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"1"" 11.11.15,;,,:I,;eg-I,1;1§1,,I, ,, Igé ,I':'1I"'I 'I I", ":5"; .." “"7, J,,1,,3[,,11, ,2",,"I,,,g,,',,gz 13:???I'M1E '1; "'1 11 m" , ,‘ 'I-z‘fil'I ' "J ., ‘" ' . "<5?ng ""1 i I . I W, ,"’3‘": ""33 I , f ,.. .,,1,I,,. 5% "R1,, 1, .,, , , ,5; ,_ "I"'"I1'I"III' I"' ~. . "t? I,‘I:", “11,111,111F1 , , , . .,.,,.1,!’f[.,"‘,,t" 3 I, . ’ 3‘ n'r . ; , ' "EIII'IIIIIIPI" 1, 4H),: 3;, 1,131,111 ‘ g, ' I" 'I'II 1,” I2, "’""‘."I "1'1"? .,. THESIS INNIHIIHIIll!HIIHIUIUIWIIHIUUNIHIHIUNIUHM 1293 01716 4215 This is to certify that the thesis entitled Rheological Behavior And Microstructure 0f Developed And Undeveloped Wheat Dough presented by Emily Joann Schluentz has been accepted towards fulfillment of the requirements for M.S. degree in Biosystems 'Engineering Jam 98/44: Major professor // Date December 15, 1997 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution LIBRARY Michigan State Unlversity PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MEWE MTE DUE DATE DUE h "3 1M W14 RHEOLOGICAL BEHAVIOR AND MICROSTRUCTURE OF DEVELOPED AND UNDEVELOPED WHEAT DOUGH By Emily Joann Schluentz A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Agricultural Engineering 1997 ABSTRACT RHEOLOGICAL BEHAVIOR AND MICROSTRUCTURE OF DEVELOPED AND UNDEVELOPED WHEAT DOUGH By Emily Joann Schluentz A study was conducted to investigate the role of shear and extensional deformation on wheat dough development. Undeveloped dough samples were strained in controlled shear and extensional flow between parallel plate sensors in a Haake RSIOO rheometer. After deformation, samples were prepared for structure evaluation using scanning electron microscopy, or rheological testing. Micrographs of wheat dough were subjected to numerical digital image processing to quantify image texture related to protein development. These results were compared to dynamic rheological properties to evaluate the influence of deformation on microstructure. The viscoelastic behavior of wheat dough showed that developed dough had the greatest amount of structure formation, followed by extensionally strained and shear strained samples, respectively. Undeveloped dough showed the lowest levels of structure development and viscoelastic moduli. Image analysis results were statistically different between deveIOped and undeveloped samples; however, results were not significantly different between shear and extensionally strained samples. Copyright by Emily Joann Schluentz 1997 DEDICATION To my parents, Jack and Joann; and to my sister, Mandy. iv ACKNOWLEDGMENTS I am extremely grateful to Dr. James Steffe for his guidance and support throughout my entire academic career. I am thankful to have had the opportunity to work in the Food Rheology Laboratory as an undergraduate, which provided a foundation for graduate studies. I am also appreciative for the funding provided by Dr. Steffe through his United States Department of Agriculture National Research Initiative Competitive Grant (USDA-NRICGP No. 9601952). Without his financial and technical support, this thesis would not have been possible. I appreciate the direction from my committee members: Dr. James Steffe (Department of Agricultural Engineering and Department of Food Science and Human Nutrition), Dr. Perry K.W. Ng (Department of Food Science and Human Nutrition), and Dr. Daniel Guyer (Department of Agricultural Engineering). I would like to acknowledge the use of the following research facilities at Michigan State University: Food Rheology Laboratory, Center for Electron Optics, Cereal Science Baking Laboratory. My sincere gratitude to Dr. Danny Campos for his patience working with me in the Food Rheology Laboratory; and to Dr. Stanley Flegler, Ewa Danielewicz, and Carol Flegler at the Center for Electron Optics, for teaching me the unique world of scanning electron microscopy. Thank you to the faculty, staff and students of the Agricultural Engineering Department. Thanks to fellow students in the Rheology Laboratory for the camaraderie: Julie Abraham, Spencer Breidinger, Nathan Hough, and Caroline Tobey. I also appreciate the support of my friends at Michigan State University: Brenda Becker, Laura Bix, Scott Cassar, Tako Inagaki, Mary Veremis, Mary Kathryn Vredevoogd. Especially, to Joe Oberlee for his support, patience, and understanding. A very special thank you to my entire family for their never-ending support, encouragement, and love. Finally, I thank God for all the Opportunities, successes, good health, and friends. vi TABLE OF CONTENTS Page LIST OF TABLES x LIST OF FIGURES xii NOMENCLATURE xv CHAPTER I INTRODUCTION 1 CHAPTER II LITERATURE REVIEW 4 2.1 Dough Development Process 4 2.2 Rheological PrOperties of Wheat Dough 6 2.3 Electron Microsc0py 10 2.3.1 Principles of Scanning Electron Microscopy 11 2.3.2 Scanning Electron Microscopy Sample Preparation Methods for Wheat Dough 11 2.3.3 Image Analysis of Electron Micrographs 14 CHAPTER III MATERIALS AND METHODS 17 3.1 Materials 17 3.1.1 Flour vii 17 3.1.2 Dough Preparation 3.2 Experimental Procedure to Develop Dough 3.2.1 Shear Deformation 3.2.2 Extensional (Biaxial) Deformation 3.2.3 Oscillatory Testing after Shear and Extensional (Biaxial) Deformation 3.3 Dough Preparation for Scanning Electron Microscopy 3.3.1 Chemical Fixation and Ethanol Dehydration 3.3.2 Ethanol Dehydration Withough Chemical Fixation 3.3.3 Vacuum Desiccation 3.3.4 Freeze Drying 3.3.5 Cryogenic Techniques 3 .4 Data Collection 3.4.1 Scanning Electron Microscopy Images 3.4.2 Image Analysis 3.4.3 Rheograms CHAPTER IV RESULTS AND DISCUSSION 4.1 Scanning Electron Microscopy Sample Preparation Techniques 4.2 Development of the Protein Matrix in Dough oooooooooooooooooooooooooo 4.2.1 Dough Development in Shear Flow viii 19 21 21 25 25 26 26 27 27 27 28 28 29 33 33 40 45 4.2.2 Dough Development in Extensional (Biaxial) Flow 4.2.3 Dynamic Rheological Properties after Shear and Extensional (Biaxial) Deformation CHAPTER V SUMMARY AND CONCLUSIONS CHAPTER VI RECOMMENDATIONS FOR FUTURE RESEARCH APPENDICES Appendix A Appendix B REFERENCES ix 45 50 61 65 67 84 94 Table 3 .1 4.1 4.2 4.3 4.4 B.1 B2 B3 8.4 B5 B6 LIST OF TABLES Farinograph properties of soft white and hard red winter wheat flour. Page 20 Power law constants for G*, determined from linear regression analysis. 