PHYSICAL AND RHEOLOGICAL CHARACTERIZATION OF A SUBSTITUTED DOUGH SYSTEM USING A YEAST PROTEIN ISOIATE Dissertation for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY THERESA ANGELA VOLPE 1976 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIZIIIIII 3 1293 10406 / /’ " This is to certify that the thesis entitled PHYSICAL AND RHEOLOGICAL CHARACTERIZATION OF. A SUBSTITUTED DOUGH SYSTEM USING A YEAST PROTEIN ISOLATE presented by Theresa Angela Volpe has been accepted towards fulfillment of the requirements for Ph.D. degreein Food Science \ /7 ("A //’7 . ‘. /" , .r'»\ I, , . ' “1/162; / x.’ , ‘~—/ ' 1 < 4.x». ,4CL I - Major prokssor Date OCtOber’ 19; l976 0-7639 . I PLACE N RETURN BOX to romanthlc checkoutfrom your neon. TOAVOD FINES Munonabdmddodn. DATE DUE DATE DUE DATE DUE MSU I. An Aflmdlvo AdloNEqual Opponunlty Im emu-nu: L ‘3‘.) .‘J‘ '\1 '\' ' ." ‘ ()va T 7’ ABSTRACT PHYSICAL AND RHEOLOGICAL CHARACTERIZATION OF A SUBSTITUTED DOUGH SYSTEM USING A YEAST PROTEIN ISOLATE by Theresa Angela Volpe The effects of single cell yeast protein (SCP) substitu- tion were observed in a hard red spring wheat dough system. Oxidant, surface—active agent, salts and heated protein were used to determine potentials of each as individual and com- bination treatments. Farinograph, extensigraph and baking studies implementing volume and sensory evaluation were used as indices for the functionality measure of the SC? substi— tuted dough. Additional evaluations of the dough mixing characteristics following treatment with various chemical reagents were made. The dough structure with and without the SC? and surface active agent (SSL) was studied by scan- ning electron microscopy. Low levels of SCP substitution (0 and 3%) yielded simi- lar results for the farinograph, extensigraph and baking study with all additives. The higher levels (6 and 12%) caused considerable decreases in dough characteristlc's qual- ity with the 12% SCP dough being generally very poor in Theresa Angela Volpe performance. For the 6% and 12% SCP dough the farinograph absorptions were particularly high, the stabilities tended to be shortened, the extensigraph extensibilities decreased considerably as did the resistance to extension. Final bread quality was generally only fair for the 6% SCP system and was poor for the 12% SCP product. When oxidant and surface active agent were included in the treatment, the 6% SCP system's overall performance tended to be improved, combinations of these or with the salt or heat treatment yielded improved results. Testing of dough mixing characteristics under treatment with various chemical reagents showed weakening of the dough by blocking amide groups with succinic anhydride. Urea at higher concentrations had a weakening effect on both doughs but interference of the H-bonds at low levels seemed to strengthen the unsubstituted dough. Higher percentages of total sulfhydryl were involved in mixing tolerance than were total disulfides. Very low percentages (approximately 2%) of the disulfides were actively involved in dough development: Scanning electron microscopy of the dough showed the surface active agent (SSL) to yield a very fine transluscent gluten texture. Extensibility and sheeting of the gluten seemed finer than in doughs without the SSL. It appeared that SCP was carried by the gluten proteins. Doughs with 6% SCP were thick and spongy; the gluten seemed to lose its extensibility with the SCP addition. Use of the surface active agent with the SCP seemed to improve the extensibility Theresa Angela Volpe and sheeting character which had been lost. However, the fluid draping thin gluten was not recovered. PHYSICAL AND RHEOLOGICAL CHARACTERIZATION OF A SUBSTITUTED DOUGH SYSTEM USING A YEAST PROTEIN ISOLATE by Theresa Angela Volpe A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1976 ACKNOWLEDGEMENTS Sincere thanks are extended to all members (both students and faculty) of the Food Science and Human Nutrition Depart- ment at Michigan State University who have been helpful throughout the process of completing this research project. To members of my committee, Dr. J. R. Brunner, Dr. R. F. McFeeters: I extend my deepest thanks for all the advice and help they provided. To Dr. Everett Everson: I appreciate his being a listening ear when I needed one so badly for all of my far flung ideas. Lastly, there are no words suitable to describe all that has been done for me by my major Profes- sor, Dr. Mary Zabik - except perhaps, "Thank you. I know I have been one thing always: difficult." Lastly - there are two people who deserve my deepest acknowledgement for this accomplishment. It is they who were always there when most direly needed. Mom and Dad, thanks and love. TABLE OF CONTENTS List of Tables List of Figures. INTRODUCTION . REVIEW OF LITERATURE . Flour Proteins Protein Bonding . Chemical and Physical Aspects of Breadmaking. Physical Testing of Protein Substituted Doughs. Additives Used in Bread Making EXPERIMENTAL PROCEDURE . Apparatus and Equipment Chemicals and Ingredients EXPERIMENTAL DESIGN Farinograph Testing . Extensigraph Testing. Baking Study. . . pH of Dough. . . . Sensory Evaluation . Chemical Analyses Moisture . KJeldahl- -Total Nitrogen. Lipid. . . . . . . Ash. . . . Sulfhydryl Groups. Total Sulfhydryl Chemical Modification of Doughs during Mixing. Estimation of Reactive Sulfhydryl and Disulfides . Scanning Electron Microscopy Studies. RESULTS AND DISCUSSION . Increasing SCP Levels and Single Treatments Farinograph Studies. Extensigraph Studies Baking Study . ii \O-UUU U) Interaction of Additives. . . . . . . . . . . . . . . 6O Farinograph Studies. . . . . . . . . . . . . . . . 60 Extensigraph Studies . . . . . . . . . . . . . . . 6U‘ Bread Volume . . . . . . . . . . . . . . . . . . . 68 Sensory Evaluation . . . . . . . . . 70 Chemical Modification of Bonding Systems - . . . . . 73 Urea . . . . . . . . . . . . . . 73 Succinic Anhydride . . . . . . . . . . . . . . . . 75 Sodium-dodecyl-sulfate . . . . . . . . . . . 78 Reactive Sulfhydryl and Disulfide - . 8O Scanning Electron Microscopic Investigation (SEM) 86 SUMMARY AND CONCLUSION . . . . . . . . . . . . . . . . . 95 PROPOSALS FOR FUTURE RESEARCH. . . . . . . . . . . . . .100 REFERENCES . . . . . . . . . . . . . . . . . . . . . . .102 APPENDIX . . . . . . . . . . . . . . . . . . . . . . . .110 I. Bread Score Card . . . . . . . . . . . . . . . .110 iii Table No. 1 2 10 LIST OF TABLES Page Formulation for Test Pan Bread . . . . . . . . . . 3O Reagents and Quantities Used for Evaluation of Bonding System Functionality in Flour and 6% SCP Substituted Dough Systems . . . . . . . 39 Farinograph Data for Doughs Prepared with 0,3, 6, and 12% Single Cell Yeast Protein Supplemented Flour Under Varied Treatments . . . . . . . A3 Means of Extensibility Measures (mm) for Dough Prepared with O, 3, 6 and 12% Yeast Single Cell Protein Supplemented Flour Under Varied Treatments A8 Means of Extensigraph Resistance to Extension Measurements (BU) for Dough Prepared with 0,3, 6 and 12% Single Cell Yeast Protein Supplemented Flour Under Varied Treatments . . . . A9 Water Absorption (ml/%) Used in Preparation of Bread from Flour Supplemented with O, 3, 6, and 12% Single Cell Yeast Protein Under Varied Treatments . . . . . . . . . . . . . . . . . . . . 50 Mean and Standard Error of Volume (cc) of Bread Prepared with 0, 3, 6, and 12% Single Cell Yeast Protein Supplemented Flour with Varied Treatments. 55 Proximate Analysis of Bread Prepared with 0,3, 6, and 12% Yeast Single Cell Protein Supplemented Flour. . . . . . 56 Mean and Standard Deviation of Sensory Evaluation of Bread Prepared with 0,3, 6, and 12% Single Cell Yeast Protein Supplemented Flour Under Varied Treatments . . . . . . . . . . . . . . . . . . . 58 Farinograph Data for Doughs Prepared at the 6% Single Cell Yeast Protein Substituted Flour Testing for the Effect of Interaction of Oxidant, Salt, Conditioner and Heat . . . . . . . . . . 61 iv Table No. 11 l2 13 1A Means of Extensibility Measures (mm) for Doughs Prepared with the 6% Single Cell Yeast Protein Substituted Flour Testing for the Effect of Interaction of Oxidant, Salt, Conditioner and Heat . . . . . . . . . . . . . . . . . Means of Resistance to Extension Measure (BU) for Doughs Prepared with the 6% Single Cell Yeast Protein Substituted Flour Testing for the Effect of Interaction of Oxidant, Salt, Conditioner and Heat . . . . . . . . . . . . . . Means of Volumes (cc) and Sensory Evaluations of Bread Prepared with the 6% Single Cell Yeast Protein Substituted Flour Testing for the Effect of Interaction of Oxidant, Salt, Conditioner and Heat . . . . . . . . . . . . . . . . . . . . . Chemical and Physical Mixing Properties of Flour and 6% BYP Substituted Flour as a Function of Total and Reactive Thiol and Disulfide Page 65 67 69 81 LIST OF FIGURES Figure No. Page 1 pH of O, 3, 6 and 12% SCP Substituted Doughs as a Function of Time Through Fermentation a) Control (untreated) System . . . . . . . . . 51 b) Oxidant System . . . . . . . . . . . . . 51 c) Sodium Chloride System . . . . . . . . 52 d) Sodium Stearoyl- 2- -1acty1ate System . . . . . 52 e) Heat System. . . . . . . . . . . . . . . 53 2 Bread Prepared with O, 3, 6 and 12% SCP Substi— tuted Flour a) Control (untreated) System . . . . . . . . . 59 b) Oxidant System . . . . . . . . 59 0) Sodium Stearoyl- -2- -1acty1ate System . . . . . 59 d) Sodium Chloride System . . . . . . . . . 59 e) Heat System. . . . . . . . . . . . . . . . . 59 3 Bread Prepared with 6% Single Cell Yeast Protein Substituted Flour Testing for the Effect of Inter- action of Oxidant, Salt, Conditioner and Heat Double Combinations a) Salt-Conditioner. . . . . . . . . . . . . . 71 b) Heat-Conditioner. . . . . . . . . . . . . . 71 c) Oxidant-Conditioner . . . . . . . . . . . . 71 d) Heat-Salt . . . . . . . . . . . . . . . . . 71 e) Salt-Oxidant. . . . . . . . . . . . . . . . 71 f) Heat-Oxidant. . . . . . . . . . . . . . . . 71 Triple Combinations a) Salt-Oxidant-Conditioner. . . . . . . . . . 72 b) Salt- Heat- Conditioner . . . . . . . . . . . 72 c) Salt- Heat- Oxidant . . . . . . . . . . . 72 d) Heat- Oxidant- Conditioner. . . . . . . . . . 72 A Effect of Various Concentrations of Urea on Con- sistency (BU) of Dough as a Function of Time (min) a) Control. . . . . . . . . . . . 7A b) 6% SCP Substituted Dough . . . . . . . . . . 7A vi Figure No. 5 Effect of Various Concentrations of Succinic Anhydride at pH 9.A on Consistency (BU) of Flour Dough as a Function of Time (min) a) Control b) 6% SCP Substituted Dough Effect of Various Concentrations of Sodium- dodecyl Sulfate on Consistency (BU) of Flour Dough as a Function of Time (min) a) Control b) 6% SCP Substituted Dough Determination of Sulfhydryls Involved in Mixing Tolerance by Treatment of Flour Dough with N-ethylmaleimide a) Control b) 6% SCP Substituted Dough Determination of um01Szfllow for both intra- and inter-molecular hydro- phobic bonds (102). The importance of ionic bonds in gluten proteins is demonstrated by the addition of salt to doughs. Theoreti- cally, the ions influence their environment in several ways, the most important being the complexing with ionic amino acid side chains of the gluten (102). The ions enhance associa- tion and dissociation of dough components. In practice the former prevails, since upon salt addition dough rigidity increases and extensibility reduces (13). About 1.A percent by weight of the amino acids of gluten are either cysteine or cystine (100). The disulfide of cys- tine can link together portions of the same or different poly— peptide chains and contribute to dough firmness. A network with permanent crosslinks cannot show viscous flow; this requires opening of rigid crosslinks. And if these crosslinks do open, avoiding loss of cohesion demands reformation of crosslinks at a similar rate at other sites (15). By reacting with available sulfhydryl groups disulfides can interchange and provide mobility (100). The effects of oxidizing agents on the rheological properties of dough may be qualitatively explained by breaking disulfides and concurrent reformation by exchange reactions with other-sulfhydryl groups (15). In practice, these effects are complicated by variations in sulfhydryl-disulfide reactivity. The reactivity depends on: the size of the molecule of which the groups are a part, stearic availability of the groups, and interactions with protein and non-protein components (99). Baking quality has been compared to both protein content and total disulfide-sulfhydryl ratio (SS/SH). For a given protein level, optimum baking results are obtained with a total SS/SH ratio around 15 (11). The SS/SH ratio increases with flour storage (102). During dough mixing only 1-2 per- cent of gluten disulfide bonds are exchanged with free sulf— hydryl groups (6A), indicating that only a few reactive disulfide bonds are critical to rheological properties (5A). Total and reactive sulfhydryls and their ratio decrease with increasing flour strength. Total disulfide content decreases only slightly, and reactive disulfide groups also decrease with increasing mixing strength. Thus, mixing strength appears tn) be inversely related to reactive sulfhydryl and disulfide contents (95). Chemical and Physical Aspects of Breadmaking Three major phases of breadmaking are in one way or another present in the production of every loaf of bread. They include mixing, fermentation or proofing and baking. The mixing will vary by type of mixer, time mixed and speed of mixing; in some methods fermentation may be very brief or completely absent and there may be variations in the temper- ature, the time or the relative humidity. Conditions of baking can also vary. But for all three basic principles still remain the same. Mixing is the process of converting flour and water into a dough by blending and distributing the ingredients with development of the gluten into a continuous phase. When water is added the flour particles are wetted and slowly hydrate. As the water penetrates it weakens starch- protein bonds and upon mixing, several changes occur. The flour-water mass gradually becomes cohesive, loses its wet, sticky appearance and becomes a smooth dough. The dough's resistance to extension increases during mixing until a maximum 10 is reached. At the peak of resistance, mixing time is con- sidered optimal. In addition to the visual and physical changes, reactions are also occurring at the molecular level. Hydration, a rather slow process, is accelerated by mixing. The proteins and other dough constituents are hydrating and free water is decreasing. As this proceeds, the dough actu- ally feels drier, resistance to extension increases, consis- tency increases and dough mobility decreases. The water is penetrating the flour particles and the mixing action promotes removal of hydrated layers of protein and starch from the flour particles. The hydrated mass continues to lose more of its remaining free water to yet unhydrated flour particles. The outer layer of these particles hydrate and the process is repeated, until all of the particles are thoroughly wetted and form a homogeneous-appearing dough. When the dough has become a coherent-mass, continued mix- ing begins to alter the protein structure, stretching it and developing it into a continuous phase capable of retaining gas (l,A,lA,A6,5A,63,77). Mixing causes a uni-directional orientation of long chain molecules and a concommitant decrease in random chain entanglements. In a properly developed dough, the gluten forms an orderly continuous, three-dimensional net- work in which are embedded the starch granules. These struc- tural flour proteins combine into minute elongated platelets which are partially laminated with flour lipids forming slip- 131anes. When stress is applied, slippage can occur along tflaese fat planes between protein platelets (92). Good 11 handling properties may be defined by the ability of this structure to hold together against subsequent physical strains imposed in