmmuee m¢.m om.m mN.o mNm.m moe.e emm.m ee.m cp.m m_.m em.m Ne.e e¢.e o.N o_.e mN.e Nm.m eueNc.N ueem.e eumm.w ap.m mm.¢ mm.m oo.o mN.e mm.m o._ u u_mm Nw.m m_.m em.e eNa.N uemm.o eeeNN.e mN.m mm.m No.m Pm.e FN.m eN.m o.o m.F m.o o.e m.p m.e e.o m.p m.o o.o m.~ m.e o.o an —e>upv oN eeezw o—le o—ue epic o_-o xuwpwemueeoe< emeuxew co omemeu hepeu mzppmee>c ewmce mzumeee mzameeu eee am we pmcouemcmee eewum=~m>e meemeom we» emcee gee—w ememeeuuee ee mo>wuweem e3» eeezuee meewuomeeuew we uoewwe mew .m_ epemw 110 30 ppm potassium bromate combination. Bread baked with three combinations of additives showed their significant (p30.0005) effect on crust character and grahT texture (Table 14). The best crust character was obtained with the combination of 0.5% conditioner, 1% salt, 50 ppm potassium bromate and the combination, 1.5% conditioner, 1% salt, 50 ppm potassium bromate (Table 16). Comparing the effect of single additives and the effect of double or triple combinations of additives on bread, it is obvious that there was an interaction between the additives when added together, and it caused improvement to the physical characters of bread (Figure 9A, B, C). Wheat/cottonseed flour breads have not been yet com- mercially produced in spite of the extensive research that has been done in this area. Some of the reasons that hinder their commercial production are: the physical bread character (volume, grain texture), flavor and color. In this part of the study the optimum levels of each additive (oxidant, conditioner, salt) were determined to produce optimum 8% cottonseed bread characters. The data were subjected to the multiple regression analysis where the regression equation was used: ¢= 80 + B] X] + 82 X2 + B3-X3 + 8]] X12 + 2 2 822 X2 + 833 X3 + 812 X1x2 + 613 xlx3 1 B23 X'2X3 111 .eeeeleemesee ewmemN .eweuleewew ewmemp .umew mOemm ewewuwez m.emoeeO mO.Owe um acmemwwwe appemewwwemwm pee eem meepuew eEmm we“ we eezepwew meme: mON.m emmN.e emmm.m -mON.m mme.m mON.e m.P emOm.e emOO.e emNO.e mmm.m mem.m mmw.e m.O ARV eewm.m eNP.O emm.O mmm.m m.w.e mmm.m 0.0 ON eeezw ARNV Fe>ew ppmm eeNO.N eO0.0 mP.Nm , emwO.e eemNm.m eeOm.m m.w eeOO.N eeeNN.w mOO.e eemNO.m eemOm.m emNm.e m.O ARV eeeeN.O emmO.m eewm.O mOP.e eepm.O eewm.O 0.0 ON eeezw Axwv Fe>epiupmm eFO.N eeO.w memw.m eeme.m eemo.O eeOO.m m.w emmw.m emNm.m eeON.N . emOO.e emew.m eeOm.O m.O “we mwm.e eme.O eewm.O eepm.m eeOO.m me.e 0.0 ON eeezw ANOV we>ew ewmm Om Om O cm on O Aseev mumseem Eewmmmeee OPTO OFTO meeuxew eepomemeu Newmew wumeeu -m:—m>e Oeemeem we» ee mm>weweem meme» eemZeee meeweemeeuew we ueewwe emeee Lee—w eeemeeuuee eOe NO we mempememee cow» emes mew .Ow ewemw Figure 9A. Effect of additives on 8% LCP cottonseed bread Single Additives a) Control Untreated Bread b Potassium Bromate 30 ppm c Potassium Bromate 50 ppm d) Salt 1% e) Salt 2% 112 Figure 9B. Effect of additive interaction on 8% LCP cottonseed bread Double Combinations a Control Untreated Bread b Conditioner 0.5% + KBr 30 ppm c Conditioner 1.5% + KBr 50 ppm d) Conditioner 1.5% + KBr 30 ppm e; Conditioner 0.5% + KBr 50 ppm f Salt 1% + KBrg 30 ppm 9) Salt 1 + Con itioner 0.5% h 1 J k 1 m 3 Salt 1 + KBrg 50 ppm Salt 1 + Con itioner 1.5% 1 Salt 2 + KBrg 30 ppm Salt 2% + Con itioner 0.5% ) Salt 2% + KBrg 50 ppm Salt 2% + Con itioner 1.5% 