51 Power law constants for 6', determined from linear regression analysis. 52 Power law constants for G", determined from linear regression analysis. Protein texture values from numerical digital image analysis. Protein texture values from numerical digital image analysis of developed soft white winter wheat dough. Protein texture values from numerical digital image analysis of soft white winter wheat dough subjected to shear deformation. Protein texture values from numerical digital image analysis of sofi white winter wheat dough subjected to extensional deformation. Protein texture values from numerical digital image analysis of undeve10ped soft white winter wheat dough. 53 58 85 86 88 89 Protein texture values from numerical digital image analysis of developed hard red winter wheat dough. 9O Protein texture values from numerical digital image analysis of hard red winter wheat dough subjected to shear deformation. X 91 8.7 B8 Protein texture values from numerical digital image analysis of hard red winter wheat dough subjected to extensional deformation. .......................... Protein texture values from numerical digital image analysis of undeveloped hard red winter wheat dough. xi 93 Figures 2. 1 3.1 3.2 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 LIST OF FIGURES Flow diagram of the powder method of making “undeve10ped dough.”(Campos et al., 1996) a. Developed soft white winter wheat dough. b. Edges of developed soft white winter wheat dough detected by the Sobel operator. ~..o.ococoooo-oo-‘on a. Undeveloped soft white winter wheat dough. b. Edges of undeveloped soft white winter wheat dough detected by the Sobel operator. Developed sofi white winter wheat dough chemically dehydrated followed by critical point drying. Developed soft white winter wheat dough chemically fixed and dehydrated followed by critical point drying. Undeveloped soft white winter wheat dough chemically dehydrated followed by critical point drying. a. F reeze-dried developed soft white winter wheat dough. b. Freeze- dried undeveloped sofi white winter wheat dough. a. Vacuum-desiccated developed soft white winter wheat dough. b. Vacuum-desiccated undeveloped soft white winter wheat dough. vvvvvvvvv Developed soft white winter wheat dough. Undeveloped soft white winter wheat dough. Developed hard red winter wheat dough. xii Page 31 32 34 35 36 37 39 41 42 4.9 4.10 4.11 4.12 4.13 4.14 4.15 4.16 4.17 A.l A2 A3 A.4 A5 A6 A.7 A.8 Undeveloped hard red winter wheat dough. Soft white winter wheat dough subjected to shear deformation. ooooooooooo Hard red winter wheat dough subjected to shear deformation. oooooooooooooooooo Sofi white winter wheat dough subjected to extensional deformation. Hard red winter wheat dough subjected to extensional deformation. Oscillatory behavior of developed, extensionally strained, shear strained, and undeveloped soft white winter wheat dough. Oscillatory behavior of developed, extensionally strained, shear strained, and undeveloped hard red winter wheat dough. Soft white winter wheat dough. a. Developed. b. Undeveloped. c. Extensional deformation. (1. Shear deformation. Hard red winter wheat dough. a. Developed. b. Undeveloped. c. Extensional deformation. d. Shear deformation. Developed soft white winter wheat dough. Developed soft white winter wheat dough. Undeveloped soft white winter wheat dough. Undeveloped soft white winter wheat dough. Developed hard red winter wheat dough. Developed hard red winter wheat dough. Undeveloped hard red winter wheat dough. Undeveloped hard red winter wheat dough. xiii 47 48 49 55 56 59 6O 68 69 7O 71 72 73 74 75 A.9 A.10 A.11 A.12 A.13 A.14 A.15 A.16 Soft white winter wheat dough subjected to shear deformation. Soft white winter wheat dough subjected to shear deformation. Hard red winter wheat dough subjected to shear deformation. Hard red winter wheat dough subjected to shear deformation. Sofi white winter wheat dough subjected to extensional deformation. Soft white winter wheat dough subjected to extensional deformation. Hard red winter wheat dough subjected to extensional deformation. Hard red winter wheat dough subjected to extensional deformation. xiv oooooooooooooooooo 0...... ooooooooooo ooooooooooooooooo 76 77 78 79 80 81 82 83 any? 00 Yo Yo NOMENCLATURE constant defined by Eq. [4. 