further processing. The most evident change in the dough during fermentation is its progressive increase in volume. As fermentation pro— ceeds, the dough rises to about five times its original bulk and assumes a light, spongy character (A7). Causing this change are yeast cells uniformly dispersed throughout the dough, acting upon the available sugars and transforming them into the principal end products: carbon dioxide and alcohol. For proper aeration of dough, about .035 lbs of fermented sugar must be available per pound of flour; any sugar not con— sumed in fermentation will show in the product as residual (80). Yeast is a living organism requiring certain foods and envir— onmental conditions before it can function properly. Environ- mental factors such as moisture, temperature, pH and ionic strength are the basic requisites for a proper fermentation. The yeast in the course of active functioning alters conditions by consumption of necessary fermentable substances and produc— tion of wastes in the form of C02, alcohols, esters and acids. These result in a highly complex system whose study is extremely difficult. Yeast does not ferment all available sugars at the same rate or time; it displays distinct preferences for less com- plex sugars. Early fermentation is supported by utilization of free glucose and sucrose furnished by the flour and added ingredients. The third sugar utilized, maltose, results 12 mainly from B-amylase hydrolysis of starch and its fermenta- tion is slower than that of glucose or sucrose so its content rises initially (53, 72). Conversion of maltose to glucose prior to fermentation is not detected and therefore, it has been concluded that maltose is acted directly upon by yeast (72)- Doughs are mixed so they may enter fermentation at 25° to 27°C. During a 3-A hr fermentation the temperature usually increases by 3-A°C. Proof box temperatures of 35-37°C are usual, but temperatures up to 50°C have been successfully tested (81). Initially, the gassing power (m1 C02/10 min.) of yeast is quite high during the initial period of fermenta- tion of free sugars. This is followed by a decided decline in fermentation as the free sugar supply is exhausted. Sub— sequently, gassing increases as it adapts to fermentation of maltose which can be depleted in about three hours. The rate of these reactions becomes more rapid with increases in tem- perature from 27.5° to 35.0°C (38). Gas production rate increases with higher temperatures up to 38°C, above which it starts to decline (3A). Gassing rate is not solely effected by temperature; sugar content, pH, alcohol concentration and other variables are constantly changing and no one condition has been individually assessed. Research has shown that maximal gassing activity is attained between pH values of A and 6 (86) with a sharp decline in production occuring around pH 3.0 (33). This, however, can be effected by substrate, molasses displaying a 13 lower pH than cane sugar at which gas production declines. In the alkaline range, fermentation activity drops off gradu- ally, beginning above pH 6. Generally, the fermentation activity of yeast is fairly constant over a range represent- ing a lOO-fold change in hydrogen-ion concentration, pH A to 6. This is the pH range encountered in most straight dough, sponge dough and other baking processes (81). Again, the effect of pH is not unidimensional, time and temperature of exposure also have an effect on gassing power with decreases occuring after longer or higher temperatures of exposure at extremes in pH (18). Ethanol, a yeast waste product, reduces fermentation rate by about 10 to 20% toward the end of fermentation when the alcohol concentration is the highest (33). In continuous fermentation by S. cerevisiae, ethanol was not inhibitory to gassing until aconcentration higher than 7% by volume was reached; concentrations below this had little or no effect (36). The alcohol content of baked bread is usually about 2.33 g ethanol/1 lb loaf or 0.8% based on flour weight (107). Alcohol concentrations in baked bread depend on baking tem- perature, size of loaf, cooling cycles, etc. While yeast growth and gas development are the primary reactions of fermentation there are concurrent alterations in the rheological properties of the dough. Some factors respon- sible for this such as starch and gluten hydration are not dependent upon the yeast. Others, however, such as decreasing pH, alcohol's effect on gluten, physical working of the dough 1A due to CO2 expansion, and slackening of the dough by enzyme catalyzed reactions are a function of yeast fermentation (81). One enzyme catalyzed slackening effect is due to yeast reductase acting through substances such as thioctic acid in the flour. These reducing compounds, are involved in a cleavage of the flour's internal disulfides producing a slack dough (26). Additional weakening may result from catalytic disruption by yeast glutathione reducase of internal dough disulfides and reaction of the newly freed SH groups with a naturally occuring reduced glutathione equivalent of the flour (55). These and other secondary reactions appear to be responsible for changes in the visco-elastic properties of the dough but their full extent is as yet undetermined due to the difficulty in controlling research conditions. When dough is placed in the oven a thin, but expandable surface film forms. For the first few minutes of oven time the dough continues its volume increase or "oven rise" (A2). The next important reaction is "oven spring" or expansion of dough volume by about one-third of its original size. Under the influences of oven heat, gases expand and liquids such as 002 amialcohol volatilize to produce further pressure (A2). Since crust formation has already been initiated, one result of oven spring is the characteristic "break and shred" on the side of the loaf in pan bread. As oven spring proceeds there is a marked drop in inter— nal pressure and at this point starch swelling begins and volume increase ceases (9). While oven spring is occuring 15 gluten softening also sets in but this is offset by starch swelling; the extent of which is restricted by limited water (85). The individual granules remain intact but are quite flexible due to gelatinization (83). At this time some amy- lose diffuses out of the granules and into the surrounding aqueous areas where it becomes concentrated as granule expan- sion continues. During cooling this linear fraction sets up into a gel and appears to play a significant role in bread staling (85). In addition to control by available moisture, gelatinization is also effected by temperature. Within a loaf, the starch in the outer areas which was subjected to higher temperatures for a longer time was gelatinized to a greater degree than that in the loaf core (106). In the immediate crust area, gelatinization is restricted due to highly insuffi- cient moisture (106). Gluten coagulation is initiated at about 7A°C and con— tinues until the end of baking. During this process the gluten matrix surrounding the gas cells is transformed into a semi- rigid film. As the gas cells expanded, the starch granules embedded in the protein elongated and the gluten film became thinner; rupture of this film may occur without collapse (83). The major phenomena that does occur during gluten coagulation is a loss of its water to the starch phase, thereby facilita- ting gelatinization (21). Throughout the baking period other reactions such as enzyme inactivation, crust formation, carmelization and Maillard reaction are occuring; all of these interact to 16 yield a product with the familiar and desirable organoleptic properties of baked bread (80). Physical Testing of Protein Substituted Doughs The farinograph and extensigraph are the most commonly used instruments in measuring and recording the physical prop- erties of dough. They are used in testing flour for quality and performance in baking. The farniograph measures dough viscoelasticity as a function of continuous mixing. It out- lines the mixing characteristics of a flour with respect to absorption, dough development and the ability of the flour to produce a maximum consistency in mixing and stability to pro- longed mixing (A3, 22, 17, 18). The extensigraph demonstrates the extensibility and elasticity characteristics of a dough; the effects of ingre- ients, particularly oxidants are studied. Retention of strength after experiencing mechanical stress as in continuous mixing, kneading and rest periods is important in machinabil- ity of doughs (17, 22, A3). Farinograms and extensigrams measure functional char- acteristics and baking quality of flour gluten proteins. With increases in protein content there are increases in farino- graph absorptions, mixing requirements, tolerance to mix and extensigram dimensions (2, 35, 60). Yeast protein isolates like most protein supplements for l7 bread contain neither gliadin nor glutenin-like proteins of the wheat gluten type. Therefore, they do not contribute to the visco-elastic breadmaking properties of flour. Farino- gram and extensigram patterns reveal marked changes upon addi- tion of yeast protein supplement to wheat flour (l7). Gener- ally, water absorption increased, mixing requirements for optimum development decreased and mixing tolerance was reduced (2, 35, 60). The degree of these changes was determined by the type of supplement, processing of the supplement, level of replacement of wheat flour and the particle size of the supplement (7, 1A, 52, 62, 82). Heat treatment of the supple- ment can significantly effect mixing tolerance and dough stability. Apparently, a factor present, probably a protein that adversely interferes with gluten development is denatured and rendered less harmful by the heat. Additionally, control of supplement particle size, strength of the wheat flour, baking procedure and use of dough stabilizing compounds are significant effectors of final product quality (52, 82). Resistance to deformation and extensibility in extensi- grams decreases in dough supplemented with concentrations of chickpea flour from 2-20% (87). Extensigrams of doughs with twelve percent defatted soy flour showed increased resistance to extension and lower extensibilities (l9). Cottonseed protein supplements that had been wet pro- cessed and dried at 6.8 pH showed large increases in dough water absorption (27). Farinograph water absorption increases significantly with replacement of wheat by four oilseed flours 18 at levels of 17.5 and 20%; heat treatment of the oilseed flour increases the water absorption even further. Although the type of oilseed does not influence water absorption, eval- uation of the mixing curves reveals differences in the effects on dough properties. Sunflower and peanut weaken dough struc- ture while cottonseed destroys dough stability while sesame shows only slight weakening of mixing strength (82). In baking tests sunflower and cottonseed dramatically reduced loaf volume; peanut and sesame flours were more compatable for bread volume and interior properties (82). Comparison of NFDM solids, fish protein concentrate, soy flour and spray-dried yeast cell contents shows that rheolo- gically the yeast performed the poorest in peak time and stability in farinograms and in extensibility and resistance in extensigrams. However, in baking tests the performance of yeast was second only to that of NFDM solids (57). When soluble yeast protein extracts were tested their functionality in bread decreased compared to the dried whole cells (56). Baker's yeast protein disperses in water rather than dissolves. Solubility through the pH 3 to 7 range is limited to an extractable nitrogen level of approximately 5%. A waterzprotein absorption ratio of 3:1 and a fat:protein absorp- tion ratio of 1:1 is displayed by yeast protein. The yeast protein may form stable emulsions or gels of varying firmness; it may be extruded or spun. These characteristics point out the functionality of this creamy-colored, nutty-flavored pow- der in food product systems (88). The protein powder has 19 shown good results when used in crackers, cookies and other baked goods (88). Additives Used in Bread Making A food additive may be defined as a substance added to food either directly and intentionally for a functional pur- pose, or indirectly during some phase of production, process- ing, storage or packaging without intending that it remain in or serve a purpose in the final product (5). The addi- tives in the following discussion are of the former group. They are ingredients used in relatively small quantities in baked foods. However, it is only in the sense of quantity that these may be considered "minor" components. In terms of effects on the sensory qualities and physical character- istics of the products, they are important, even indispen- sible ingredients (68). Among the additives that may be studied in relation to baked products are malts, enzyme preparations, mold inhibitors, yeast foods, minerals and buffers, gelatin, salt, oxidizing agents and dough condi- tioners. This section reviews the last three. Salt is a basic additive of dough systems under the Standards of Identity. The 2% salt in most dough systems functions to improve taste but also plays an important role in dough rheology. It aids in: stabilizing dough fermen- tation, toughening wheat gluten, retarding enzymatic acti- vity, which can effect gluten (68). 20 Low levels of sodium chloride stiffen doughs and makes it less sticky. The increased stiffness has been measured with the extensigraph; curves of doughs treated with salt show higher resistance (32, 37). However, farinograph studies usually fail to show this trend and in fact decreases in consistency (viscosity) have been reported (37, Al, A3). Variation of temperature, pH and other conditions have dis- played increased farinograph consistencies in tests with increasing salt concentration but these are usually exceptions (59). The decreased consistency is generally explained as decreased stickiness rather than stiffness (32, 37). Not only do extensigrams of salted doughs show increased resistance but they also indicate increased extensibility. This differs from oxidizing agents which usually decrease the dough extensibility while increasing resistance. .The explana- tions of salt effects are incomplete. Some of the effects of salts in dilute acid solutions can be explained in terms of electrostatic effects (25, 59). In doughs the concentra- tion of salt is usually higher than in solutions and there- fore electrostatic effects are an insufficient explanation. Oxidizing agents exert a two-fold effect on flour. They are: l) flour improvers; and 2) bleaching agents. The bromates and iodates function only in the former role which may be defined as changing the rheological properties of the dough. Oxidation shows minimal effects in mixing or farino- graph studies but clearly demonstrates its role in extensigraph 21 tests (58, 69). In extensigrams oxidants increase resistance and decrease extensibility depending on the type and amount of oxidant used and reaction time. A corresponding decrease of thiol and increase of disulfide occurs (39, A0, 107) with the use of oxidants. Bromates form reactive sites at the disulfide linkages that interchange during structural activa— tion such as mixing; in a resting dough these groups are too far removed from one another and will react only when they are brought into a closer position (77). The reaction of bromate is slow except at elevated temperatures (28) while the rate of reaction of iodate is more rapid (16, 20). Doughs treated with bromate tend to show increased relaxation con- stants with time as oxidant is still available to react. Doughs dreated with iodate have higher relaxation constants in the initial testing which tend to decrease with time as not much oxidant is left to react (27). The most recently introduced surfactants are used primarily for their dough strengthening