113 ,'_M.Om_‘ Figure 9C. Effect of additive interaction on 8% ‘cottonseed bread ' Triple Combinations Untreated Bread a b c d e i l f) 9 do: ) Control Salt Salt Salt Salt Salt Salt Salt Salt 1% 1% 1% 1% 2% 2% 2% 2% Conditioner Conditioner Conditioner Conditioner Conditioner Conditioner Conditioner Conditioner +-++-+-t+-+-+ 0.5% 1.5% 0.5% 1.5% 0.5% 1.5% 0.5% 1.5% +-++-+-t+-+-+ KBr KBr KBr KBr KBr KBr KBr KBr LCP ppm mm mm mm mm mm ppm ppm . -m‘“"-— 114 115 where X1, X2 and X3 are salt, conditioner and potassium bro- mate respectively and X12, X22, X32 are the second order terms and X1X2, X1X3, sz3 are interactions between salt X conditioners, salt X oxidant and oxidant X conditioner. 80 - 823 are coefficient of regressions. The predicted value was calculated for each dependent variable (Y) of interest in this case bread texture and specific volume. The optimum values were calculated using the equation 2 w = 80 + 81 X1 + B X this equation was solved to 2 2 find the 1st derivative .dY - . = a; — 282 X + B.' + 0 0 Example calculation for optimum level of salt: m = 38.972 + 6.579 x1 + 2 (-4.918) x2 = 0.67% The three independent variables were calculated and were found to be: salt 0.67%, conditioner 0.4% and oxidant to be 37 ppm. An optimum bread was then baked using the optimum levels of additives. Bread was evaluated by 16 panelists for crust color, crust character, grain tenderness, flavor, overall acceptability and volume. The data presented in Table 17 116 mm>wewmme we mpe>ep seswuee wee me mewe eeeww emee: 3m: emuemeuxe amm we eemzm ewemaeeeem 1- epempeeeemee .zuwwwemaeeeem.wwmee>oe weepweexeieme eeeeeusemeeu mppee seewweetee>eee ememee m e eeeeeu uwemlzeeeeeg eeweem eemelepmeF Oe.m mN.m OO.N ON.O O—.m Ow.m OO.m mOe «Feeeeeo ee.m Op.m NO.m ww.m OO.¢ N0.0 N—.m one eeemeeue: OO.¢ mw.e me.m mO.m oe.m Oe.O mo.e Omm seaweeO O_-O OP-O OF-O O\ee . . eeeuxew genomemeo eePeO esewe> ee emmem we O < O mee>mpm emmeeeeeeew mewmew Numeeu Pemeeu ewwweeem maewe> peesumeew meee eeumeeue: eem emeee eemmeepuee eOe NO Eeswuee we meme eewumewm>e Oeemeem .Nw ewemw 117 are means of 64 taste panel scores. Optimum bread was served to the panelists along with a sample of 8% LCP cotton- seed untreated bread and a control of HRW wheat bread made of extracted flour and, all with the optimum levels of additives to minimize the color factor in scoring the cottonseed flour breads that always underscored any plant protein substituted bread if tested against white bread. Volume and specific volume were higher than untreated bread. Sensory evaluation data indicate that optimum bread was scored better than the untreated bread. The tenderness, flavor and overall acceptability effectively improved with optimum combination of additives over the untreated system. The optimum cottonseed flour scored better than the control bread in volume, specific volume, crust color and grain texture (Figure 10A, B). Egyptian "Baladi" Bread Many attempts have been made to improve the protein quality of various types of Western bread by substituting with different protein sources and synthetic amino acids. However the studies on the substitution of Arabic bread with protein sources are few (Shakir et al., 1960; Maleki et al., 1968; Dalby, 1970; Shehata, 1970). Egyptian bread, which is a typical