1], Pa sb constant defined by Eq. [4.1], dimensionless complex modulus, Pa shear storage modulus, Pa shear loss modulus, Pa gap width, mm initial gap width, mm outer plate radius, mm phase shift or phase angle, radians biaxial extensional strain, dimensionless strain amplitude, dimensionless maximum shear strain, dimensionless stress amplitude, Pa sweep angle, radians XV CHAPTER I INTRODUCTION Rheology is the science of deformation and flow of matter. It is how a material responds to applied strains and stresses and is an important tool applicable to process engineering, quality control, shelf life testing, ingredient functionality and sensory analysis. Materials are rheologically classified according to their flow behavior. The two simplest materials are ideal elastic solids and viscous liquids. An ideal solid (Hookean solid) is characterized by a shear stress proportional to strain, indicating the ability of a material to fully recover when the stress is removed. Newtonian fluids are ideal viscous liquids where stress is proportional to strain rate. However, most materials do not exhibit ideal flow behavior. Wheat dough is viscoelastic, exhibiting both liquid-like and solid- like behavior. Rheological characterization of wheat dough is important because the rheological properties of the material continuously change throughout processing stages (Szczesniak, 1988). Wheat varieties are usually classified as soft or hard. These terms generally correspond to protein content: sofi wheat products are associated with cakes, cookies, and crackers; and hard wheat products with bread and pasta. When wheat flour is combined with water, and energy is applied, a basic dough will form. Traditional mixing systems such as the farinograph and mixograph uniformly distribute flour and water, and add energy by rotating the mixing elements. Water causes the proteins to swell and mechanical energy promotes organization of the proteins into a continuous matrix, giving dough a unique viscoelastic structure (Scholfield and Scott Blair, 1932). In this research, “development” is associated with the energy input element of mixing and “undeveloped” refers to a homogeneous, hydrated flour that has formed in the absence of mechanical energy input. Traditional mixing systems combine distribution and energy input functions in a manner that provides limited fundamental information on dough development. Qualitative information obtained by these instruments cannot be used for fundamental computations because the results pertain to dough as it is mixed and not to the flow characteristics of mixed dough (Campos, 1996). When using these traditional instruments, the influence of shear and extensional deformation on protein development is uncontrollable. The current research allows for separate evaluation of shear and extensional flow on dough development. The flour and ice teChnology developed by Campos et al. (1996) decouples hydration and energy input in dough development allowing the amount of shear and extensional strain to be controlled and the amount of structural development to be rheologically tested. Scanning electron microscopy will provide meaningful images of b) dough development after different levels of deformation. Furthermore, digital image analysis techniques will use edge enhancement and thresholding to numerically quantify image texture reflecting the level of protein development. Understanding how the protein matrix develops from controlled deformation will improve equipment design, process design, and process Optimization. :1. . The objectives of this research are: a) to use controlled shear and extensional deformation to investigate the role of deformation on dough development; b) to prepare dough samples for scanning electron microscopy in a way that minimizes artifacts and maintains the integrity of the starch and gluten matrix; c) to quantitatively determine the degree of image texture related to development (structure formation) of proteins in wheat dough from image analysis of electron micrographs; d) to evaluate the influence of controlled deformation on the dynamic rheological properties of wheat dough. CHAPTER II LITERATURE REVIEW 2.1 Dough Development Process Understanding the role of mixing is an essential step toward Optimizing wheat dough development. However, the processing effects have been difficult to ascertain because there is a lack of instrumentation that measures the amount of protein development. Dough, in the most basic form, is a combination of water, flour, and energy (Campos et al., 1996). Water causes the proteins to swell and mechanical energy organizes the proteins into a continuous matrix, giving dough viscoelasticity (Schofield and Scott Blair, 1932). Lindborg (1995) describes three Objectives of dough mixing: blend the ingredients into a homogeneous mass, develop the protein capable of holding gas, and include air cells into the dough. Bloksma and Bushuk (1988) separated mixing into three distinct elements: distribution of materials, hydration, and energy input to stretch and align protein molecules. The energy input phase of mixing, which involves shear and extensional deformation, is an important aspect of protein development. MacRitchie (1986) describes the alignment of the largest gluten molecule in the direction of shear, as peak development time associated with mixing. 5 Mixing is important because it significantly changes the rheological properties of wheat dough. Dough formation is achieved through unknown amounts of shear and extensional deformation using industrial or laboratory bench top mixers. Typically, the mixed dough is then tested to determine physical properties such as viscosity, strain or shear stress history, or temperature and time dependence. Depending on the type of mixing equipment and mixing time, large deformations stretch and align gluten proteins giving dough the ability to store energy. Lindborg (1995) discusses several dough mixers and characterizes the type of deformation acting on the dough. Single-arm mixers exert pushing and pulling on the dough to develop the proteins. Lindborg (1995) also describes shaking, lifting, folding, and stretching movements in a twin-arm batch mixer, and a lifting and swiveling action in high speed mixers. Besides intensity, mixing time is also important to dough development and ranges from 1.5 min in a high speed mixer to 15-20 min in a single arm mixer. The farinograph and mixograph are the preferred lab instruments for evaluating wheat flour. They use mechanical energy to distribute the ingredients and develop protein. When