capabilities and to some extent their usage levels are determined by the strength of the flour. Historically surfactants were used for their antifirming effect in bread but the development of new com- pounds has changed this aim. The dough strengtheners or conditioners have been found to provide increased tolerance of the dough to mechanical abuse during processing. This tolerance is particularly important when conditioners are used in conjunction with weaker flours or with protein supplements that have a weakening 22 effect (96). Calcium and sodium stearoyl-2—1actylate have an improving effect on loaf volume, increase dough absorption, improve mixing tolerance and machinability of the dough, accelerate proofing, improve grain and texture, tenderize the crust and extend shelf life (61). They significantly strengthen soy, cottonseed and fish protein supplemented bread doughs (96). The mechanism of the stearoy1-2-lactylates is only partially understood. Evidence indicates that it reacts with only the flour protein to alter gluten structure of the dough similar to the effect of the flour lipids. The stearoyl- 2-lactylates act to retard the swelling of starch during bak- ing. This has the effect of decreasing the association of the starch granules in the continuous protein film that makes up the crumb structure, thus yielding a less firm initial consistency to the crumb (l). EXPERIMENTAL PROCEDURE Apparatus and Equipment For bread production dough was mixed in a Hobart Kitchen- aid K5-A mixer and proofed in a Ores-Cor Model 120-1828 fer- mentation cabinet. The dough was sheeted and molded with a National Manufacturing Company sheeter and molder. Bread was baked in a General Electric deck oven, Model #CN16, equipped with a Honeywell Versa-Tronik Indicating and con- trolling potentiometer, Model #R761B. Bread volume was measured using a National Manufacturing Company loaf volu- meter. Bread was sliced on a Hobart-Model A10 slicer and stored in a Puffer-Hubbard Sa-F-lS-l-SC freezer. Farinographic studies were completed using a C. W. Brabender Instruments, Inc. Farinograph equipped with a Type Pl-2H Dynamometer and a Type 3-S-300 Measuring Read. A Bra- bender Extensigraph Type E-l was used in visco-elastic studies. Temperature of these instruments was controlled by a Heat-Transfer Circulator Type T-60-B (C. W. Brabender Instruments, Inc.). Lab Con-Co digestion rack #21621 was used for micro- Kjeldahl sample preparation. A Scientific Glass Associates distillation apparatus equipped with 100 ml non-transfer 23 2A Kjeldahl flasks was used for nitrogen determinations. For ashing, a Temco Muffle furnace Model #Fl7A0 manu- factured by ThermoElectric Manufacturing Company was used. Temperature was controlled by a Thermolyne Corporation Bar- her-Colman Model CPS-AO32P thermostat. A Precision Scientific Model 18 Thelco drying oven and a Hotpack #633 vacuum drying oven were used for sample and glassware drying and moisture determinations. In the Ellman's sulfhydryl determinations a Corning PC- 351 Hot Plate Stirrer was used for heating. Following color development samples were centrifuged in a Sorvall GLC-l cen- trifuge equipped with a Type GSA rotor. Spectrophotometric measurements were made with a Beckman DB-G Grating spectro- photometer equipped with visible and ultraviolet light sources. Samples were analyzed in l—cm pathlength quartz cuvettes. All pH measurements were made on a Beckman Expandomadic pH meter. Chemicals and Ingredients The principal ingredients and chemicals used in this study are listed below. All chemicals were reagent grade unless otherwise stated. The water used was distilled for baking and rheological studies and distilled and deionized for all chem- ical studies. Ingredients used in dough tests: the flour was Balancer, Code 2831, a hard red spring wheat flour donated by the 25 Pillsbury Company, Minneapolis, Minn. Red Star Active Dry Yeast was supplied by Universal Foods, Milwaukee, Wisc. Sugar and salt were purchased from Michigan Sugar Company, Saginaw, Mich. and Mallinckrodt Chemical Works, St. Louis, Mo., respec- tively. The protein supplement (SCP) was Baker's Yeast Pro— tine (BYP) donated by Anheuser-Busch, Inc., St. Louis, Mo. Patco, a division of C. J. Patterson Company, Kansas City, Mo. donated sodium stearoyl-2-lacty1ate (Emplex). Analytical reagent potassium bromate and potassium iodate were purchased from Mallinckrodt Chemical Works. Chemicals used in nitrogen (micro—Kjeldahl) determina- tions included: tryptophane for recovery studies purchased from Sigma Chemical Company, St. Louis, Mo. Potassium sul- fate was obtained from J. T. Baker Chemical Company, Phillips- burg, N.J. Mercuric sulfate was supplied by Fisher Scientific Company, Fairlawn, N.J. Potassium biiodate used for titration was purchased from Sigma Chemical Company. Chemicals used in the sulfhydryl groups determinations: Ellman's reagent, 5, 5'-dithiobis (2-nitrobenzoic acid), also known as DTNB, was purchased from Aldrich Chemical Co., Inc., Milwaukee, Wisc. The disodium salt of ethylenediamine tetra- acetic acid, di-NaEDTA, was supplied by Mallinckrodt Chemical Works. Sodium lauryl sulfate was brought from Fisher Scienti- fic Company. Chemicals used in the sulfhydryl groups plus disulfude determinations were: DTNB and di-NaEDTA came from the same sources as above. Urea was purchased from Aldrich Chemical 26 Company, Inc., and the sodium borohydride was manufactured by Fisher Scientific Company. L—cysteic acid monohydride was purchased from Sigma Chemical Company. In the reactive sulfhydryl determinations the N-ethyl- maleimide was purchased from Aldrich Chemical Company, Inc. The dithiothreitol for the reactive disulfide test was supplied by Aldrich Chemical Company, Inc. In the bonding interaction studies on the farinograph the succinic anhydride was bought from Eastman Kodak Company, Rochester, N.Y. while the urea and sodium lauryl sulfate were obtained from the above noted sources . EXPERIMENTAL DESIGN This research project was divided into four separate studies and for continuity will be discussed as such. The initial section included an overall physical and rheological characterization of a hard red spring wheat sub- , stituted at O, 3, 6 and 12% of flour weight with a commercial yeast protein isolate (SCP). Five treatments were investi- gated at each level: no additive; 50 ppm Potassium Bromate (KBrO3)-Potassium Iodate (K103) (3:1); 0.5% sodium stearoyl- 2-lacty1ate (SSL); 2% sodium chloride (NaCl); and a 1.50 min microwave heat treatment. Testing in this section included farinograph, extensigraph, baking, sensory evaluation, pH of dough, and proximate analysis. The second section was an investigation of the potential for interaction among the four additives of the treatments on the 0 and 6% SCP substituted systems. Testing included farino- grams, extensigrams of doughs and volume and sensory evalua- tion of baked bread. The third sequence of testing was a farinograph investi- gation of the effects of different chemicals on flour bonding during dough mixing. Among the reagents tested were sodium dodecyl-sulfate, succinic anhydride, urea, N-ethylmaleimide and dithiothreitol. 27 28 The final test was a scanning electron microscopic examination of four different dough systems. The 0 and 6% SCP untreated doughs and the,D and 6% SCP sodium stearoy1-2— lactylate treated doughs were mixed to peak dough development and then evaluated. \ Farinograph Testing Farinograph measurements were made according to the AACC Constant Dough Weight Procedure 5A-21B (3). In this test variable weights of flour and water, deter- mined by the absorption, were selected by estimate from AACC Farinograph Table 5A-28A (3). The flour was premixed in the farinograph 300 g bowl for 1 min at 63 RPM and then it was titrated with the water in 20 sec. Bowl sides were scraped and the cover was replaced. The dough was allowed to mix for 15 min. If the curve did not center on the 500 B.U. (Bra— bender Unit) 1ine adjustments were made on the basis of 20 B.U. = 0.6% absorption as read from Table 5A-28A. The curve was then repeated with corrected absorptions until a curve centering on the 500 B.U. line was achieved. The apparatus temperature was maintained at 30°C. Triplicate curves of each sample were made for the variable testing and the inter- action study. The curves provided values for absorption (%), arrival time, development time, stability and departure time. 29 Extensigraph Testing AACC Extensigraph Method 5A-10 (3) was the basis for extensigraph procedures. A dough for testing was prepared in the farinograph 300 g bowl. Three hundred grams of flour sample plus 6 g NaCl were premixed at 63 RPM and titrated with a sufficient amount of water to yield a peak centered on the 500 B.U. line; the dough was mixed for l min at which time the machine was shut down and the dough allowed to rest for 5 min. Mixing was continued and then stopped at the pre- determined peak time. The dough was scaled into two pieces of 150 g. It was rounded on the extensigraph rounder at a rate of 20 revolutions per piece and formed into a cylinder on the shaping unit. Cylinders of dough were clamped into the lightly greased dough holders. The dough was proofed in the humidified chamber for A5 min. Next it was placed in the sample balance arm and the chart pen adjusted to 0 B.U. The stretching arm was started on the down stroke and con- tinued until the test piece broke. Following testing, the same dough was reshaped and replaced in the humidity chamber. Testing was repeated at 90 and 135 min. Each variable was tested in triplicate. Test curves were analyzed for extensi- bility (mm) and resistance to extension (B.U.). 3O Baking_Study Bread was baked using the above basic formula (Table l) with conditions of mixingfermentation, bench rest, proofing and baking optimized for the 0% formula according to AACC Method 10-10 (3). Table 1. Formulation for Test Pan Bread. Ingre- Level of SCP (%) dient 0 3 6 l2 % g 8 g 8 Flour 100 200 19A 188 176 BYP O,3,6,12 - 6 12 2A Yeast 3 6 6 6 6 Salt 1.5 3 3 3 3 Sugar 5 10 10 10 10 Water (2A0C) - variable variable variable variable The yeast was hydrated in the mixing bowl for 5 min after which the sugar, salt and flour were added. The ingredients were blended at low speed for l min and then the dough was mixed at speed 10 for 9 min. Mixed doughs were fermented for 60 min at 30°C and 85% R.H. Doughs were degassed with a dough sheeter, scaled to 150 g and given a 10 min bench rest at room temperature. The loaf was molded and then panned; the loaf 31 was proofed for 35 min at 30°C and 85% R.H. Proofed dough was baked at 218°C for 20 min. Baked bread was wrapped in plastic wrap after cooling. Loaf volume was measured approximately 10 hr after baking by rapeseed displacement on a loaf volumeter. Loaf volume (cc) represented the average of three replications of two loaves per treatment baked on three different days. An interval of six days occurred between replications. pH of Dough The pH electrode was inserted directly into the dough and 0.5 min was allowed for equilibration before pH of the dough was measured at 0 time and at 10 min intervals during fermentation and at 0, 15 and 35 min of the proofing. Sensory Evaluation The quality of the bread was evaluated according to a descriptive system designed by Solle (90). In this procedure loaf characteristics of primary interest were listed and given a qualitative range of short descriptive terms. Each term was assigned a value from 1 to 5. The resulting table con- stituted a descriptive matrix (Appendix I). With all characteristics described they were grouped into broad classifications (i.e. shape, grain, etc.). The classi- fications were given weighing points dependent on their inher- ent importance. The total value of the points equaled 100. 32 Within each classification another 100 points was divided among the characteristics depending on their relative impor- tance. The characteristic weighings were determined by mul- tiplying characteristic values times classification values ‘and dividing by 100. A realistic target loaf was outlined by selecting the appropriate descriptive level for each characteristic on the matrix. All test loaves were evaluated according to this theoretical reference and Quality assessment was determined by difference from the profile of the target loaf. The score obtained was a deviation from the target by the test loaves. A perfect match would be a score of zero while departure would result in penalty. All departures are positive numbers which are multiplied times the characteris— tic weighing to give the penalty. The penalties of all characteristics were summed to give a final value, "The non- conformity Index" (NCI), a total of the products deviation from target or desired quality. The larger the NCI the greater the deviation from target quality. Under scoring procedure, each panelist was presented with one slice of bread and a whole loaf on which they could make their judgments. The variable testing bread was scored for four replicate bakes while the bread from the interaction study was scored for duplicate bakes. 33 Chemical Analyses Moisture Moisture content of the bread and ingredients were determined in triplicate by AACC Method AA-AO (3). A 2 g sample was weighed to the tenth mg into an aluminum sample dish that had been previously dried at about 168°C for 30 min, cooled in a dessicator and weighed after attaining room temp- erature. The sample was dried at 90°C for about six hours under vacuum at pressure equivalent to approximately 25—30 mm Hg. The samples were reweighed after cooling to room tem- perature in a dessicator. Percent moisture was computed as grams of weight lost divided by total sample weight time 100. Kjeldahl-Total Nitrogen Total nitrogen was determined using the Kjeldahl method described by McKenzie (65). This consisted of digesting a dry sample of approximately 30 mg with 1.5 g of powdered potassium sulfate, 1.5 ml of sulfuric acid and 0.5 ml of mer- curic sulfate solution. The mercuric sulfate solution was prepared by dissolving 13.7 g mercuric sulfate in a total volume of 100 ml of 2 M sulfuric acid. After digestion the flasks were allowed to cool and each digest was diluted with aPproximately 20 ml of ammonia-free water. The flasks were transferred to a micro-kjeldahl distillation apparatus, 10 ml 3A of sodium hydroxide—sodium thiosulfate solution were added and the mixture steam distilled into 5 ml of boric acid indi- cator solution contained in a 50 m1 beaker. The sodium hydrox- ide-sodium thiosulfate solution was prepared by dissolving 200 g of sodium hydroxide and 12.5 g sodium thiosulfate in A00 ml water. The boric acid indicator solution was a mixture of 20 g boric acid in 800 ml water; 6.67 mg methylene blue dis- solved in 50 ml water; and 13.3 mg methyl red dissolved in 10 ml ethyl alcohol; all of these were combined and brought to a liter. Distillation was continued until about A0 m1 had been distilled. The beaker was lowered from the condenser tip which was rinsed with a few ml of water and about 5 m1 more of distillate was collected. Distillation was then halted. The distillate was titrated to a grey-lilac end-point with potassium biiodate which was prepared by dissolving in 1 liter of water 3.899A g of potassium biiodate that had been dried over dessicant. Recoveries of nitrogen were affirmed with dl-tryptophane which had been dried over dessicant for one month. Through- out testing a blank was run to correct for nitrogen contamina- tion in the system. The mg of nitrogen were calculated by multiplying ml of potassium biiodate titrant by .1A01. 