type of bread con- sumed in the Middle East was substituted with 0, 4, 8, 12 and 16% LCP cottonseed flour. The data in Table 18 show Figure 10A: Effect Of optimum levels Of additives on LCP cottonseed bread ' a) Untreated 8% LCP cottonseed bread b) Salt 0.7% + Conditioner 0.4% + KBrg 37 ppm 103: Comparison between Optimum bread made Of white flour, dark flour, and 8% LCP cottonseed flour a) Bread made of 85% extracted HRW wheat flour b) Bread made of 8% LCP cottonseed substituted flour c) Bread made Of straight grade HRW wheat flour. 118 119 Table 18. Effect LCP cottonseed flour on Egyptian bread volume and specific volume Cottonseed Loaf Loaf level Volume** Specific Volume* % cc cc/g c b o 581.00$105.01 4.57:0.97 4 640.13$110.71e 4.99:0.90a 8 594.67$105.39d 4.65$0.80b 12 550.00:101.41a 4.34$0.82b 16 556.44$105.69b 4.41$0.80b a r Value followed by the same letter are not significantly different at p‘0.05 D-ncan's Multiple Range Test * p<0.05 **p<0.001 120 a decrease in loaf volume and specific volume at the high levels of cottonseed (12 and 16%). At the levels of 4 and 8% cottonseed, the volume and specific volume increased indicating a better product than the control loaf. The bread was tasted by 6 Middle Eastern panelists for crust color, crumb color, flavor, texture, aroma and acceptability. The results indicated (Table 19) a signifi- cant decrease in crust and crumb color ratings indicating that a darker product was produced when the flour was substituted with higher levels Of cottonseed flour. All the sensory data show a significant decrease in flavor, texture, aroma and general acceptability scores with increasing level of cottonseed bread. Nevertheless, the bread prepared with 4 and 8% LCP cottonseed flour was scored in the acceptable range for all the sensOry characters tested except color (Figure 11). Sensory data suggest that the adverse color scored may have influenced the judges scoring on the other attributes even though some of these attributes received acceptable scores. Effect of Four Dough Conditioners on the\Mixogragh Progertieg. The mechanism of action of dough conditioners was discussed in a previous section. The comparison between the effect Of dough conditioners: Tween 60, Polyoxyethylene lO-stearyl ether, Tween 20, and Tendem 552 on peak time, 121 .ueeP—eexe mw N eem eeee zem> mw P meme: N e» N mw ewmem mew .eewee wmeseme N on zeppex eeme emweeeee P mw epmem eepee eseeu .emp peOwF mw N eem ezeee esme Nee> mw P meme: Nip Eeew mw epmem eepee pmeeu m N — um.owmm.m eN.FHON.m um.OHON.m om.omom.m up.FwON.N em.Pwew.N OP eem puma m _ ep Pwmm m ee Fame e ueN owmm m uem prm m euN OHNN m NF one OHeN e em OHNP e ep Fwom e ea FHON e eme OHON m uem OHOO m m em owee e em OHmN e UN PHNO m eo Fwoe e em NHON m eN owwm e e mF.OHON.m mm.owoe.m mp.owNm.m mF.mee.m m¢.ommm.m m~.omom.o O a m zewwwemueeeé m 28.2 m 23x3. m ee>mE Leweu eePeO 3.6.. wmeeeeO . NeEeeO Fpmeeu eeemeeuaeu mewweeeeee Oewemsemeee ee Leeww eeemeeueee eOe we weewwm .Ow ewemw Figure 11A: A typical loaf of Egyptian bread. 