using these traditional instruments, the amount of shear and extensional deformation contributing to protein development is uncontrollable. MacRitchie (1992) relates development time to protein composition. High dough mixer shear rates remove the outer layer of flour particles and stretch the components into layers as they become hydrated (Lindborg, 1995); however, Cheng (1979) concluded that the relationship between dough strength, hydration, and energy input from shear and extensional deformation is unclear. Lindborg (1995) agrees that there is little known about dough 6 mixing and points out the complicated nature of mixing velocity (shear rate) as an example. In describing the effects of mixing on protein development, information in published literature can only relate the characteristics of a piece of equipment (with undefined flow fields) to an optimally developed dough. This correlation does not represent a true optimum, only a crude feature of the mixing system. Campos et al. (1996) developed a method, using a flour and ice powder technology, to enhance understanding of the mechanical factors involved with protein development. This technology decouples hydration and energy input in dough development. An “undeveloped” dough is a homogeneous, hydrated flour (Campos et al., 1996) that has formed in the absence of mechanical energy (Figure 1.1). The advantage of an undeveloped dough matrix is that the contribution of shear and extensional flow fields can be separately analyzed in controlled deformation experiments. Past research efforts have also tried to produce dough in the absence of mechanical energy (Olcott and Mecharn, 1947, Davies et al., 1969); however, these studies only focused on lipid binding. The “undeve10ped” dough method of Campos et al. (1996), achieved a uniform distribution of water in the dough system by combining flour and ice particles. This technique allows for separate evaluation of shear and extensional flow fields that are otherwise, undefined in traditional dough mixing systems. 2.2 Rheological Properties of Wheat Dough Rheological properties of dough prepared in traditional mixing systems may not accurately reflect the dough development process because the amount of shear and 7 Break solid CO2 (dry ice) in Waring blender I Add ice to blender with crushed CO2 4°C Walk-in Iv Cooler Pulverize ice I Sieve ice and CO2 mixture collecting the particles with size range of 150-250um Hold mixture in freezer allowing sublimation of CO2 and retention of ice particles l Combine known amounts of flour and ice in a centrifuge tube t -8°C Walk-in Distribute powder using Freezer vortex mixer l Place mixture in moisture resistant container l Hold at 25°C for 24 hr allowing ice to melt and flour to hydrate Figure 2.1. Flow diagram of the powder method Of making "undeve10ped dough." (Campos et al., 1996) 8 extensional deformations (which may have a significant influence on molecular alignment during processing) are unknown. Mechanical instruments only describe the mechanical properties of dough (Szczesniak, 1988), not the basic rheological prOperties Of stresses and strains related to protein development and structural characteristics of the material. Rheological flow characteristics Of wheat dough provide powerful knowledge and understanding to improve wheat dough handling and processing. Some prOperties can be measured using standard rheological methods (Steffe, 1996). Wheat flour dough behaves as a viscoelastic material, partly due to unique protein characteristics (Scholfield and Scott Blair, 1932). Viscoelastic materials have both solid and liquid properties. Campos (1996) used the Maxwell, Kelvin, and Burgers mechanical analogs to describe the rheological behavior of wheat dough. These models combine springs and dashpots, representing elastic and viscous behavior, respectively, to better visualize the viscoelastic nature of wheat dough. Muller (1975) also used the spring and dashpot model to describe thixotropy of wheat dough. The rheological properties of dough are largely controlled by the molecular weight distribution Of the gluten protein (Castell—Perez and Steffe, 1992). Recent developments of dynamic rheometers have allowed the evaluation of storage and loss moduli in dynamic oscillatory flow. Wheat dough samples are subjected to a sinusoidal deformation and the time dependent response of the material is related to the viscoelastic behavior of the material. The amount of energy stored in wheat dough is related to G' (storage modulus) and the viscous dissipation is related to G" (loss modulus). Both 9 moduli are functions of frequency and can be expressed in terms of amplitude ratio and phase shift (Steffe, 1996): G’ = [9i] 0055 [2.1] 1’ o Oajflqmm pm 7 o where 0'0 is stress amplitude, yo is strain amplitude, and 5 is phase shift relative to strain. The ratio of viscous to elastic moduli is equal to the tangent of the phase shift: tan5 = E— [2.3] G! Complex modulus, G‘, is an additional frequency dependent material function: ° CSo I 2 N 2 G: =flc)+@) pm To Previous research efforts agree that linear viscoelastic behavior of wheat dough occurs only at low strains. Campos (1996) found linear behavior for undeveloped dough up to a shear stress Of 50 Pa, corresponding to a 0.2% strain level. The 50 Pa maximum shear stress was applied to the sample in the frequency sweep mode of a dynamic oscillatory test. A frequency dependence of both 6' and G" was Observed. Furthermore, G' was consistently greater than G", throughout the frequency range tested, indicating greater elastic properties of wheat dough at 50 Pa. This behavior is typical for structured 10 materials like wheat dough. Campos (1996) also obtained similar rheological results for dough prepared in a farinograph. 