35 Lipid Lipid material was determined as crude fat according to AACC Method 30—10 (3). After moistening 2 g of sample in a beaker with 95% ethyl alcohol it was mixed with 10 ml of HCl. The H01 was a mixture of 25 parts concentrated HCl to 11 parts of water. The mixture was held in a water bath at 70-80°C for 30—A0 min and was stirred frequently. Following heating, the mixture was cooled in an ice bath and 10 ml of 95% ethyl alco- hol was added to it. The mixture was transferred to a Mojon- nier fat extraction flask with 25 m1 of ethyl ether divided into three portions. The stoppered flask was shaken vigor- ously for l min. After adding 25 ml of petroleum ether (b.p. below 60°C) the flask was again shaken for l min. Following centrifuging of the mixture for 20 min at approximately 600 RPM, the upper fat-ether layer was filtered through a glass wool plug into a dried preweighed 125 m1 flask. The material remaining in the flask was reextracted twice more according to the above procedure modified in using only 15 m1 of each ether in extraction. The ether-fat solution in the flask was dried slowly on a steam bath and then the flask was dried in a drying oven at 100°C to constant weight (about 90 min). The flasks were air cooled for 30 min and then reweighed. A blank determination was run to correct for reagent residue. The percent crude fat was calculated as the weight of extract remaining divided by sample weight times 100. 36 ;> m 3' I Determination of ash was made according to AACC Method 08401 (3). Approximately 3 g of dry sample were weighed into porcelain or vicor ashing dishes that had been dried and cooled in a dessicator prior to preweighing. The samples were ignited before being placed into a muffle furnace preheated to 575°C. Incineration was continued for 25 hr. The samples were cooled in a dessicator until reaching room temperature at which time they were reweighed. Ash was calculated as weight after ash- ing divided by sample weight times 100. Sulfhydryl Groups Sulfhydryl groups were determined by a modification of the procedure developed by Ellman (30). To approximately 10 mg dry sample were added 5 m1 of a 0.01 M sodium phosphate buffer, pH 8.0, containing 1.0% sodium lauryl sulfate and 0.A% diNa-EDTA. This solution was boiled for 30 min, cooled and then 0.2 m1 of a 5,5'dithiobis-(2-nitrobenzoic acid) solution, DTNB, was added. The DTNB solution was prepared by dissolving A0 mg DTNB in 10 ml 0.1 M sodium phosphate buffer, pH 7.0. The color was allowed A5 min to develop, and the sample was centrifuged for 10 min at 1000 RPM. Absorbency at A12 nm and 600 nm was read in a spectrophotometer. Concentration of the sulfhydryl groups was determined using an extinction coeffi- cient of 12,000. A blank was run along with the protein sam- ples. 37 Total Sulfhydryl In this test disulfides were first reduced with sodium borohydride to form sulfhydryl groups. The reducing agent was destroyed with acid and acetone and the concentration of sulfhydryl groups was determined. The method is a modifica— tion of one developed by Cavallini et a1. (23). About 3 mg dry sample was placed in 1 ml 0.05 M sodium phosphate buffer, pH 7.A containing 10 ml 0.02 M di—Na-EDTA per 200 m1 of buffer. To this solution was added 2 ml 1- octanol, an anti-foaming agent, and 1 ml of a urea-sodium borohydride solution. This solution contained 10 g urea, 0.25 g sodium borohydride, and 10 ml of water. The protein, urea, borohydride mixture was shaken and incubated in a A0°C water bath for 30 min. After cooling to room temperature 0.5 ml of a low pH buffer was added. It consisted of 13.6 g KH2P0u plus 1.66 ml concentrated HCl brought to a final vol- ume of 100 ml with water. This solution was introduced drop— wise to prevent excessive foaming. The walls of the reaction vessel were wetted by the low pH buffer. A reaction time of five min was allowed and 1 ml acetone was added to complete borohydride destruction. Again the mix- ture was shaken to wet the walls of the reaction container. Finally 0.02 ml DTNB (as used in the sulfhydryl determination) was added and absorbency at A12 nm and 600 nm was measured after A5 min. The extinction coefficient of 12,000 was used. A blank determination was run parallel to the test 38 solution to check for complete destruction of sodium boro- hydride. Undestroyed borohydride would reduce DTNB causing the blank to yellow and absorb at A12 nm. Chemical Modification of Doughs During Mixing In this test 300 g of flour was titrated with water to develop a farinograph curve of 500 B.U. The percent absorp- tion of the dough was maintained constant throughout testing. Table 2 lists all chemicals and quantities used to determine effects of bonding systems on the mixing characteristics. Solid reagents were added dry to the systems unless otherwise noted while liquids were pipetted into the dough mass at the time of water titration. The volume of liquid reagents was used on a substitution basis for the water. All curves were evaluated for overall effect on mixing and points of particular effect by the chemical modifying agent, among which may be included arrival time, departure time, stability or 30-minute drop. Estimation of Reactive Sulfhydryl and Disulfides In the determination of rheologically active sulfhydryls and disulfides the procedure of Jones et a1. (A9) was followed. All experiments were carried out at 30°C in the large bowl of the farinograph for sulfhydryl and the small bowl of the farinograph for disulfides, using 300 g and 50 g of flour or 39 Table 2. Reagents and Quantities Used for Evaluation of Bonding System Functionality in Flour and 6% SCP Substituted Dough Systems Reagent Quantity Comments Acetic anhydride 0.5, 1.0, 1.5 liquid 2.0, 2.5, 5.0 10.0 ml Sodium-dodecyl 1 0, 1.5, 2.0 dry powder sulfate 5 0 g Succinic anhydride 0.1, 0.5, 1.0 dry powder 5.0, 10.0 g pH 9.A Urea 0.1, 0.5, 5.0 g liquid 1.0M, 5. M ' N-ethylmaleimide Dithiothreitol see reactive sulfhydryl. see reactive sulfhydryl A0 6% SCP substituted flour for each mix. The percent absorp- tion of the control systems was determined. This volume of water was maintained constant for all testing. The speed of the mixing blades was set at 63 RPM. The duration of the mixing required to bring the dough to a maximum resistance was reported as development time. The loss of B.U. at the end of 30 min mixing was recorded as loss of resistance to mixing. Reactive sulfhydryls in the dough were determined by N-ethylmaleimide (0-A00 umoles), which was added in approxi- mately 0.1 — 1.0 m1 of ethanol to the total volume of H20 titrated at zero time. The dough was mixed for 30 minutes at which time the resistance to mixing was measured. Reactive dough disulfide was measured by dithiotreitol (0—300 umol) added in 0.1 m1 of ethanol to the total volume of H20 titrated at zero time. The dough was mixed for 30 minutes at which time the resistance to mixing was measured. Scanning Electron Microscopy Studies Doughs were prepared with hard red spring wheat flour with and without yeast single cell protein (SCP) and with and without the surfactant, sodium stearoyl—2-lactylate (SSL). Supplementation levels were at 6.0% for SCP and 0.5% for SSL, both based on the weight of the flour (100 grams). Dough samples were prepared by mixing components in a fork—type mixer to optimum development. A1 Immediately after mixing, dough samples were prepared for scanning electron microscopy (SEM) using methods devel— oped by Hooper (A5). Thin strips of dough were excised with a pair of scissors from a smooth freshly exposed surface. Each strip was divided into shorter segments (about 3 mm long), with care being taken to not disturb the surface of the dough, and immersed in .1 M phosphate buffered glutaraldehyde (5%). Fixation time was 2A hours at 10°C followed by stepwise dehy- dration in graded ethanol and critical point drying with 002 as the ambient liquid. Specimens were mounted on aluminum stubs with television Tube Coat (liquid carbon compound), coated with gold, and viewed at 10 KeV in an ISI-Super—Mini scanning electron microscope. All sample preparations for SEM were duplicated and the areas representative of each dough sample were selected for photography after careful study by the microscopist. RESULTS AND DISCUSSION Increasing SCP Levels and Single Treatments Farinograph Studies Absorption is defined as the amount of water required to center the farinograph curve on the 500 B.U. line for a flour-water dough. Research has shown that when the 580 B.U. line is used for the optimum consistency, the absorption obtained from the farinograph agrees within 1% of those determined in the bake shop (70). The height of the farino- graph curve at the maximum develOpment time increased with the percent protein of the flour; that is, employing optimum absorptions from baking tests, the plasticities of the far- inograph increased with an increase in protein content (66). In studies with defatted heat treated soy flour,farinograph absorptions increased with raising levels up to 5% supplemen- tation (73). In the single cell yeast protein system increasing absorption (Table 3) is a function of the high water-binding capacity of yeast protein, three grams of water per one gram of SCP. A measure of the rate at which water is taken up by the flour is the arrival time. Generally, as protein content A2 A3 Table 3. Farinograph Data1 for Doughs Prepared with 0, 3, 6 and 12% Single Cell Yeast Protein Supplemented Flour Under Varied Treatments2 Farinograph Level of SCP Substitution Measure Treatment 0% 3%: 6% 12% Arrival time None (control) 1.9 1.5 1.5 2.3 (min) Oxidant 5.1 2.0 1.5 1.6 Salt 3.1 2.1 1.8 2.5 Conditioner 1 6 1.8 1.8 2.0 Heat — 3.0 3.0 3.1 Peak time None (control) 6.1 3.0 2.5 2.9 (min) Oxidant 9.1 7.1 2.0 2.1 Salt 15.0 15.0 A.0 5.3 Conditioner 3 0 3.3 3.3 2.8 Heat - 5.6 5.1 5.3 Stability None (control) >13 0 12.5 3.0 1.0 (min) Oxidant 9.1 12.5 2.0 0.8 Salt 15.0 15.0 >13.1 8.5 Conditioner >13.3 10.3 2.0 2.1 Heat - 7.0 5.6 A.5 Absorption None (control) 65.6 69.7 75.8 92.0 (%) Oxidant 66.6 69.u 7u.3 83.9 Salt 63.9 68.3 7A.2 82.3 Conditioner 63.A 68.5 73.A 8A.3 Heat - 69.3 72.5 73.3 1 Average of three replications. 2 Treatments: None'(control) Oxidant - KBrO Salt - NaCL; 2 :KIO3 (3:1); 50 ppm Conditioner - Sodium stearoy1-2—lactylate; 0.5% Heat - Microwave heat on SCP; 1.25 min AA increases, the arrival time increases. Single cell protein showed varied effects on arrival depending on treatments (Table 3). With the control (no additives) system, the arri- val decreased from 1.9 min for 0% SCP dough to 1.5 min for the 3% and 6% SCP levels but increased to 2.3 min for the 12% SCP dough. In the oxidant and salt treated systems the arrivals decreased as the SCP increased to 6%. However, the time increased slightly for the highest level. The SSL treated system increased in arrival time from 1.6 to 2.0 min as the supplement increased from 0 to 12%. The arrival times of the heat-treated system were not effected by supplement level. The peak or dough development time is the time from first addition of water to the development of the dough's maxi- mum consistency or minimum mobility. A variety of effects resulted depending on treatment and substitution level (Table 3). In the control system as the SCP increased from 0 to 6% the peak time decreased. A slight increase was observed for the 12% SCP substituted system. This also occurred in the oxidant, salt and heated systems. However, in the SSL system, the peak time increased as the supplement increased from 0 to 6% and decreased at the 12% level. One study found only a 0.27 correlation between the far- inograph peak and the baking mixing time on 186 samples con— taining eight different varieties of winter wheat (67). Another pointed out that the time just before the peak of maximum plasticity on the farinograph curves appeared to be A5 the state of optimum development of a dough as far as baking quality is concerned when the dough mixed in the farinograph contained the "baking formula" (91). The stability of a dough which indicates the flour mix- ing tolerance is the difference between arrival and departure times. Except for the oxidant system, as the percentage of SCP rose the stability of the dough decreased with very small differences between the 0% and 3% levels but with considerable drops after that point (Table 3). Under oxidant treatment the stability of the 3% SCP dough increased considerably over the 0% but declined progressively at 6% and 12% SCP. The mixing properties of the dough are effected only slightly by substitution to the 3% SCP level but 6% and 12% substitution showed rather negative effects. It may be that the lower levels of SCP acted as an enhancer to some of the chemical reactions of dough development while the higher levels of SCP acted as a diluent of the flour gluten and thus resulted in a weakened dough and inferior final product. The farinograms of the control and SSL systems at 0 and 3% SCP had curves with short development time and long stabil- ity characteristics. The salt treated systems showed short to medium development with long stability at the 0, 3 and 6% SCP levels and medium stability at the 12% SCP level. Heat treatment resulted in medium peak time and medium stability for 3% SCP substitution but short stability at 6% and 12% EMSP levels. The oxidant treated system had long development and stability at the 0% level but short development and long A6 stability at the 3% SCP level. The 6 and 12% SCP levels dis— played short development and stability. Most farinograph mixing curves are of seven general types, which can indicate to the baker their overall mixing characters. These types of curves include: 1) short development; short stability 2) short development; long stability 3) medium development; short stability A) medium development; long stability 5) long development; short stability 6) long development; long stability 7) double peak, sway back, dip in early curve. Generally, if a curve indicates a higher absorption, longer peak time and longer stability, the flour will be strong and tolerant. It will require more mixing and will withstand more mechanical abuse. It is with this knowledge that bakers may make appropriate flour or ingredient system selections depending on the final product desired. Extensigraph Studies The length (mm) of the extensigram indicates extensi- txility of the dough and the height (B.U.), its resistance t<) extension. The extensibility of a dough is an expression of‘ the ease of stretching or dislocating structure; the ress'stance to extension is the inverse of the energy required to Estretch or dislocate that structure. A soft, flowy A7 dough will yield a long and low extensigram, while a tight dough will result in a high narrow curve. As indicated in Table A, in all treatments except oxi- dant, the dough's extensibility decreased as substitution increased. In the oxidant system however, the extensibility for the 3% SCP dough increased above that of the 0% system for all three test times (A5, 90, 135 min). Except for the 0% control and the 0, 3 and 12% SSL variables Huaextensibility decreased with time at all levels and treatments. The resistance to extension consistently decreased as the level of substitution increased for all treatments (Table 5). Except for the 3% and 12% SCP control, 0% SCP with oxi- dant, 0% SCP with salt and 6% SCP with SSL variables, the resistance to extension increased with time. The decrease 1J1 dough extensibility with increased sub- stitution and time may mean that at the molecular level the dough became increasingly tighter or less flowy chm? to bond- ing forces. The SCP inhibits the smooth unfolding and stretching of the proteinaceous sheets in the gluten structure so that easy tearing of the sheets occurred. This was accompanied by decreasing dough resistance as SCP level increased and indicated mounting interference at the molecu- liar level of bonds that were less strong than in the all flour (Lough. With time, this interference or bonding increased qiuantitatively such that while tightening was occuring the: forces responsible for this were of a less energetic nature. A8 Table A. Means1 of Extensibility Measures (mm) for Dough Prepared with 0, 3, 6 and 12% Yeast Single Cell Protein Supplemented Flour Under Varied Treat- ments Time of Level of SCP Substituted M _ Treatment efiifiie 0% (mm) 3% (mm) 6%*zmm) 12% (mm) (min) None A5 237 235 211 137 90 251 225 201 115 135 256 210 193 103 Oxidant A5 2A0 2A5 207 132 90 195 217 181 121 135 171 200 170 108 Salt A5 295 278 230 170 90 275 257 216 1A2 135 233 217 203 1A1 Conditioner us 316 258 231 106 90 295 263 207 126 135 305 2A5 187 1A6 Heat A5 267 230 155 90 2A7 208 1A2 135 2A3 196 131 1 Average of four replications. 