122 Figure 11B. Effect of LCP cottonseed flour on Egyptian bread characters 0, 4, 8, 12 and 16 represent percent levels of substitution with cottonseed flour 123 124 peak height, height at 9 minute point and the area under the curve were studied. From the results shown in Table 20, it can be seen that the conditioner and its level affected significantly all the mixograph characters (p<0.0005). Peak time increased with the addition of these dough con— ditioners, ranging from 3.72 minutes for the control to 4.87 minutes with the addition of the higher level of mono and diglycerides of Polysorbate (60) (Tandem 552) (Table 21). This dough conditioner used at the 0.5% level also signifi- cantly increased the mixograph height at 9-minute point, which indicates its strengthening effect of flour proteins. The area under the curve also was significantly affected by dough conditioners (Table 21): polyoxyethylene (60) sorbitan monostearate (Tween 60) produced the largest area when it was added at 0.5% level; while polyoxyethylene 10- stearyl ether at 0.5% level and polyoxyithylene (20) sorbitan mono laurate, at 0.5% level had the second largest influence on the area. These data show that the maximum effect Of dough conditioner on the mixograph characters was reached when the conditioner level was 0.5% Of flour weight. The term dough conditioner refers to those adjuncts, usually surface active, that possess the ability to strengthen the gluten structure of dough and thus improve its gas retaining ability. Langhans et al. (1971) found that the addition of 0.2% polysorbate 60 (polyoxyethylene (60) sorbitan monostearate) imparted a dry consistency to dough 125 mooo.oVQ«ww OO wmpew OOOO.N mmw0.0 mmm0.0 OO_O.O . me eewwm meewmee «mmeOm.em «mmmme0.0 ONm0.0 mmmwmww.O Nm weeewuweeeu x eeemeeuuee memeOe0.0N «mm—ON~.O «mmwON0.0 «mwOOON.w O weeewuweeeu «memmm.wNOw mmweeew.ww mmmOONe.m «mmmewm.w e we>ew eeemeeeuee so so so ewe . Eeeemww eewemwem> Owee Owe O O: eeOwem emee eeww eeee we meeemeO we eeeeem . we>ew eeeewuweeee eem mew -eewuweeee we>mp eeemeeupee we emueewwm mm memuomwmeo eememexwe we eemeem cmm: .ON ewemw 126 emew eOemm ewewewez m.OmeeeO mO.Owe um peewewwwe appemewwwemwm we: eem wmpuew esmm me» me emzewwew meewmew r OeOmeN eeemm.e eem.m mNm.e m._ eemm.NN mmm.e eemO.m emN.e m.O Nmm Oeeeew eewm.mw eOOm.e emON.m eemm.m m.w eeNmOm eemmm.e Oeem.m eww.m m.O ON Oeezw amem.mh omp.e unm¢©.m mtuom.m m.F swcpm FXmeum op OONN.Om eeemm.e Oemmm meOO.O m.O eeewmeeemxemwee n—¢.¢N unhp.¢ unpm.m QNN.¢ m.F mmm.mm eeemm.e eeNN.m eeew.e m.O Om Oeezw mom.~m amN¢.¢ mmo.m wNn.m 0.0 smcowuwucou oz Ee so uewwe: Hawk eeeewmweeeO meeeewuweeeu e.< ewe O O: eeee eeee we wesee OOOeO . memeee weeww eeemeeeuee eueNumeez 2m: we mewuweeeee eemwmexwe ee memeewuweeee emeee we poewwewemmee>m mew .FN ewemw 127 as measured by Farinograph; Knightly (1962) reported a similar effect using polyoxyethylene sorbitan. Surfactants contain two distinct types of functional groups, a hydrophilic and a hydrophobic group. The hydro- phobic group is usually a hydrocarbon in nature while the hydrOphilic one may be ionic or polar non-ionic group (such as polyhydrol or polyoxyethylene). Individual surfactant molecules tend to orient themselves in air-water or Oil- water interfaces so that hydrophilic group has maximum and hydrophobic group has minimum contact with water. In this mode they lower the surface tension between immisible phases. The surfactants that work as dough conditioners must be anionic compounds or if not anionic, they should contain polyoxyethylene