2.3 Electron Microsc0py Electron microscopy (EM) is the science of using an electron beam to produce magnified images of minute specimens (Flegler et al., 1993). Two EM techniques are commonly used: transmission electron microscopy (TEM) and scanning electron microscopy (SEM). An advantage of EM, compared to light microscopy (LM), is an increased resolving power because electrons have a shorter wavelength than photons. The ability to distinguish fine detail for a light microscope is at best 200 nm; for TEM and SEM, the resolving power is 0.2 nm and 3 nm, respectively (Flegler et al., 1993). Two other advantages to EM include a better depth of field, and analytical methods to determine the composition of structures containing variOus metallic elements. Food microscopy is a practical science which has been applied to flours, dough, bread, proteins, yogurt, and ice cream. Better than any other technique, EM has allowed scientists to collect data based on high resolution images. Both TEM and SEM systems require high vacuums. There are many reasons a vacuum system is mandatory: to reduce electron beam instability, to reduce reactive gases that could btun out the electron filament, to decrease the occurrence of electron beam scattering which lowers image contrast, and to inhibit specimen-beam interactions that may contaminate or alter the specimens. One disadvantage of EM is that specimens 11 often require more extensive preparation which is critical to image quality in EM (Flegler et al., 1993), than those required with LM. 2.3.1 Principles of Scanning Electron Microscopy The first commercial scanning electron microscope was introduced in 1965 (Flegler et al., 1993). Since then the instrument has been recognized by food scientists as the primary source of microstructure information (Aguilera and Stanley, 1990). In SEM, the surface of the sample is scanned with an electron beam and the digital image observed is created by secondary electrons emitted from the specimen. The microscope’s electron gun emits a beam of electrons that traverses through a high vacuum column and is focused by the objective lens on the Specimen. Scanning coils create a magnetic field that deflects the electron beam across the specimen surface in a repeated raster format, releasing secondary electron from the specimen surface. The secondary electrons are detected by an Everhart-Thornley scintillator-photomultiplier system, and the cathode-ray tube displays a digital image of the specimen surface (Flegler et al, 1993). 2.3.2 Scanning Electron Microscopy Sample Preparation Methods for Wheat Dough The advantages to SEM, compared to TEM, include a very large depth Of focus, the possibility of viewing larger sample sizes, and easier sample preparation techniques (Aranyi and Hawrylewicz, 1968). There are three SEM requirements for biological samples: 1) the sample must not contain any volatile chemicals that may decrease the 12 vacuum or contaminate the microscope, 2) the sample must be firmly mounted to a stub, 3) the sample must be electrically conductive. Since wheat dough contains water, it must be dehydrated. After dehydration, the sample must be firmly mounted to an aluminum stub using an epoxy resin or adhesive tabs. Finally, the dough sample must be sputter coated with a thin layer of gold particles to make it conductive. Several researchers have used the SEM to observe the ultrastructure of bread and bread dough (Aranyi and Hawrylewicz, 1968, 1969; Berglund et al., 1990, 1991; Chabot et al., 1979; Evans et al., 1977, 1981; Khoo et al., 1975; Parades-Lopez and Bushuk, 1982; Pomeranz et al., 1984; Pomeranz and Meyer, 1984; Variano-Marston, 1977). Others have observed the ultrastructure of wheat gluten (Cumming and Tung, 1975; Freeman et al., 1991), wheat flour tortillas (McDonough et al., 1996), and bagels (Umbach et al., 1990). Berglund et al. (1990), reported that even though freeze drying or chemical fixation and dehydration are the most common sample preparation techniques, these methods may produce artifacts, mask surface detail or cause alteration of the microstructure. Variano-Marston (1977) explored many SEM preparation techniques for wheat dough specimens including four dehydration procedures: freeze drying at -65°C for 48 hours, vacuum desiccation at room temperature for 24 hours, air drying at room temperature for 24 hours, and acetone dehydration followed by critical point drying. The effect of chemical fixation with osmium tetroxide (OsO4) vapor or buffered glutaraldehyde, postfixed with buffered 0504 were considered, as well as frozen and 13 unfrozen dough samples. Variano-Marston (1977) concluded that freeze drying (frozen dough state) and vacuum desiccation (frozen and unfrozen dough state) gave the best resolution and depth of field. Furthermore, it was concluded that air drying and chemical fixation, followed by critical point drying gave poor results with wheat dough. Latter work by Chabot et a1. (1979), however, did not conclude that air drying caused more structural distortions than freeze drying in bread samples. Aranyi and Hawrylewicz (1968) used vacuum desiccation for wheat dough specimens and described the Observed image as a veillike networking of protein covering an even distribution Of starch. Evans et a1. (1981) used freeze drying to observe optimally developed dough and characterized the dough as having a continuous and strong adhering gluten layer covering the starch granules. Researchers described dough and bread samples exposed to chemical fixatives as not having a continuous protein covering over the starch granules (Aranyi and Hawrylewicz, 1969) and found severe rupturing in gluten sheets at the starch-protein