2 Treatments: None (control) ‘ Oxidant - KBrO3zKIO3 (3:1); 50 ppm Salt — NaCL; 2% Conditioner - Sodium stearoyl—2-lactylate; 0.5% Heat — Microwave heat on SCP; 1.25 min A9 Table 5. Means1 of Extensigraph Resistance to Extension Measurements (BU) for Dough Prepared with 0, 3, 6 and 12% Single Cell Yeast Protein Supplemented Flour Under Varied Treatments Treatment M62:u::— Level of SCP Substitution ment 0% (BU) 3% (BU) 6% (BU) 12% (BU) (min) None A5 570 A26 316 307 90 601 A8A 38A 325 135 700 A65 A16 315 Oxidant A5 8A6 616 516 A90 90 1001 787 660 52A 135 995 907 752 52A Salt A5 762 65A A95 315 90 937 72A 579 3A1 135 925 791 60A 355 Conditioner A5 6A0 AA9 399 261 90 667 A90 A21 270 135 750 512 A16 270 Heat A5 A9A 286 266 90 50A 336 285 135 526 375 289 Average of four replications. 2 Brabender Units. 3 Treatments: None (control) Oxidant — KBrO3zKIO3 (3:1); 50 ppm Salt - NaCL; 2% Conditioner — Sodium stearoyl-2-lactylate; 0.5% Heat - Microwave heat on SCP; 1.25 min 50 Baking Study The water absorptions used in the baking tests (Table 6) were higher by about 3% than those determined by farinograph measure. In the dough fermentation and proofing it was found that the SCP had a buffering effect on the dough's pH. The data graphed in Figures 1 A-E indicate that with increasing levels of SCP the final pH of the dough increased. The rate of pH decrease was similar but there were instances in the control variables where the rate of pH drop of a higher sup- plemented dough exceeded that of the immediately preceeding lower level. In the case of the initial pH of the doughs there was only about 0.05 pH unit difference among all levels for the control, salt and SSL treated systems. The oxidant Table 6. Water Absorption (ml/%) Used in Preparation of Bread from Flour Supplemented with 0, 3, 6 and 12% Single Cell Yeast Protein Under Varied Treat- ments Treatment Level of SCP Substitution 0% 3% 6% 12% None 137/68.6% 1A5/72.7% 158/86.9% 180/95.0% Oxidant 139/69.6% lA5/72.A% 155/77.3% 17A/78.8% Salt 13A/66.9% 1A3/71.3% 15A/77.2% 171/85.3% (Conditioner 133/66.A% lA3/71.5% 153/76.A% 175/87.3% Iieat 1A5/72.3% 151/75.5% 153/76.3% :1 Treatments: None (control) Oxidant - KBrO3zKIO3 (3:1); 50 ppm Salt - NaCL; 2% Conditioner - Sodium stearoyl-2-lactylate; Heat - Microwave heat on SCP; 1.25 min 0.5% Figure 1. pH as a) b) C) d) e) of 0, 3, 6 and 12% SCP Substituted Doughs a Function of Time Through Fermentation Control (untreated) System Oxidant System Sodium Chloride System Sodium Stearoy1-2-lactylate System Heat System as: est m 00 Om OW AEEVoEE. C . . o 0.m o.m oo 51 o\oN_ III I o\o® O\Om Ila-Ila- $0 .238... ' I... .Illllfl’o . ow ON a O In 52 see 2.: a 3,5 2:: a 0w 0.0 0.0 0% 0m 0 00 00 00 0? Om 00.0 l 0.0 53 6.0 b .\ \. x 5.2 r- 5'00 ab 46 so so IOO E Time (min) 5A and heat treatments showed higher differences at initiation of fermentation. In all instances the final pH of the 0 and 3% SCP doughs fell below 5.3, while the 6 and 12% SCP vari— ables did so only after oxidant and SSL treatment. It is noteable that despite final pH differences which were within 0.01 pH units, the SSL treated doughs at 0, 3 and 6% SCP had a difference of about 90 cc in loaf volume. The loaf volumes of breads containing 0, 3, 6 and 12% SCP are shown in Table 7. Bread with 3% SCP had a similar loaf volume to the 0% control, oxidant and salt treatments. The higher SCP levels of these treatments yielded loaves of reduced volumes. However, the 6% SCP oxidant treated loaf had a volume higher than those of the 0 and 3% levels with salt. The breads treated with SSL had acceptable volumes. With 0 and 3% SCP the volumes were quite different at A95: 28.21 and A51.25:7.A9 cc. The volume of 6% SCP SSL treated bread improved compared to the control 6% SCP loaf with respective volume of A06.25:51.23 and 3A8.75:38.A3 cc. Heat treatment resulted in a volume decrease for 3% SCP bread as compared to the control 0% SCP loaf. In two cases, the salt treated and oxidant treated breads with 30% SCP, there were improvements over the 0% SCP salt and oxidant variables. In all treatments use of 12% SCP resulted in bread of highly reduced volumes, ranging from 228.12 cc to 296.8 cc. Early research had shown that the use of full fay soy decreased loaf volume (19) but later this effect was found to be overcome by increasing bromate concentrations in the 55 Table 7. Mean1 and Standard Error of Volume (cc) of Bread Prepared with 0, 3, 6 and 12% Single Cell Yeast Protegn Supplemented Flour with Varied Treat- ments Level of SCP Substitution Treatment 0% 3% 6% 12% CC CC CC CC None A73.12 A63.12 3A8.75 265.00 :37.A9 :29.95 :38.A3 120.51 Oxidant A98.12 A98.75 A35.00 252.50 :21.73 :33.00 150.0A :12.A1 Salt A10.62 A21.20 380.60 296.80 :18.07 129.75 :10.68 :25.76 Conditioner A95.00 A51.25 A06.25 272.50 :28.21 i 7.A9 151.53 :56.08 Heat AA7.50 355.60 228.12 :22.A5 :22.02 + 8.50 1 Average of 2 loaves of each of four replications. 2 Treatments: None (control) Oxidant - KBrO3zKI03 (3:1); 50 ppm Salt - NaCL; 2% Conditioner — Sodium stearoyl-2-lactylate; 0.5% Heat - Microwave heat on SCP; 1.25 min 56 bread formulas (31). Pollack and Geddes reported that 1% unheated soy improved loaf volume but higher levels were deleterious (73). With the SCP substitute, oxidant performed as it does with soy to improve loaf volume. The salt treat- ment appears to have decreased loaf volume in the 0% and 3% SCP samples over that of the control 0% and 3% SCP bread possibly by tightening of the gluten structure. This is verified by data in Table 6 which indicates increased resis- tance to extension for the salt variables. A proximate analysis of the bread crumb shows the the protein content of the bread increased as the SCP increased (Table 8). The 3% SCP bread contained 17.61% protein (as is moisture) which would permit its acceptance as high pro- tein bread. In turn, the higher two SCP levels were more than within the commonly accepted 15% protein level for high protein bread status. Table 8. Proximate Analysis1 of Bread Prepared with 0, 3, 6 and 12% Yeast Single Cell Protein Supplemented Flour Component Level of SCP Substitution 0% 3% 6% 12% Moisture 39.26 A0.60 A2.36 A5.A9 Protein 1A.35 17.61 20.31 23.A9 Fat 2.30 2.57 2.85 3.63 Ash 1.9A 2.13 2.13 2.22 Carbohydrate (by difference) A2.15 37.09 32.35 25.17 1 Average of triple replications. 57 The moisture levels of all the breads were above the federal standard of 38%. This would require some adjust- ment by formula manipulation but lower moisture for adequate dough handling could probably be achieved. Data on sensory evaluation (Table 9) shows that the most acceptable product is the 3% SCP bread treated with SSL. Despite its failure to improve loaf volume the SSL effected crumb color and grain positively, yielding a very acceptable product (Figure 2). Generally, most of the 3% SCP bread scored either better or nearly as good as the 0% SCP in the individual treatment series. Most of the 12% SCP bread scored higher than 100 in the sensory evaluation, this was the cut off for a minimally acceptable product. A loaf with a score of 90 could be described as fairly acceptable. The 6% SCP control and heat- treated variables scored minimally acceptable while the 6% SCP SSL, salt and oxidant breads scored fairly acceptable. 58 Table 9. Mean1 and Standard Deviation of Sensory Evalua- tion2 of Bread Prepared with 0, 3, 6 and 12% Single Cell Yeast Protein Supplemented Flour Under Varied Treatments3 Level of SCP Substitution Treatment 0% 3% 6% 12% None 68.21 68.15 99.25 112.2A :16.35 111.79 :13.69 :18.08 Oxidant 73.68 72.31 89.31 117.93 :15.97 115.86 :22.91 :10.65 Salt 71.A6 66.62 86.A9 12A.56 :17.09 111.07 :10.91 :15.62 Conditioner 72.96 63.2A 89.03 119.01 :15.62 :15.A0 :12.23 :13.72 Heat 70.28 96.90 126.A9 :18.A9 :16.67 i20.15 Average of 2 loaves of each of four replications. See Appendix I for components of score. The number reflects the degree of deviation from a standard bread. 3 Treatments: None (control) Oxidant - KBrO3zKIO3 (3:1); 50 ppm Salt - NaCL; 2% Conditioner - Sodium stearoy1-2-lactylate; 0.5% Heat - Microwave heat on SCP; 1.25 min Figure 2. Bread Prepared with 0, 3, 6 and 12% SCP Substituted Flour a) b) c) d) e) Control (untreated) System Oxidant System Sodium Stearoy1—2-lacty1ate System Sodium Chloride System Heat System 59 A—B C—D 59 60 Interaction of Additives Farinograph Studies A combination of two additives in the 6% SCP doughs had a variety of effects on mixing characteristics. Outlined in Table 10, the arrival times for doughs containing combina- tions of two additives show several noticeable changes com- pared to doughs in which the additives were individually tested (Table 3). The arrivals for doughs with a double combination of additives ranged from 1.0 - 1.8 minutes. In single additive doughs the arrivals ranged from 1.5 - 3.0 minutes with the 3.0 minute arrival time being in the heat treated system. In the testing of doughs with double addi- tive combinations, however, the arrivals of the variables which included a heat treatment had at least halved in time (p:0.001). Thus, the other additives have an apparent effect of facilitating hydration. In practical terms, a baker would expend less time and energy on hydration of his mix thereby conserving mix time. Doughs with double com- binations including salt as one of the additives exhibited the longest arrival times (p50.001)(Tab1e 10). The longest being 1.8 min or equal to the time needed to develop the 6% SCP supplemented dough with salt alone. The salt heat pair- ing had the longest arrival for all combinations with a heat treatment.. The salt may have had a dehydration effect on the flour protein bonding sites or it may have acted as a blocking agent. 61 Table 10. Farinograph Datalftn°Doughs Prepared at the 6% Single Cell Yeast Protein Substituted Flour Testing for the Effect of Interaction of Oxi- dant, Salt, Conditioner and Heat A rival Peak Treatments2 Time Time Stability Absorption min min min min DOUBLE ADDITIVES Oxid-cond l.A 3.A 8.0 71.8 Heat-oxid l.A A.0 6.8 72.5 Heat-cond 1.0 2.0 A.0 73.8 Salt-cond 1.5 2.5 5.0 69.7 Salt-oxid 1.8 5.5 9 8 71 0 Salt-heat 1.5 2.5 3 6 7A.0 TRIPLE ADDITIVES Salt—oxid-cond 1.8 3.5 6.5 72.8 Salt-heat—cond 2.0 3.8 5.5 73.7 Salt-heat-oxid l.A A.5 9.1 71.5 Heat—oxid-cond 1.0 2.5 8.0 72.5 1 Average of 2 Treatments: Oxidant — KBrO3:KIO3 (3:1); 50 ppm Salt - NaCL; 2% Conditioner - Sodium stearoyl-2-lacty1ate; 0.5% Heat - Microwave heat on SCP; 1.25 min 62 The peak times display an interesting trend with decreases to 2.0 and 2.5 min respectively for the heat lacty- late and heat salt treated doughs (Table 10). This compares with a peak time of 5.1'min for the heat treated dough. The least decrease in peak time was for the oxidant heat treated dough. An examination of the other oxidant pairings showed that peak times of doughs with oxidant lactylate and salt oxi- dant combinations were not decreased at all; in fact, they showed slight increases (p:0.001). This could be predicted because the bromates have little effect during early mixing times. Doughs which contained heat treated SCP and any other additives had lower stabilities (p50.001) (Table 10). The salt oxidant treatment yielded the most stable curve at 9.8 minutes, the salt lactylate was less stable with a measure of 5.0 minutes. This leads to a search of the other treatments for improving effect. And while the lactylates do not seem to be involved in any trends,the oxidant combinations with all the other treatments yielded the longest stabilities (p:0.001). This indicated that sometime during the first 5 ‘to 6 minutes the oxidants became reactive and after that they Ixromoted sulfhydryl-disulfide interchange. In turn, this :resulted in a stronger more durable dough. The absorption trends for dough with the double addi- txive combinations (Table 10) showed that the combinations vnmich include heat treatment have slightly higher percent atuworptions. In general, the percent absorptions ranged 63 from 69.7% to 7A.0% which was similar to individual applica- tions of the treatments where the range was 72.5% to 7A.3% (Table A). For the series of doughs containing triple combinations of treatments, the arrival times do not seem to follow any set pattern (Table 10) except that those doughs containing a salt treatment displayed longer arrival times than those without. The range of arrival times was 1.0 to 2.0 min. The heat oxidant lactylate treated dough exhibited a short arri- val time, l.0 min. When salt was added to the heat treatment in combination with either lactylate or oxidant, a slow down in hydration occured with arrival times of 2.0 and l.A min respectively, the lactylates seemed to have less ability (p:0.001) to overcome the time lengthening effect of the salt than did the oxidant. Triple combinations of additives that contained salt showed the longest peak times (Table 10). Treatments with salt and heat along with either oxidant or lactylate resulted in.peak times of A.5 and 3.8 min, respectively. The lacty- late seemed to be more effective than the oxidant in decreas— :Lng peak development. The heat oxidant lactylate combination :resulted in a dough with a peak time of 2.5 min compared to a {Meak time of 3.8 min for the doughs containing the salt heat lactylates (p:0.001). Lactylate reduced the peak time when ccnnpared with doughs containing oxidant. In the double addi- txive combinations the heat oxidant peak time was A.0 min ccnnpared with 2.0 min for the heat lactylate doughs. 6A In stability doughs with combinations including salt or oxidant were more stable (p:0.001), however, combination of these two did not result in the greatest stability (Table 10). The salt oxidant heat treated dough was the most stable (9.1 min) while the lactylate oxidant heat treated dough ranked second in stability (8.0 min) and the lactylate salt oxidant treated dough was third in stability. Oxidant treat- ment again showed strengthening of the dough in later mixing. The percent absorption data for the triple combinations showed a range from 71.5% to 73.7% with no significant trends (Table 10). Extensigraph Studies Double combinations of additives in doughs had little effect on extensibility (mm) after fermentation (Table 11). The range of dough extensibilities after individual treat- ment by the additives at the A5 min test time was 207 to 231 mm (Table 5) while the range of extensibilities in doughs with double combinations of treatments was 210 to 230 mm. There were no extensive changes in extensibility at 90 min- utes. However, at 135 min the extensibility for the doughs lNith double combination of additives decreased. Treatment (of dough with oxidant and lactylate decreased the extensi- loility to 150 mm at the 135 min test time (p:0.001). Doughs twith the other possible combinations including oxidant also :flaowed lower extensibilities, neither of which were signifi- carn;. This supports the oxidant treatment data of 170 mm, 65 Table 11. Means1 of Extensibility Measures (mm)for Doughs Prepared with the 6% Single Cell Yeast Protein Substituted Flour Testing for the Effect of Interaction of Oxidant, Salt, Conditioner and Heat Treatment2 Time of Extensibility Measure A5 min 90 min 135 min DOUBLE ADDITIVES Oxid—cond 210 185 150 Heat-oxid 230 200 190 Heat-cond 210 205 200 Salt-cond 220 195 185 Salt-oxid 215 190 170 Salt—heat 225 215 205 TRIPLE ADDITIVES Salt-oxid-cond 220 210 200 Salt-heat—cond 225 210 200 Salt—heat-oxid 230 205 200 Heat-oxid—cond 235 225 215 1 Average of three replications. 