compounds because of their actions to insolubilize gluten proteins. The effectiveness of these surfactants is related to the hydrocarbon chain length since the longer the chain, the more effective the surfactant becomes. The introduction of the continuous-process breadmaking - in Egypt necessitates the use of dough strengthener to overcome the weakening effect of continuous mixing on the gluten. These dough conditioners also serve as an anti- staling agents to extend the bread shelf life. The effect of dough conditioners: Polyoxyethylene (60) Sorbitan Mono Stearate (Tween 60), Polyoxyethylene (10) Stearyl Ether, Polyoxyethylene (20) Sorbitan Mono Laurate 128 (Tween 20) and Mono and Diglycerides of Polysorbate (60) (Tandem 552), on bread volume and specific volume, is pre- sented in Table 22. There is a significant effect Of con- ditioner, level of conditioner and level of cottonseed on bread volume (p<0.0005). The interaction between the three factors on volume was also significant (p<0.01). Condition- ers Tween 60, Tween 20 and Tandem 552 were the most effec- tive in increasing loaf volume, especially with high levels of substitution with cottonseed (Table 23). In 0% cotton- seed flour bread both levels of Tween 20 increased loaf volume slightly over the untreated bread. At 8% level of cottonseed both levels Of conditioner Tween 60 increased both volume and specific volume significantly over the control. Tween 20 and Tandem 552 significantly increased loaf volume over bread made with POESE and the control bread. At the 12% level Of cottonseed, Tween 20 at 0.5% was very effective in increasing bread volume, while at tht 16% cottonseed level, all three conditioners except POESE were effective in increasing volume, as well as specific volume. Effect of four dough conditioners on retaining bread softness after 3 and 6 days Of storage is presented in Table 24. The bread tenderness (Figure 12)decreased sig- nificantly after 3 and 6 days of storage (p<0.005). The rate Of bread staling was higher at the 0% cottonseed flour level. The effect of cottonseed on dilution of starch contents can explain that effect. 129 mooo.ovaw«¥ mOO.Ove w. - mO.Ove . Omm weeew wmwe.mw OOOm.O Oem eewewwszO .ewwm weeewmee eeemeeuuee we we>mw x OOmO.ww .wOmmO.w em weeewewmeee we wesew e weeewwweeee .wmmmmO.~m .m.emmm.e m eeemeeeeee we we>ew e weeeweweeee mmww.mw .meOm.w Nw eeemeeweee we we>ew e weeewwweeee wmmm~.mm .w..OmO.O m weeewwweeee we we>ew e weeeweweeee .mNmO.Om ...mm~m.mw e eeemeeweee we we>ee .wwmmNO.ewe wewmmmm.e m weeewwweeee we we>ee emmO.Om .wwmwwe.ww m weeewwweeee .mwww. .EMN.> .demwmwm .5...... .. .eeeom ewwweeem emewe emwweamm we msewe> ewwwemem we maewe> ee mweeewuweeee :33 we poewwe we eeemwwm> we mwmmwmcm we mewmeem emez .NN ewemw. 130 maepe> ewwweeemN oe 2.5.3,P e N¢.¢ o mmom eOO.e eNNm mOO.v emOOO emOO.m eeNO a OO.¢ OONm m.O e “OO.¢e emNpO wer.e eNcm MOOO.¢ emOOO OON.¢ ONFO OWOO.O eONm m.O Nmm sweemw emONHc uememm onNne eNem eeONUe eeOpO emwenm emOOO mOeum mOOO Onw UDGNO G UQOONW Ema Q QQNO QGN m “cam UDOP m. DNmO ammo m 00mm m C ON :flwlh eOO.¢ eo—m eeON.¢ epem eeOm.¢ eeeom epO.m OOOO OOO.m eme m.w weeae pxwmeum Op emm.m ammo emm.m eOmO mmw.m «ONO mO0.0 emmm eeww.e emmm m.O eeewmewemeemwee ON.¢ NFO OO.¢ ONO Om.e «Om mmN.m NNO OO.