interface (Evans et al., 1977). Chabot et a1. (1979) concluded that ethanol dehydration before drying altered the specimens by further compacting the structure causing it to appear dense. Cumming and Tung (1975) also concluded that fixation and dehydration removed the veillike protein from the starch, which permitted evaluation of starch morphology. It was suggested by Variano-Marston (1977) that chemical fixatives caused discontinuities in the protein film. 14 Water is an important component in wheat dough. Traditional SEM preparation techniques require the removal of water by the aforementioned drying techniques which, unfortunately, cause changes in ultrastructure. Berglund et al. (1990) compared two cryogenic preparation techniques for low temperature SEM on frozen bread dough. Bread dough was frozen at -23°C and sampled using two methods: 1) samples were placed on specimen holders at 22°C to allow partial thawing, 2) samples were kept frozen with dry ice during mounting on specimen holders (Berglund et al., 1990). The cryogenic preparation used by Berglund et a1. (1990), involves plunging the specimen and holder into a nitrogen slush at -196°C in the freezing chamber, transferring the specimen under vacuum to the cold stage (-180°C) using a shroud (prevents warming and contamination), fracturing the sample with a cooled knife and observing the fractured samples in the scanning electron micrOSCOpe as it sublimes on the cryostage. Berglund et a1. (1990) concluded that thawed specimens produced patterns due to recrystalization of water: samples kept frozen during mounting did not exhibit these patterns. Berglund et a1. (1990) also found that smaller samples had a greater tendency to thaw during mounting, causing recrystalization upon refreezing in the nitrogen slush. 2.3.3 Image Analysis of Electron Micrographs Digital image processing and analysis is a powerful technique applicable to manufacturing, medicine, military, and agriculture. Digital image processing and machine vision have been used in plant identification (Guyer et al., 1986), bread crumb grain evaluation (Zayas, 1993), extruded yellow corn puff evaluation (Gao and Tan, 15 1993), raisin grading (Okamura et al., 1993), snack quality evaluation (Sayeed et al., 1995), and wheat classification (Zayas et al., 1996). Whittaker et a1. (1984) describes digital image processing in steps: capture of a digital image with a sensor, segmentation, description, recognition, and interpretation. An image is a source of information about the physical object it-represents (Whittaker et a1, 1984). Physical properties may include size, shape, color, and texture of a specimen whereby, meaningful data can be derived from the image using image analysis techniques. A monochrome digital image is a two dimensional array Of pixels (picture elements). Each pixel represents the gray level or brightness at each specific location of the image. In SEM, a photomultiplier tube detects or senses secondary electrons emitted from the specimen under observation. The digital image of the specimen, subsequently produced on a monitor, is ready for digital image processing. Castleman (1979) defines digital image processing as subjecting numerical representations of objects to a series of Operations in order to obtain a desired result. In this research, the result will be a quantification of image texture reflecting the level of protein development. There are 256 gray levels in a digital image, where 0 is black and 255 is white. Segmentation, the first processing technique, includes the following: edge detection, thresholding, contrast stretching, and smoothing (Whittaker et al., 1984). Scanning electron micrographs can be subjected to edge detectors which identify gradients in gray levels. Gradient operations compute an angle indicating the direction of maximum gray level change, and a magnitude to describe the steepness of change. Thresholding uses l6 contour lines of the gray level intensities as boundaries for segmentation (Whittaker et al., 1984). After segmentation, image properties such as percent area, can be computed to describe the image. Typically, algorithms are used to calculate shape features from the segmented image. The final two steps in digital image processing are recognition and interpretation. Recognition assigns labels to an Object and interpretation is the final decision based on derived information about the specimen (Whittaker et al., 1984). CHAPTER III MATERIALS AND METHODS 3 .1 Materials 3.1.1 Flour Two types of commercial wheat flours were used in this research study: soft white winter and hard red winter (King Milling Co., Lowell, MI). These flours have distinct physical and chemical characteristics. Typically, soft wheat flours are used in cakes, cookies, and crackers; and hard wheat flours are used in breads and pasta (durum wheat). Campos (1996) characterized the same flour types (King Milling CO. soft white and hard red winter wheat flour) for their protein content, moisture content, ash content, and falling number. Since the same lot of flour was used in this research, only moisture content and falling number were retested and compared to the Campos (1996) results to determine if there were any significant chemical changes in the materials. A farinograph test following standard American Association Of Cereal Chemists (AACC) procedures was also conducted (AACC, 1995). Campos (1996) reported higher protein levels for hard wheat flour (12.36%) than soft wheat flour (10.73%). The higher protein content in hard wheat flour is usually 17 l8 expected, and is responsible for the elevated viscoelastic structure Of the dough. The ash content does not affect physical characteristics of flours (Hoseney, 1994) but