2'I'reatments: Oxidant - KBrO3:KIO3 (3:1); 50 ppm Salt - NaCL; 2% Conditioner - Sodium stearoyl-2-1acty1ate; 0.5% Heat - Microwave heat on SCP; 1.25 min 66 the lowest value, for the individual additive testing. Data from the triple additives testing did not affirm this observation (Table 11). Over the 135 min the range (235 — 200 mm) of decrease in extensibility amounted to only 35 mm. Data in Table 12 indicates that in the double combina- tions of additives,dough systems that included a heat treat- ment had the lowest resistance to extension at the A5 min testing. The heat lactylate treatment dough had the lowest resistance, A25 BU (p50.001) while the neat oxidant and heat salt variables followed with respective values of 525 (p50.001) and 5A0 BU. The lactylate oxidant followed with a reading of 690 BU (p50.001) while the lactylate salt and oxidant salt (p50.001) followed with respective values of 780 and 900 BU. In order of degree of effect in tightening the molecular structure and resulting in increased resistance to extension the additives ranked: heat < lactylate < oxi- dant < salt. With increasing fermentation the oxidant and salt treatment showed some interchangeableness with respect to degree of effect on the tightening of the gluten. These observations were supported by the triple combina— txions of treatments in doughs (Table 12). The salt oxidant lactylate treatment displayed the most resistance to exten- :sion at all times. The second most resistant system at all tximes was the salt oxidant heat treated dough. The other two cxnnbinations, salt lactylate heat and oxidant lactylate heat, remaked third and fourth in degree of effect on resistance to 67 Table 12. Means1 of Resistance to Extension Measure (BU) for Doughs Prepared with the 6% Single Cell Yeast Protein Substituted Flour Testing for the Effect of Interaction of Oxidant, Salt, Condi- tioner and Heat 2 Time of Measure of Resistance A5 min 90 min 135 min Treatment DOUBLE ADDITIVES Oxid-cond 690 785 895 Heat-oxid 525 650 720 Heat-cond A25 AA5 A75 Salt-cond 780 835 860 Salt-oxid 900 1065 1150 Salt-heat 5A0 610 655 TRIPLE ADDITIVES Salt-oxid—cond 865 1075 1100 Salt-heat-cond 590 650 700 Salt—heat-oxid 675 870 975 Heat-oxid-cond A05 620 780 1 Average of three replications. 2 Treatments: Oxidant - KBrO3zKIO3; 50 ppm Salt — NaCL; 2% Conditioner - Sodium stearoyl-2-1actylate; 0.5% Heat - Microwave heat on SCP; 1.25 min 68 extension at A5 and 90 min of fermentation but they reversed order at 135 min. Thus, it seemed that the doughs were tightened more by salt and oxidant while the degree of effect by the lactylate and heat was considerably less. Bread Volume The effect of the combination of additives can be seen in the bread volume data (Table 13). All double combinations resulted in loaf volumes considerably higher than the individ- ual treatment systems for 6% SCP breads (Table 7). Volumes of bread with double combinations of additives were higher than any of the 0 and 3% SCP substituted breads (Table 7). Heat in combination with the other three dough treatments yielded loaf volumes greater than 500 cc in each case. Use of the heat treated SCP in combination with oxidant, salt and lactylate in doughs yielded loaves with respective volumes of 570, 555 and 5A5 cc. Two of these systems yielded the highest extensibility measures for doughs at A5 min, the heat oxidant and heat salt. Support for the idea that extensibil- ity may be a measure of possible volume was found in that the lowest extensibility was for the oxidant lactylate treated dough which also had the lowest bread volume. In the triple combinations of treatments the heat treat- ment along with all possible combinations of the other addi- tives ranked first, second and third in loaf volume measure- ment (Table 13). The heat oxidant lactylate loaf had the Table 13. Means tions Singl Flour of Ox 69 1 of Volumes (cc) and Sensory Evalua- 2 of Bread Prepared with the 6% e Cell Yeast Protein Substituted Testing for the Effect of Interaction idant, Salt, Conditioner and Heat Treatment3 Volume Sensory Score cc DOUBLE ADDITIVES Oxid-cond A55 71.65 Heat-oxid 570 56.35 Heat-cond 5A5 A9.05 Salt-cond A60 70.95 Salt-oxid A55 63.25 Salt-heat 555 51.05 TRIPLE ADDITIVES Salt-oxid-cond A75 62.50 Salt-heat-cond 5A0 60.00 Salt-heat-oxid 555 56.A5 Heat—oxid—cond 591 60.20 1 Score is devi 3 Treatments: Average of three replications. ation from norm. Oxidant - KBrO3zKIO3 (3:1); 50 ppm Salt - NaCL; 2% Conditioner - Sodium stearoyl-2- lactylate; 0.5% Heat - microwave heat on SCP; 1.25 min 70 highest volumes of all double and triple combinations with a value of 591 cc. The salt heat oxidant and salt heat lacty— late loaves ranked second and third with respective volumes of 555 and 5A0 cc (p:0.001). The improvement in volumes for all of the loaves with combinations including oxidant was between 20 and 160 cc over the A35 cc volume for the oxidant treated 6% SCP substi— tuted loaf. Also it seemed that the heat treatment effected some unidentified factor, possibly a protein in the SCP, and in conjunction with the other additives resulted in bread of considerably higher volume. Sensory Evaluation Sensory evaluation data (Table 13) for the 6% SCP bread with combinations of treatment resulted in scores that were considerably lower (better) than in any individual treatment products (Figure 3). Following the trends of the volume data, the bread prepared with heat treated SCP plus other additives scored first, second and third. The heat lactylate (p50.05), heat salt (p:0.001) and heat oxidant (p:0.001) loaves scored respectively A9.05, 51.02 and 56.35. The salt oxidant (p:0.01) and salt lactylate (p:0.001) bread ranked fourth and fifth with scores of 63.25 and 70.95. The poorest scoring product was the bread with oxidant and lacty- late (p50.001). In triple combinations the heat combined with the other Figure 3. Bread Prepared with 6% Single Cell Yeast Protein Substituted Flour Testing for the Effect of Interaction of Oxidant, Salt, Conditioner and Heat Double Combinations a) Salt-Conditioner b) Heat-Conditioner c) Oxidant-Conditioner d) Heat-Salt e) Salt-Oxidant f) Heat-Oxidant Triple Combinations a) Salt-Oxidant-Conditioner b) Salt—Heat-Conditioner c) Salt-Heat-Oxidant d) Heat-Oxidant—Conditioner 73 additives again scored first, second and third (p:0.001) (Table 13). The salt also seemed to be a factor in high ranking in this factorial level. It was heat plus salt plus a third additive which ranked first and second. In the single treatment systems,a salt treated loaf ranked first while heat treated loaves ranked last (Table 9). Thus an improving effect by the other additives on the heat treatment was indicated. Chemical Modification of BondingrSystems Urea The 0% and 6% SCP substituted dough systems were treated with urea to evaluate the significance of hydrogen bonding on dough structure. In both the doughs, the 5 M urea showed strong denaturation of the wheat proteins as demonstrated by extremely low farinograph resistance (BU) readings (Figures AA and B). The effect of the urea at all concentrations was more detrimental to the 6% SCP dough system. In this dough the 1 M urea also tended to solubilize or denature the pro— tines as demonstrated by a rather rapid drop in the mixing resistance after the first five minutes. This was not evi- denced in the flour system. The curve for the 1 M urea 0% SCP dough required 30 min of mixing with urea to achieve the same consistency that occurred at five minutes in the 6% SCP dough. The SCP may be more easily denatured than the gluten by the urea . Thus, the SCP is unable to bind the water Figure A. Effect of Various Concentrations of Urea on Consistency (BU) of Dough as a Function of Time (min) a) Control b) 6% SCP Substituted Dough 7A see 2.: 0.». ON 0. 2 80.. .......... 288.0 I: s. 88.0 I I s. 986 II s. 8.0 I CON 00m 00¢ 000 3.5 2.: 00m .00m 00¢ 00m (n9) Kauaisgsuog 75 necessary to maintain a higher early peak resistance. At the lower concentrations of urea (0.0016, 0.0083, 0.083 M) in the flour system (Figure AA) the urea either had no effect at earlier times (five and ten min at 0.0016 M and 0.0083 M) or it displayed a strengthening (0.083 M) compared to the control (0.0 M). A similar trend was observable in the SCP dough when at early times the 0.0083 and 0.083 M urea treat- ments showed an initial strengthening of the dough but the viscosity dropped rather quickly in this system (10 min). In both the 0% and 6% SCP doughs, the 0.083 M urea had a greater strengthening effect than the 0.0083 M urea. It may be that the gluten was partially denatured, unfolding the chains and making available bonding sites that had associative and thus strengthening effects. The 6% SCP dough (Figure AB) was effected to a greater degree through loss of resistance than was the 0% SCP dough at the constant 10 minute measurement. Succinic Anhydride In testing the effect of the high concentrations of amide (—NH3) groups in the wheat protein, succinic anhydride was applied at a variety of concentrations to the dough. Succinic anhydride reacted with protein as shown: R-CH—C = O 0 \0 + P-(NH3)n /\ CH-C O P-(NH-C-CH-R _ + 2yH+ CH-COO ) 76 In this testing it was necessary to mix at a higher pH than normal. Usual farinograph dough pH was approximately 5.9, but in this testing the dough was titrated with N NaOH to a consistent dough pH of 9.A. Degradation effects may have been a problem thus controls at 5.9 and 9.A pH are displayed (Figure 5A). Some early degradation of protein may have occured in the alkaline system but after 20 minutes of mixing this was equilibrated. As evidenced in Figures 5A and 5B the higher concentrations of succinic anhydride (5.0 and 10.0 g) tended to have extremely detrimental effects on the farino- graph mixing pattern of the 0% and 6% SCP substituted doughs. The 0.1 g treatment with succinic anhydride had virtually no effect; in the 6% SCP dough the 0.5 g treatment had no effect. But the 0% SCP dough with 0.5 g succinic anhydride showed a strengthening in the mixing pattern at 20 min and had a final resistance, 60 BU greater than the untreated system. After treatment with 1.0 g succinic anhydride, the 0% SCP dough was virtually uneffected; however, the 6% SCP dough displayed a delayed weakening with a final resistance 50 BU less than the untreated system. The succinic anhydride at all concentra- tions (Figures 5A and B) except one (5 g) showed a more detri- mental effect on resistance of the 6% SCP dough than on the 0% SCP dough at the 10 minute time measurement. Figure 5. Effect of Various Concentrations of Succinic Anhydride at pH 9.A on Consistency (BU) of Flour Dough as a Function of Time (min) a) Control b) 6% SCP Substituted Dough 77 20 500. 400r- “5“... F- J J l I O 8 O O S e 8 8 <7 02 03 In E 3.; of 'f ’ \ ~.c' ' b x 7 (n9) Kauaisgsuoo 30 IO 20 IO Time (min) Time(min) 78 Sodium—dodecyl-sulfate The effect of sodium dodecyl sulfate (SDS) was observed on two characteristics of each dough system: mixing resis- tance and arrival time. Concentrations from 1.0 - 2.0 g SDS in the 0% SCP dough showed that as the concentration increased the resistance increased (Figure 6A). The rate of resistance increase at 1.0 and 1.5 g was parallel but decreased at 2.0 g. This was further substantiated by checking the rate of increase with 5.0 g SDS which demonstrated a much slower development to maximum resistance than at lower treatment levels. The 1.0, 1.5 and 2.0 g SDS tests in the 0% SCP dough seemed to plateau in resistance after 10 to 15 min of mixing. In the 6% SCP dough the mixing trends were not as clear as in the 0% SCP dough (Figure 6B). The doughs with 1.5 and 2.0 g SDS had approximately the same curve as the control (0.0 g SDS) dough up to 15 min at which time they displayed some weakening. The 6% SCP dough treated with 1.0 g SDS seemed stronger initially (5 min) with a fairly rapid and consistant drop in resistance occuring thereafter; no plateau effect was seen at any level of SDS. The 5.0 g SDS treat- ment of the 6% SCP dough caused high initial resistance measurements with subsequent decreases but at no time did the resistance drop below that of untreated 6% SCP dough. If in the 0% and 6% SCP doughs the SDS was weakening the hydrophobic bonds it was making available other hidden bonding sites that may interact. This unfolding and associa- tion at previously unavailable sites was most obvious at the Figure 6. Effect of Various Concentrations of Sodium- dodecyl Sulfate on Consistency (BU) of Flour Dough as a Function of Time (min) a) Control b) 6% SCP Substituted Dough 79 30 20 Time (min) J D Ion-Av (09w) emu IO- é a 3 S S (n3) Kauaisgsuoo IOOP o LES 2:5 275 5.0 Cone. SDS :0 IO Time (min) 80 5.0 g concentration of SDS. The effect of the SDS on the 6% SCP dough was more intense than on the 0% SCP dough; the SDS additions up to 2 g caused increased mixing resistance in the 0% SCP dough with a drop in the resistance at the 5.0 g level. The 6% SCP dough displays the opposite effect from SDS treatment with the mixing resistance decreasing with addition up to 2 g SDS and then increasing with the 5.0 g SDS addition. Reactive Sulfhydryl and Disulfide Sulfhydryl (-SH) and disulfide (-SS-) bond importance in rheological properties of doughs is a long established belief (A9). It has been determined however, that not all sulfhy- dryls and disulfides are reactive and display an effect on the rheological parameters of a dough (A9). As shown in Table 1A, the 0% and 6% SCP doughs displayed total sulfhydryl counts of 123.5 and 210.A mol/50 g respectively. Of these, 22.5 and A6.6 mol/50 g were involved in the mixing toler- ance of the respective 0% and 6% SCP doughs (Figures 7A and B). Thus, only 19.2% and 22.1% of the total sulfhydryl groups were reactive in the respective systems. The remaining -SH groups were in all liklihood either buried inside of the protein molecule or unreactive due to some other chemical or physical force. The total disulfide counts of the two systems (0% and 6% SCP dough) measured 888.0 umols/50 g and 1057.0 umol/50 g. 81 Table 1A. Chemical and Physical Mixing Properties of Flour and 6% BYP Substituted Flour as a Function of Total and Reactive Thiol and Disulfide Flour 6% BYP Protein (%) 1A.35 19.31 Water absorption (%) 57.2 72.5 Development time (min) 6.1 2.5 Total thiol (umol/50 g) 123.5 210.A Thidlinvolved in mixing tolerance (umol/50 g) 22.5 A0.6 Total disulfide (umol/50 g) 888.0 1057.00 Disulfide involved in develop- ment (umol/50 g) 11.5 25.0 Disulfide involved in resistance to mixing (umol/50 g) 82.5 92.5 Mixing SS/ mixing SH 3.66 2.28 Total SS/ SH 7.190 5.0223 Figure 7. Determination of Sulfhydryls Involved in Mixing Tolerance by Treatment of Flour Dough with N-ethylmaleimide a) Control b) 6% SCP Substituted Dough Determination of umols of (-SS-) Disulfides Effecting Development Time of Flour Dough 0) Control d) 6% SCP Substituted Dough Determination of Disulfides Involved in Mixing Tolerance by Treatment of a Flour Dough with Dithiothreitol e) Control f) 6% SCP Substituted Dough 82 j 50 I00 ‘I60 200 260 300 400 O 21 L_ll 4 l L A I00 3 99 0 0 I 2 o 0 3'? U) 0 a: “6 g 300 O _.| 200- B IOOI O O 50 IOO I50 200 250 3007 400 N- Ethylmaleimide (,1. mols) 83 Actively involved in dough development were 11.5 umol/50 g and 25 umol/50 g of the disulfides (Figures 70 and D). This was about 2.08% of the total groups in the 0% SCP dough and 2.56% of the total content of the 6% SCP system. The reactive disulfides involved in mixing tolerance were 82.5 umol/50 g or 9.29% for the 0% SCP dough and 92.5 umol/50 or 8.75% for the 6% SCP dough (Figures 7E and F). These values were slightly low as the reported range is 11-13% (A9) but they are still viable. The mixing SS/mixing SH of the 0% SCP dough was 3.66 versus 2.28 for the 6% SCP dough. In contrast, the total SS/SH ratios were somewhat higher, 7.19 and 5.02 respectively. It had been reported that of the sulfur containing pep- tides endogenous to flour only gluathione was acting as a determinant of dough properties. As can be seen from Table 1A, the content of total sulfhydryl and disulfide in the 6% SCP flour dough increased considerably over that of the all flour dough. Because of the non-gluten nature of the SCP and yet the arrarent reactivity of some of its -SH and ~SS- groups it may be suggested that the SCP could function as does gluta- thione, a non-structural sulfur bearer, which may enter into the sulfhydryl-disulfide interchange. In turn because of its inability to aggregate with the gluten network other than through these -SH and -SS- groups the SCP in the dough tended to be a weakening factor in the overall mixing, extensibility and resistance to extension characteristics (Table 3 and A). 