¢ NFO m.w em . em em . em 6e . eee e . e em . em . eeecm e eoemmm eemv c eOOm uemN e eeeeOO emOe m mNOO eeNc e eONm O O OO eeezw eOOp.¢ eeomm emOm.e emOOm eO¢.¢ eOmm mOO.m mmmN emNN.m One 0.0 weeewuweeee ez O— Np O n we>ue seepw eoemeeaueu ONOO oe ONOO oe ONOO uu ONOO ue ONOO ee a N.>.m —e> N.>.m —e> .>.m we> N.>.m we> N.>.m we> weeewuweeeu weeewuweeeu wmee mee wmee mee Nwmee mee wmee mee wmee mee we we>ee eOeeO weeww ememeeeuee eOe new eem Np .O .O .O euwz meme emowe emwueamm we maepe> uwwwoeem eem usepe> wme. ee mweeewuweeeo we weewwu .ON O—emw 131 Table 24. Effect of conditioners on bread tenderness after 3 storage time Cottonseed Storage period (days) legel 0 3 6 Tween 60 0 20.36, 15.64 14.87 4 16.69 15.60 14.12 8 16.18 13.69 13.74 12 16.29 15.22 14.35 16 16.15 14.72 14.36 Polyoxyethylene 10 Stearyl Ester 0 18.05 15.19 14.28 4 17.11 14.97 14.07 8 18.09 15.43 15.77 12 19.21 16.79 15.03 16 17.23 13.34 13.99 Tween 20 0 18.71 17.43 14.39 4 17.21 15.99 ' 13.04 - 8 17.23 16.50 14.27 12 .13.39 17.25 15.04 16 17.28 16.65 15.96 Tandem 552 0 18.10 16.94 12.88 4 17.47 16.54 12.83 8 16.77 15.73 12.98 12 16.61 16.55 13.49 16 17.34 16.53 13.31 Tenderness was measured as the resistance of a round piece of bread to shear by a single blade. The softer and elastic bread the more the resistance to shear. Expressed as g/cm (diameter of test piece Of bread). Figure 12A. Tenderness Of Egyptian bread substituted with cottonseed flour after 0, 3 and 6 days of storage . Tenderness / g /5 cm 19 17- 15- 13 132 fi—dr—dr 0% LCP Cottonseed Flour .—.—. 8% . OOOOOOO .0 ...... . ‘27. ¢ 0 3 6 Storage/days 133 When dough conditioners were added to bread. the rate of staling significantly decreased. Tween 20 and Tandem 552 were the most effective as anti-staling factors, (Figure lZB). Tween 20 A retained the softness after 6 days of storage better than any other dough conditioner studied, with higher reading for softness. Bread staling starts directly after the bread is taken out of the oven. It was reported that softness can be affected by the moisture content of loaf but loss of mois- ture of bread has no significant relationship wiUItherete of crumb firming. The staling phenomenon according to I Lindet (1902) due to starch retrogradation during which moisture migrates from the swelling starch granules to the gluten (Knightly et al., 1973). The crystallization of amylopectin was profoundly affected by long chain fatty acids. Mono and diglycerides specially fully saturated mono- glycerides at 0.3% of flour weight produced the optimum results on retardation of bread staling and increasing loaf volume. Polyoxyethylene mono stearate reduced bread firm- ness during aging. Surfactants (dough conditioners) were reported to slow the rate of firmness by forming a complex with amylopectin fraction within the starch granules. The ability of satu~ rated fatty acids to complex with amylopectin increases with increasing carbon chain length up to C-l6 (palmitate) Figure 128. .Effect of dough conditioners on substituted bread after 0, 3 and 6 days of storage Tenderness ./g '5 cm Tenderness / g /5 cm 134 POESE T Tweenao 20- 18L 16- 14- I 18- 17- 16- 1s 14- 13. L ‘i Tendem 552 135 (Knightly et al., 1973). Supplementation value of cottonseed flour on Egyptian "Baladi" bread. In the Middle East. the