does indicate the presence of bran. Again, the ash content of hard wheat flour (0.52%) was slightly greater than soft white flour (0.45%) (Campos, 1996). The moisture content Of the flours was determined by adding ten grams of flour to an aluminum weighing dish. The dish was placed on an Ohaus Moisture Determination Balance (Ohaus Scale Corporation, Florham Park, NJ) and the weight was tared. The scale was placed under intense heating until the scale reached equilibrium (approximately five minutes) and a final weight could be recorded. The final weight on the scale corresponded to the moisture percentage of the flour sample. The average of three replications was the moisture content for hard wheat (14.00%) and soft wheat (14.70%) flours. Campos (1996) reported a moisture content of 14.24% and 14.84% for hard wheat and soft wheat flours, respectively. The falling number test measures Ot-amylase enzymatic activity and indicates the degree of sprouting in wheat grains. A significant degree of enzymatic activity will change the physical properties of wheat flour over time. The falling number test is the total time in seconds from the immersion of the viscometer tube into the water bath until the viscometer stirrer has fallen the prescribed distance through the gelatinized suspension (AACC, 1995). Low or-amylase activity is indicated by a falling number value above 300 s; and a high Ot-amylase activity is indicated by a falling number value 19 below 150 s. The approved AACC Method 56—81B (AACC, 1995) was used to obtain an average (three replications) falling number value of 383 s and 249 s for hard and soft wheat flours, respectively. Campos (1996) reported falling number a value of 478 s for hard wheat flour, and 259 s for soft wheat flour. Rheologically, a high falling number value (low Ot-amylase activity) is indicative of viscosity of the gelatinized suspension. An increase in activity enzymatically decreases the ability of starch to gelatinize, therefore becoming less viscous. Farinograph tests were also conducted for the hard and soft wheat flours following the approved AACC Method 54-21 (AACC, 1995). Fifty grams of flour were placed into the 50 g mixing bowl of a Brabender Farinograph (C.W. Brabender Instruments Inc., South Hackensack, NJ). Water absorption, development time, arrival time, stability time, departure time, and mixing tolerance results are reported in Table 3.1. 3.1.2 Dough Preparation “Undeveloped” dough, prepared using the method of Campos et a1. (1996) described in Figure 2.1, achieved a uniform distribution of water in the dough system by combining flour and ice of similar particle size (150-250 pm). The powder mixtures were prepared and stored in a -8°C freezer. Campos et a1. (1996) allowed samples to thaw for 24 hours; however, Campos et a1. (1997) later found that holding flour and ice mixtures at room temperature for at least 3 hours was adequate to achieve an equilibrium condition. In this study powder samples were thawed for approximately 3 to 8 hours, 20 Table 3.1 F arinograph properties of soft white and hard red winter wheat flour. Farinograph Properties Soft White Winter Hard Red Winter Wheat Flour Wheat Flour water absorption 50.0 53.9 (% wt. basis) arrival time (min) 0.25 0.75 dough development time 0.50 2.0 (min) stability time (min) 1.75 9.25 mixing tolerance index (BU) 160 40 departure time (min) 2.00 10.0 21 forming a dough in the absence of mechanical energy. This technique allows for separate evaluation of shear and extensional flow fields that are otherwise undefined in a traditional dough mixing system. 3.2 Experimental Procedure to Develop Dough 3.2.1 Shear Deformation A Haake Model RSlOO RheoStress (Haake, Paramus, NJ) controlled-stress rheometer was used to rheologically test dough samples. Serrated parallel plates, 20 mm in diameter, were used in all measurements and connected to a load cell with a 5 N-cm torque capacity. The rheometer was interfaced with a computer for measurement control and data acquisition, using software developed by Haake. Shear deformation was created by rotating parallel plates to a maximum predefined strain in a creep test. Maximum strain at the outer rim of the plates, was determined from the following equation (Steffe, 1996): R r. = —-W [3.1] where yo is maximum strain, R (m) is outer radius of plate, w is the sweep angle in radians, and h is the distance between the parallel plates (mm). For this study, R=10 mm, tuzrr radians, and h=2 mm. Following Eq. [3.1], the maximum shear deformation achieved was 1570% strain. Preliminary experiments established 7t radians (180°) of plate rotation as the maximum possible movement before dough samples “rolled out” of 22 the gap between the plates. To eliminate artifacts due to the edge of the plate, dough samples for SEM were taken at R/2, resulting in a 785% shear strain for each sample. Flour and ice powder mixtures were leveled and thawed flat in a small petri dish lined with parafilm, for 3 to 8 hours at 25°C, prior to testing. After the powder mixture thawed, the parafilm and dough were lifted from the petri dish and cut into quarters with scissors. A quartered section of dough was removed from the parafilm with a spatula, and placed on the stationary plate of the rheometer. To prevent dough from sticking to the parallel plates, they were coated with a thin layer of corn oil. It is important to note that this technique did not create slip because the dough sample was firmly engaged by the plate serrations. Once the bottom stationary plate moved to measurement position (2 mm gap width), a thin layer of petroleum jelly was applied around the outside of the sample to prevent drying during measurement. To initiate sample deformation, a controlled stress testing program was loaded into the Haake RS100, serrated plates were attached to the instrument, and the torque sensor was zeroed. The gap between the plates was maintained at 2 mm. A creep program was set to shear the dough sample at 600 Pa until the predefined breaking strain of 1570%, equivalent to 180° rotation, was achieved. Once the shear test was complete and the sample had reached maximum strain, the sample was allowed to relax for 3 minutes in the rheometer to prevent recoil and uncontrolled deformation. After the sample relaxed, the dynamic rheological properties were immediately measured, or the dough was prepared for scanning electron microscopy. 