8A ' ‘ o fil—r—fi—H 50 75 IOOfi' 200 T” 300 I-..-.. 5‘0 '75 I00 "’L 200 '1’ 300 Dithiothreitol (FJDOIS) N U" Loss of Resistance (BU)u E200 IOO 85 Dithiothreitol ( pmols) i so 200 250 300 ' h IOO ISO 200 250 300 86 Scanning Electron Microscopic Investigation (SEM) When flour and water were mixed into an optimally developed dough the most striking observation was the envel- opment of the starch granules by a continuous sheet of gluten protein. The visual nature of this gluten network when pre- pared with and without 6% SCP and with and without 0.5% sodium stearoyl-Z-lactylate will be the topic of this section. Previously Argani and Hawrylewicz (6) described the interaction of flour proteins during dough development as form- ing a smooth veil-like network that stretched over the starch grains. The nature of this network was both visually and rheologically effected by the above listed treatments of the dough. At the lowest magnification (Figures 8, 9, 10, ll-A) the doughs appeared roughly similar. There was a pebbly charac— ter with no evidence of a trend in directional placement of the granules. Both large and small starch granules were apparent in all doughs, and they could be seen both on the surface of the dough and under the gluten sheeting. All doughs showed crater-like structures that were about the same size as the large granules. In all liklihood starch granules that were not firmly positioned in the gluten net- work had dislodged leaving these pock-like structures. Figure 80 showed a starch granule that appeared to be weak- ened in its positioning in the gluten network. At the next range of magnification (Figures 8B, C, D) Figure 8. Scanning Electron Micrographs of Flour and Water Doughs a) 200x b) 800x 0) 1,000X d) 1,600X e) 2,800X 87 a‘ Water Figure 9. Scanning Electron Micrographs of Flour, Water and Na Stearoyl-2-lactylate a) 200x b) 800X c) 1,000x d) 2,800x e) 8,000x 88 Figure 10. Scanning Electron Micrographs of 6% SCP, Flour and Water Dough a) 200x b) 800K 0) 1,000X d) 8,000X 89 Figure 11. Scanning Electron Micrographs of 6% SCP, Flour Water and Na Stearoyl-Z-lactylate a) 200x b) 800K c) 1,000X d) 7,000x 90 91 the gluten sheet looked thin with a crepe-like texture. With increasing magnification (Figure 8B) the crepy roughness in texture was diminished but the surface of the sheet was noticeably marred by very small random pockets. These pockets displayed a multiple layering phenomenon, the extent of which governed the thickness of the protein sheet. It was inter— esting to note that the diameters of these pockets were com- parable to the sizes of osmiophilic inclusions discussed and demonstrated by Simmonds (89) and Khoo et a1. (51) which they accredited to the redistribution and aggregation of membranes and organelles, a source rich in lipids. It was also noted that the most severe rupture of the gluten sheet occurred at the starch/protein interface (Figure 80), an area subject to great stress during dough manipulations. The mixing process developed and stretched the gluten into thin sheets flexible enough to snuggly con- form to the shapes of underlying starch granules. However, stress forces during mixing sometimes exceeded the elastic strength of the gluten allowing sheet breakage at points of weakness. On a microscopic level, the breakage was most obvious at the base of protruding starch granules (Figure 80). The torn areas were not clean breaks and many still exhibited finger-like projections spanning the gap between the two parting edges indicating the adhesive nature of the gluten. In addition, an affinity between granules and the membraneous protein that coats them was quite apparent in many cases (Figures BB, D, D). There was, however, in other cases a 92 definite separating space between granules and the protein. Whether the difference in the closeness of the relationship between the granule and the gluten sheet was a function of the degree of gluten development or an artifact of sample preparation was not clear. The addition of 880 to the 0% SCP dough resulted in alteration of the system (Figure 9). When examined it was similar to that of the control but subtle changes were evi- dent. The gluten sheet was extremely thin, in flung translu- cent in many areas; such that starch granule silhouettes were visible beneath the veiling protein. It appeared very exten- sible and draped fluidly over the pebbly mass of granules (Figure 2B). The sheeting character of the gluten in the emulsified dough seemed finely improved over the control; although, higher magnification (Figure 9E) showed that the smoothness of the gluten was interrupted by small pockets as observed in the control (Figure 8E). The most obvious change in dough structure was observed in the sample containing 6% SCP substitute (Figure 10). The gluten structure was severely altered in the presence of SCP indicating that the supplement was carried by the gluten. The gluten did not develop into a continuous smooth network which could conform to the surface contours of starch granules as in the control (Figure 8) but, being less flexible, it was physically disrupted by the irregular shapes. There was no translucency to the malformed sheet and the outlines of under- lying granules were masked. In contrast to the control the 93 gluten mass was opaque with respect to granule contours, very thick and rough textured to the point of appearing spongy and much less coherent (Figures 10B, C, D). It appeared that the SCP gluten could not maintain or produce the sheeting typical of its nature. There was no evi- dence (Figures IOB, C) of the finger-like fibrils which seemed to indicate the cohesive quality of the gluten mass observed in the control (Figure 8B). The SCP dough with the emulsifier (SSL) exhibited improved cohesion of the gluten compared to the protein substituted dough (Figure 11B compared with Figure 10B). The texture was slightly smoother, less spongy and thick and showed increasing sheeting ability. There was still discontinuity of the gluten phase though not as extensive as without emulsifier. The gluten sheet of this preparation was thinner than that of the SCP alone but still failed to completely coat starch granules as it was stretched over their surfaces. The sheet had a ten- dency to rupture at stress points and settle around the base of granules. When different dough treatments had been tested for extensibility (Table A), it was observed that SSL added to the mixes increased extensibility while substitution of 6% SCP reduced it. SSL added in conjunction with the 6% SCP somewhat restored extensibility at the initial testing time. These results were consistent with the physical SEM observa- tions. One point of concern in a flour/water dough containing 9A additives was the problem of uniform distribution of ingredi- ents throughout the system and therefore the equal distribution and random selection of samples for SEM could lead to a misin- terpretation of the results. Ample sampling of each variable, as in this work, may minimize this problem. SUMMARY AND CONCLUSION The primary objective of this study was to observe the effects of substitution of varying levels (0, 3, 6 and 12%) of SCP on the physical and rheological effects of doughs and bread. The SCP substituted doughs and bread systems were treated with oxidants, conditioners, salt and heat to deter- mine additive functionality. In addition, interactions of these treatments in the 6% SCP substituted system were also observed. The effect of chemical reagents on the mixing char- acter of the 0% and 6% SCP substituted dough was tested and scanning electron microscopic investigation of the 0% and 6% ~SCP substituted dough with and without conditioner was per- formed. Farinograph studies of the 0, 3, 6 and 12% SCP substi- tuted doughs showed that overall mixing character of the dough at the 0 and 3% SCP substitution level were quite simi- lar for all treatments, displaying similar peak and stability times. However, inclusion of the SCP at the 6 and 12% levels in the dough generally resulted in severe decreases in these two mixing characteristics under all treatments. Consistently, as the level of SCP increased the dough absorption increased. The absorptions of all doughs above the 3% level was abnor- mally high. 95 96 Extensigraph measures showed a loss of extensibility of the doughs as the level of substitution of SCP increased. This loss of dough extensibility at the 3% SCP level was in most cases rather small but greater differences occurred_at the 6 and 12% SCP levels. All of the treatment systems resulted in greater extensibility of the 3% SCP doughs than in the untreated 0% SCP dough. In general, most systems dis- played a loss of extensibility with increasing time. Resis- tance to extension values decreased with increasing levels of SCP substitution but they concurrently showed increases with time. In most cases, even at the longest testing time, the dough at the level of SCP substitution was not able to dis- play a resistance equal to the earliest test measure of the preceeding level's dough system. In baking tests the bread volumes decreased as the SCP substitution level increased. The differences in volume be- tween the 0 and 3% SCP substituted bread was negligible but the volume loss in the 6 and 12% SCP bread, was quite exten- sive. The oxidant and conditioner treatments seemed to be the most effective additives in controlling volume decrease. As the level of SCP substitution increased in bread the protein, moisture, lipid and ash contents also rose. Sensory evaluation of the breads showed a slight pre— ference for the product prepared with 3% SCP, however, the scores of the 0 and 3% SCP bread were quite similar. Consid- erable decline in overall acceptability occurred with 6% SCP inclusion in the bread, making this product only fairly 97 acceptable. The 12% SCP bread was unacceptable under all treatments. Considerable interaction of the additives was found for the doughs prepared at the 6% SCP level with double and triple combinations of additives. Farinograph arrival time for all doughs except one with combinations of treatments halved in value. In all instances doughs treated with combinations including salt displayed the longest arrival times. Combinations of dough treatments which included heat resulted in peak times in all cases that were decreased from that of use of heat treatment alone. Pairing oxidant with other treatments resulted in later peak times for doughs com- pared to the use of oxidant alone. Doughs containing oxidant in combination with other additives were most stable while those with a heat treatment as long as it was not combined with oxidant were least stable. The extensibility measures of the 6% SCP doughs treated with combinations of additives showed very little change as compared to use of additives individually. However, combina— tions of treatments resulted in doughs that were considerably higher in resistance to extension compared to use of the additives alone. The oxidant and salt treatments were most effective in increasing dough resistance to extension. The use of combinations of treatments resulted in higher volumes for 6% SCP substituted bread than when the additives were tested alone. Most of the volumes of 6% SCP bread treated with additive combinations had volumes as high or 98 higher than the 0% and 3% SCP bread with the individual addi- tives. Sensory evaluation of the 6% SCP bread treated with com- binations of additives found the acceptability to be consid- erably improved. With the exception of two, the 6% SCP prod— ucts'hmjnladditive combinations scores were higher in accepta- bility than all of the 0% and 3% SCP bread with individual additives. The chemical reagents were generally more detrimental to the mixing strength of the 6% SCP substituted doughs than in the 0% SCP dough. The urea treatment which was being evaluated for the effect on hydrogen bonds exhibited a strenth- ening in mixing at lower concentrations while higher concen- trations showed distinct weakening for the 0% SCP dough. In the 6% SCP dough lower concentrations of urea resulted in early strengthening with subsequent weakening and higher con- centrations were definitely detrimental to the mixing strength. The succinic anhydride was generally increasinly detri- mental to the mixing character as the concentration increased in both the 0% and 6% SCP doughs. In both systems the weaken- ing effect of the succinic anhydride at the lower concentra- tions seemed to be overcome with longer mixing times. In determining the reactive sulfhydryl-disulfide levels it was found that 19.2 and 22.1% of the total sulfhydryl groups were involved in mixing tolerance of the 0% and 6% SCP substi— tuted doughs, respectively. In the 0% and 6% SCP substituted doughs, 9.29 and 8.75% of the disulfides were reactive in 99 mixing tolerance, respectively. About 2.08 and 2.36% of the disulfides were actively involved in dough development of the respective 0 and 6% SCP substituted doughs. In the scanning electron microscopic investigation of the 0 and 6% SCP doughs it was found that the SCP made the gluten sheeting appear bucky and quite thick compared to that of the dough without SCP. Addition of conditioner (SSL) to both 0% and 6% SCP doughs resulted in a thinner and smoother gluten sheet. The overall results of this study indicated that SCP may be added to bread dough up to the 3% level with individual additive treatment and it may be added up to 6% when used with combinations of the additives. The electron microscopic investigation showed that the SCP was carried by the gluten proteins but the exact nature of their interaction is uncer— tain. PROPOSALS FOR FUTURE RESEARCH Future investigation dealing with the content of this dissertation could be multiple particularly with respect to the area of increasing protein content of bread products. However, an even more timely need is seen for the technical methods of evaluating the effect of inclusion of foreign substances i.e. proteins or fiber in bread products. Methods for evaluation of objective parameters of the bread such as volume or compressibility are available but need further standardization. The ultimate decision as to product quality as done by subjective evaluation are satis- factory but again lack consistency as to importance of para- meters. This research amply demonstrated that there are food additives i.e. conditioners, oxidants, salt, etc. that have a multiplicity of effects on a system that has a protein sub- stitute in it. But rapid determination of which of these will alone or in combination maximize final product quality when using a range of concentrations of ingredients i.e. pro- tein is still beyond our technological grasp. If it could be determined what effects protein ingredients actually have on the molecular level in the dough then additives that have an improving function may be more easily and rapidly selected. 