caloric contribution of bread to the diet may be as high as 80-90% (Dalby, 1969). In 1969, Abbott stated that as much as 64% of the daily protein intake in this area is derived from cereal. The present food resources of Egypt do not satisfy domestic demand and additional quantities of certain foods, principally wheat, are imported. The protein content of bread baked with 0, 4, 8, l2 and l6% LCP cottonseed protein increased significantly with increasing the LCP cottonseed flour in bread (Table 25). The total amino acids of LCP c0ttonseed flour, 85% extracted HRW wheat flour, and cottonseed/wheat flour com- binations are presented in Table 26. Lysine is the first limiting amino acid in wheat flour, the substitution with LCP cottonseed flour increased the lysine content from 0.28% to 0.54% with 16% substitution with cottonseed flour. Lysine also is the first limiting amino acid and methionine is the second limiting amino acid in cottonseed protein for the supporting of growth in chicks (Fisher. 1965). Comparing the essential amino acid composition of all wheat/cottonseed flour mixes (Table 27) to the FAO/NHO suggested pattern of amino acid requirements of infant, school child and adult indicated that all the 136 .mm>mo_ Pogucoo so; ~.mxz ccm gsopm nmwmcoauoo mcwcpmucou mm>mo— Ppm com m~.oxzn .mwpmovpaau we mamgm>m msa-u:m mwmmn mgsumwoe we? co cmmmmsaxm mew mospm> PP< m m~.mp mn.~ -.~ mm.o~ mp n~.¢~ me.~ mm.~ mo.mp NP mm.~P mm.~ om.~ Nu.np m oo.~F m~.N me.~ o~.¢~ v mo.~P Pm.F No.~ ~m.~p o a x a a a mczamwoz . cm< and acwmpoga cameo cw ummmcouuou we Pm>m4 :Popoga ummmcouuou mo mpm>m— ucmgmmywu saw: cosmamga women mo mcomuwmoqsou mamswxoga .mm mPno» 137 ommmcouuou so; m~.oxz Lao—w anus: “.mxza m_.r oc._ ~m.c ¢~.o mm." _o.c m=.=a_apscoga mm.c mm.o _m.c e¢.c _..~ ~m.o a=_mocsp mm.r -._ o... “m.O. mm.m mm.c o=_u=ms om.c m¢.c ~¢.o o¢.c ¢~.c m¢.c u=_u=mpomH o~.o ~c.o m¢.o mm.c o~.m m_.o u=.=o_=oa= oa.° ma.o hm.o om.c ~m.~ e~.c o=P_a> .m.c mm.c ~¢.o mm.o me.~ cm.o a=_»m»u o~.o .o.o mm.o m¢.c m¢.~ mm.o o=_=a.< o~.o so.c mm.c om.o mm.~ .e.o a=_o»Pu mu.” o~.~ m~.~ cm.F. om.m. mm._ ~=_.oca we.” om.m mm.n mm.m em.~ mm.m u_u< upsaus_a om.o e~.o mm.o wo.o mo.~ mm.o mcpcmm o~.F mo._ m~.o om.c m_.o mm.o m=.=oageh om.o m~.o ~e.o .o.c mo.~ mm.o u_u< usocaam< Nm.P mp._ mm.° mm.c me.m c~.c cacaouasch _~.c Ne.o «m.O oo.o ¢¢._ hm.c m=_=_mc< em.c me.° _¢.o mm.o ma._ m~.o ~=Fu_um_: i, E, E. m; . _ . . .m. _¢.mm hm.P. c a at us a mo.¢ .e.o m~.a amo.~ amc.c_ ochuw.om ausuno. a a u a . a ago am. so; am ago ac mu_u< a: new a: “mm a: “Na a: new ago noc— az soc. mLQHQEacaa o=.e< mogsux_s L_msu new sac”; commcouuoo no; .Auzv snap; «cog: mo cowu_moqsou u_um ocpsu can :Pmuosg .mm epoch 138 Ache—V .pe um ecumeue eweuege m co—\me m.p p.¢ w.¢ mo.m mN.m mm.m pm.m m~.m mew—e> m._ ¢.e ¢.¢ ~¢.e ee.m ee.m we.¢ e~.~ m=_=oe.;w m.~ ¢.m m.e me.u~ ocwsa uwueeege —euew cm.m cm.m m~.m o~.m N—.m ocwmogah om.m mo.m mm.m mm.m ¢—.m mzwcepe—Azosm ¢.N ¢.m a.~ muwue o:_Ee eewpem peuew aN.m mp.m cc.m mm.~ mm.~ mcpumxu w cm.m mm.m wo.m c¢.~ om.— ocwcewzuez N.~ m.n N.m cm.m ~¢.m mn.m wa.~ mm.~ mcwmxA m.~ o.m o.w ma.m v¢.m No.~ cc.w o~.~ ocwusma m.— n.m m.m sm.~ m~.~ co.m cm.m mn.m mcwuszOmu ¢.p un.m m~.~ No.~ em.~ om.~ ocwuwumw: e_=e< cwuwhwme ucnwge e. em N. we came euem ence, musu< e: s meseuuee oxzxo