100 101 Further research using model systems should be developed to study the molecular bonding of gluten and to determine which are most significant types of bonding in development and strengthening the system with respect to final product quality. If the functioning of these could be realized accurately, a tool for all stages of manipulation of produc- tion would be available. Further development in scanning electron microscopic (SEM) examination of doughs and bread should also be under- taken. It may be that SEM will provide excellent informa- tion particularly with respect to structural faults that occur in products of newly devised formulations. Currently, the only real method for improving a faulty dough or bread is via trial and error and this takes more time usually than would several reliable technical evaluation procedures work- ing at a micro level of protein, starch or lipid functionality in this heterogeneous system. REFERENCES 10. 11. REFERENCES Adoo, E. 3. High protein breads: Interaction of Wheat proteins and soy proteins with surfactants in doughs and in model systems. Ph.D. Thesis, Kansas State University Library, Manhattan, KS (1972). Aitken, T. R., M. H. Fisher and J. A. Anderson. Effect of protein content and grades on farinograms, exten- sigrams and alveograms. Cereal Chem. 21:“65 (19AA). American Association of Cereal Chemists. AACC Approved Methods (formerly Cereal Laboratory Methods, 7th Ed.). The Association, St. Paul, MN (1962). Anon. Baker's yeast protein specifications. Anheuser- Busch, Inc., St. Louis, MO (1973). Anon. Food additives. What they are/How they are used. Manufacturing Chemists Assoc., Inc., Washington, D. C. (197“). Aranyi, C. and E. J. Hawrylewicz. A note on scanning electron microscopy of flours and doughs. Cereal Chem. “5:500 (1968). Bacigalupo, A., T. S. Aguilar, R. Luna de la Fuente and J. Valle Riestra. Bread enrichment with protal- peruvian cottonseed flour. Cereal Sci. Today 12: 431 (1967). Bailey, C. H. The Constituents of Wheat and Wheat Products. Reinhold Pub. Co., New York, NY (19A7). Baker, J. The effects of yeast on bread flavor. Baker's Dig- 31(5):6A (1957). Beckwith, A. C., J. 8. Wall and R. J. Dimler. Amide groups as interaction sites in wheat gluten pro- teins: effects of amide ester conversion. Arch. Biochem. Biophys. 103:319 (1963). Bilderok, B. Bedeutung der thiol and disulfid-gruppen fur die zuchtung des weizens auf backqualitat. Getreide Mehl. 17:20 (1967). 102 12. 13. 1A. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 103 Bennett, R. and J. B. M. Coxpoch. I. Dough consistency and measurement of water absorption on the Brabender Farinograph and Simon "Research" water absorption meter. Trans. Am. Assoc. Cereal Chem. 11:72 (1953). Bennett, R. and J. A. Ewart. The effects of certain salts on doughs. J. Sci. Fd. Agri. 16:199 (1965). Bennion, E. B. Breadmaking. Its Principles and Practices. Ath Ed. Oxford University Press. London (1967). Bloksma, A. Effect of potassium iodate on creep and recovery and on thiol and disulfide content of wheat flour doughs. Soc. Chem. Ind. Monograph 27. London (1968). Bloksma, A. H. Oxidation by potassium iodate of thiol groups in unleavened wheat flour doughs. J. Sci. Fd. Agri. 15:83 (196"). Bloskma, A. H. Rheology and chemistry of dough. In: Wheat Chemistry and Technology. Ed. by Y. Pomeranz, p. 523, Am. Association Cereal Chem., St. Paul, MN (1971). Bloksma, A. H. Flour composition, dough rheology and baking quality. Cereal Sci. Today 17:380 (1972). Bohn, R. T. and H. H. Favor. Functional properties of soy as a bread ingredient. Cereal Chem. 22:296 Bushuk, W. Accessible sulfhydryl groups in dough. Cereal Chem. 38:“38 (1961). Bushuk, W. and I. Hlynka. Water as a constituent of flogfi, dough, and bread. Baker's Dig. 38(6):”3 (19 ). Bushuk, W., C. C. Tsen and I. Hlynka. The function of mxing in breadmaking. Baker's Dig. 92(A):36 (1968). Covallini, D., M. T. Graziani, S. Gupre. Determination of gésulfide groups in proteins. Nature 212:29A 19 . , Crow, M. J. and J. A. Rothfus. Chromatography of proteins from wheat gluten on polyacrylamide gel. Cereal Chem. M5:A15 (1968). 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 10A Cunningham, D. K., W. F. Geddes and J. A. Anderson. Pre- cipitation by various salts of the proteins extracted by formic acid from wheat, barley, rye and oat flour. Cereal Chem. 32:192 (1955). Dahle, L. K. and R. S. Hinz. The weakening action of thioctic acid in unyeasted and yeasted doughs. Cereal Chem. “3:682 (1966). Dempster, C. J., D. K. Cunningham, M. H. Fisher, I. Hlynka and J. A. Anderson. Comparative study of the improving action of bromate and iodate by baking data, rheological measurements, and chemical analy- ses. Cereal Chem. 33:221 (1956). Dempster, C. J., I. Hlynka and J. A. Anderson. Influence of temperature on structural relaxation in bromated and unbromated doughs mixed in nitrogen. Cereal Chem. 32:2u1 (1955). Dimler, R. J. Gluten - the key to wheat's quality. Baker's Dig. 37:52 (1963). Ellman, G. L. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 33:70 (1959). Finney, K. Loaf volume potentialities, buffering capacity and other baking properties of soy flour in blends with spring wheat flour. Cereal Chem. 23:96 (19A6). Fisher, M. H., T. K. Aitken and J. A. Anderson. Effects of mixing, salt and consistency on extensigrams. Cereal Chem. 26:81 (1949). Franz, B. Kinetics of alcoholic fermentation during the propagation of baker's yeast. Die Nahrung. 5:“58 (1961). Carver, J. C., I. Navarini and A. M. Swanson. Factors influencing the activation of baker's yeast. Cereal Sci. Today 11:A10 (1966). Geddes, W. F., T. R. Aitken and M. H. Fisher. The rela- tion between the normal farinograms and the baking strength of Western Canadian wheat. Cereal Chem. 17:528 (19A0). Gray, W. D. and C. Sova. Relation of molecule size and structure to alcohol inhibition of glucose utili- zation by yeast. J. Bacteriol. 72:3A9 (1956). Grogg, B. and Diane Melms. A method of analyzing exten- sigrams of dough. Cereal Chem. 33:310 (1956). 38. 39. U0. U1. U2. U3. uu. “5. #6. U7. A8. “9. 50. 51. 105 Harbrecht, A. and J. Kautzmann. Die Branntweinwintschaft. 107:21-23 (1967). Hird, E. J. R. and J. R. Yates. The oxidation of pro- tein thiol groups by iodate, bromate and persul— phate. Biochem. J. 80:612 (1961). Hird, F. J. R. and J. R. Yates. The oxidation of cysteine, glutathione and thioglycollate by iodate, bromate, persulphate and air. J. Sci. Fd. Agri. 12:89 (1961). Hlynka, I. Influence of temperature, speed of mixing and salt on some rheological properties of dough in the farinograph. Cereal Chem. 39:286 (1962). Hlynka, I. Some rheological aspects of yeast leavened dough. Baker's Dig. A6(2):Uu (1972). Holmes, J. A review of wheat flour proteins and their functional properties. Baker's Dig. uO(6):38 (1966). Holmes, J. and D. R. Briggs. Studies on the physical nature of gliadin. Cereal Chem. 36:321 (1959). Hooper, G. A. Personal Communication. Director of Elec- tron Optics Laboratory, Michigan State University, East Lansing, MI (1975). Hosney, C. and P. L. Finney. Mixing - a contrary view. Baker's Dig. A8(1):16 (197A). Jackel, S. S. Fermentation - today and tomorrow. Proc. Amer. Soc. Bakery Engr., p. 91 (1969). Jankiewiez, M. and Y. Pomeranz. Comparison of the effects of N-ethylmaleimide and urea on rheological proper- ties of dough. Cereal Chem. U2:Al (1965). Jones, I. K., J. W. Phillips and F. J. Hird. The esti— mation of rheologically important thiol and disul- fide groups in doughs. J. Sci. Fd. Agri. 25:1 (197“). Kauzmann, W. Denaturation of proteins and enzymes. In: The Mechanism of Enzyme Action. The John Hopkins Press, Baltimore, MD (195”). Khoo, V., D. D. Christianson and G. E. Inglett. Scanning and transmission microscopy of dough and bread. Baker's Dig. A9(A):ZA (1975). 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 106 Kirleis, A. W. Some effects of soy protein concentrates on dough and bread characteristics. Master's Thesis, Kansas State University Library, Manhattan, KS (1969). Koch, R. B., F. Smith and W. F. Geddes. The fate of sugars in bread doughs and synthetic solutions undergoing fermentation with baker's yeast. Cereal Chem. 31:55 (1954). Kuninori, T. and B. Sullivan. Disulfide-sulfhydryl inter— change studies of wheat flour. II. Reaction of glutathione. Cereal Chem. 45:486-495 (1968). Kuninori, T., M. Yogi and H. Matsumoto. Glutathione leached from yeast cells. Hakko Kogabu Zasshi 46:196 (1968). Labuza, T. A. and K. A. Jones. Functionality in bread— making of yeast protein dried at two temperatures. J. Food Sci. 38:187 (1973). Labuza, T. P. and D. Barrera Santos. Concentration and drying of yeast for human food: Effect of evapora- tion and drying on cell viability. Proceedings American Society of Agricultural Engineers. Chicago, IL (1970). Locken, L., S. Loska and W. Shuey. The farinograph hand- book. Am. Assoc. Cereal Chem., St. Paul, MN (1960). Lusena, C. V. Preparation of dried native wheat gluten. Cereal Chem. 27:167 (1950). Markley, M. C., F. L. Harrington and C. H. Bailey. Effect of mixing on the physical properties of dough. Cereal Chem. 13:560 (1936). Marnett, L. F. and R. J. Tenney. Calcium stearoyl-2- lactylate. A new and versatile baking ingredient. Baker's Dig. 35(6):52 (1961). Matthews, R. H., E. J. Sharpe and W. M. Clark. The use of some oilseed flours in bread. Cereal Chem. 47: 181 (1970). Matz, S. Bakery Technology and Engineering. AVI Publish- ing Co., Westport, CT (1960). Mauritizen, C. M. The incorporation of c steine 35S, cystine 353, and n-ethylmaleimide 1 C into doughs made from wheat flour. Cereal Chem. 44:170 (1967). 66. 67. 68. 69. 70. 71. 72. 73. 74. 76. 77. 78. 107 McKenzie, H. A. Milk Proteins Chemistry and Molecular Biology. Academic Press, New York, NY (1970). Merritt, P. P. and O. E. Stamberg. Some studies on flour absorption. Cereal Chem. 18:632 (1941). Miller, B. S., B. Hayes and J. A. Johnson. Correlation of farinograph, mixograph, sedimentation and baking data for hard red winter wheat flour samples vary- ing widely in quality. Cereal Chem. 33:277 (1956). Miller, B. S. and J. A. Johnson. High levels of alpha- amylase in baking. II. Proteolysis in straight and sponge doughs. Baker's Dig. 21:111 (1947). Moore, C. L. and R. S. Herman. The effect of certain ingredients and variations in manipulations on the farinograph curve. Cereal Chem. 19:568 (1942). Near, C. and B. Sullivan. The use of the farinograph as an accurate measure of absorption. Cereal Chem. 122527 (1935). Osborne, T. B. The proteins of the wheat kernel. Carnegie Inst., Wash. Publ. No. 84 (1907). Pelshenke, P., A. Rotsch and H. J. Koeker. Sugar balance in wheat-flour dough. Biochem. Z 306:205 (1940). Pollack, J. and W. F. Geddes. Soy flour as a white bread ingredient. I. Preparation of raw and heat- treated soy flours, and their effect on dough and bread. Cereal Chem. 37:19 (1960). Pomeranz, Y. Protein composition and breadmaking poten- tialities of wheat flour. Cereal Sci. Today 11:192 (1966). Pomeranz, Y. Flour chemistry and breadmaking potential. In: Advances in Food Research. Ed. by C. O. Chichester, E. M. Mrak and G. F. Skwart. p. 335. Academic Press, New York, NY (1968). Pomeranz, Y., P. F. Finney and R. C. Hoseney. Molecular approach to breadmaking. Science 167:944 (1970). Pomeranz, Y. (ed.) Wheat Chemistry and Technology. 2nd Ed. American Assoc. of Cereal Chemists. St. Paul, MN (1971). Pomper, S. Biochemistry of yeast fermentation. Baker's Dig. 42(2):32 (1969). 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. '92. 108 Ponte, J. G., V. A. Stefanis, S. T. Titcomb and R. H. Cotton. Study of gluten properties as influenced by certain organic solvents. Cereal Chem. 44:211 (1967). Pyler, E. Bakery Science and Technology. Siebel Publish- ing Co., Chicago, IL (1973). Reed, G. and H. J. Peppler. Yeast Technology. AVI Publishing Co., Westport, CT (1973). Rooney, L. W., C. B. Gustafsoon, S. P. Clark and C. M. Cater. Comparison of the baking properties of several oilseed flours. J. Food Sci. 37:14 (1972). Sandstedt, R. M. The function of starch in the baking of bread. Baker's Dig. 35(3):36 (1961). Schachman, H. K. Considerations on the tertiary structure of proteins. Cold Spring Harbor Sumposia on Quant. Biol. 28, 409 (1963). Schoch, T. Starch in bakery products. Baker's Dig. 39(2):48 (1965). Seeley, R. D. and H. Ziegler. Yeast. Some aspects of its. fermentative behavior. Baker's Dig. 36(4): 48 (1962). Shehata, N. The effect of supplementation with chickpea flour on the protein quality of wheat. Ph.D. Thesis, Kansas State University Library, Manhattan, KS (1969). Sidoti, D. R., R. G. Harper, R. D. Seeley, E. A. Robbins and R. W. Sucher. Functional properties and food applications of baker's yeast protein. Paper #119. 33rd Annual Meeting of IFT (1973). Simmonds, D. H. The ultrastructure of mature wheat enodsperm. Cereal Chem. 49:212 (1972). Solle, H. A descriptive system of bread scoring. Baker's Dis- 46(5):55 (1972). Stamberg, O. E. and C. H. Bailey. Relationship of mix- ing speed to dough development. Cereal Chem. 15: 739 (1938). Sternberg, G. Practical gluten structure control. Baker's Dig. 47(2):34 (1974). 93- 94. 95- 96. 97. 98. 99- 100. 101. 102. 103. 104. 105. 106. 107. 109 Tanak, K., K. Furuhawa and H. Matsumoto. The effect of acid and salt on the farinograms and extensi— grams of dough. Cereal Chem. 44:675 (1967). Toledo, R., M. P. Steinberg and A. L. Nelson. Quantita- tive determination of bound H20 by NMR. J. Food Sci. 33:315 (1968). Tsen, C. C. and W. Bushuk. Reactive and total sulfhydryl and disulfide contents of flours of different mixing properties. Cereal Chem. 45:58 (1968). Tsen, C. C. and W. J. Hoover. High protein bread from wheat flour fortified with full fat soy flour. Cereal Chem. 50:7 (1973). Underhafler, L. A. Enzyme supplementation in baking. Baker's Dig. 351(5):74 (1961). Vakar, A. B., A. Pumpilyanski and L. Semanova. Effect of D20 on the physical properties of gluten and wheat dough. App. Biochem. Microbiol. 1:1 (1965). Villegas, E., Y. Pomeranz and J. A. Shellenberger. Effect of thiolated gelatins on rheological properties of wheat doughs. Cereal Chem. 40:694 (1963). Wall, J. S. Cereal Proteins. In: Proteins and Their Reactions. Ed. by H. W. Schultz and A. F. Anglemeir. pp. 315. AVI Publishing Co., Westport, CT (1964). Wall, J. S. and A. C. Beckwith. Relationship between structure and rheological properties of gluten proteins. Cereal Sci. Today 14:16 (1969). Wehril, M. P. and Y. Pomeranz. The role of chemical bonds in dough. Baker's Dig. 43:22 (1969). Wiseblatt, L. Some aromatic compounds present in oven gases. Cereal Chem. 37:728 (1960). Wrigley, C. W. Analytical fractionation of plant and animal proteins by gel electrofocusing. J. Chroma- tog. 36:362 (1968). Wu, Y. V. and R. J. Dimler. Hydrogen ion equilibria of wheat glutenin and gliadin. Arch. Biochem. Biophys. 103:310 (1963). Yasunaga, T., W. Bushuk and G. Irvine. Gelatinization of starch during bread baking. Cereal Chem. 45:269 (1968). Zentner, H. The oxidation of mechanically developed doughs. J. Sci. Fd. Agri. 15:629 (1964). APPENDIX APPENDIX 1 BREAD SCORE CARD Name Date Use the following scales: CRUST COLOR CRUST CHARACT. GRAIN FLAVOR. CRHMB COLOR TEXTURE Rich golden Soft, tender, Fine 8 dis- Mild, sl. Creamy white Sm. s1. elong. brown; very breaks easily tinct cells sweet & bright cells; even even throughout; size 8 dist. not compact Ithin cell walls Rich golden Sl. soft, ten-'Mostly fine 81. bland, Creamy inter-Huneven sm. s1. brown w/some der crust; cells with a s1. observ- ior w/yellowb elong. cells; unevenness breaks easier few coarse able flavor ish shadows thin walls 51. light; 81. tough, Coarse & fine Bland w/o Sl. grey wl Irr. sm. & lg. s1. dark; thick, rubbery cells; uneven distinguish- yellowish cells; thick & uneven thru or s1. soft dist. wlsome able flavor shadows thin walls loaf air holes 81. light; Top tough, Mod coarse Somewhat off ; Grey w/yel- Irr. cell; s1. dark; thick, rubbery cells; un- yet not lowish thick walls over full 5 mod.-dif. to even w/lg. completely shadows but not err. loaf break holes distasteful large Too light; Tough, thick, Coarse cell Flat or smug Grey, dull Lg. irr. too dark rubbery, dif. structure, distasteful cell; uneven to break uneven lg. & thick walls holes Sample CRUST CRUST CRUMB No. cows - cmc'r. cum mvon. copes TWE cams 110 HICHIGRN STATE UNIV LIIBRRR ES 1 isllLlllleHllsfll |H1|NHIII WILLIILIHITI52