23 Samples deformed for Observation in the scanning electron microscope were rapidly frozen by pouring crushed dry ice particles over the parallel plates. This hardened the dough allowing easier handling during SEM preparation, and further prevented dough from sticking to the plates causing undesirable extensional sample deformation during plate separation. After the bottom plate was lowered, a portion of the sheared dough sample was cut with a razor blade at R/2 and immediately submerged in ethanol to initiate dehydration. 3.2.2 Extensional (Biaxial) Deformation The Haake Model RS100 RheoStress (Haake, Paramus, NJ) was also used to induce lubricated squeezing flow between parallel plates and create extensional (biaxial) deformation. In biaxial extension, the upper plate is fixed and the lower plate moves vertically upward. The diameter of the dough sample in contact with the 20 mm stainless steel plates, increases as the height decreases. The extensional (biaxial) strain, for an incompressible material with a partially full gap is (Steffe, 1996) 83 = Juli] [3.2] where extensional strain, 83, is a function of the initial (ho) and final height (h). If a sample is not properly lubricated, shear flow at high strain levels is introduced (Steffe, 1996). Extensional deformation is uniform throughout the sample. 24 Flour and ice powder mixtures were leveled and thawed flat in a small petri dish lined with parafilm, for 3 to 8 hours at 25°C, prior to testing. After the powder mixture thawed, the parafilm and dough were lifted from the petri dish and cut into quarters with scissors. A quartered section of dough was removed from the parafilm with a spatula, and placed on the stationary plate of the rheometer lubricated with corn Oil. The stationary plate moved vertically upward at 5 mm/min until the sample was in contact with the lubricated upper parallel plate and a gap width of 2.5 mm was obtained. Once the measurement position was attained, the lower parallel plate moved vertically upward and induced lubricated squeezing flow at 1.5 mein until a gap width of 0.5 mm was obtained. Following Eq. [3.2], the computed extensional strain is 80.5% when h=0.5 mm and ho=2.5 mm. After biaxial deformation, the dynamic rheological properties of the cylindrical sample were measured, or the dough was prepared for scanning electron microscopy. Samples deformed for observation in the micrOSCOpe were rapidly frozen between the plates by placing dry ice particles on the rheometer. After the sample was frozen, the lower parallel plate was lowered, and the disk shaped dough sample was placed in a petri dish. NO tensile extensional flow was observed when the plates were separated. A small sample of dough was cut with a razor blade at the center of the disk and immediately submerged in ethanol for dehydration. 25 3.2.3 Oscillatory Testing After Shear and Extensional (Biaxial) Deformation The structural development of wheat dough was determined from an oscillatory test on the Haake RS100. Following shear deformation and relaxation, the complex modulus G* (Pa), storage modulus 6' (Pa), and loss modulus G" (Pa) were measured over a frequency range of 0.1 to 100 rad/s at a constant shear stress of 50 Pa. The frequency range was selected because it represents three decades of change commonly used to test the viscoelastic behavior of food. The shear stress level of 50 Pa was established fi'om previous research efforts by Campos (1996) and corresponds to a 0.2% strain level which maintains a linear viscoelastic material response. Rheograms reflect the mechanical strength, due to structural development, of wheat dough at the specified strain level. 3.3 Dough Preparation for Scanning Electron Microscopy Sample preparation is the most critical step in SEM. Poor technique can cause artifacts and produce variable results among images of the same sample. In this work, five sample preparation techniques were studied for both developed and undeveloped dough. Following each preparation technique, dehydrated samples were mounted with epoxy resin on standard aluminum stubs. After mounting, samples were sputter coated with gold particles (EMSCOPE SC500, T55-29173, Ashford, Kent, England) at 20 mA for 4 minutes. The effect of fracturing versus not fracturing a sample was also studied. Fracturing was done by shattering a sample with sharp pointed tweezers to expose inner 26 surfaces, and mounting a fractured piece on an aluminum stub for metallic coating. Both non—fractured and fractured samples were mounted and scanned. 3.3.1 Chemical Fixation and Ethanol Dehydration Chemical fixation crosslinks proteins in biological samples while ethanol dehydration, followed by critical point drying with C02, allows for complete water removal. Developed and undeveloped dough samples, approximately 4 m3, were cut from frozen samples. Dough was fixed by submergence in 4% glutaraldehyde, buffered with 0.1M phosphate buffer (pH 7.4), for 0.5 hr at room temperature. Fixed samples were rinsed with phosphate buffer for 10. to 15 minutes. After buffering, samples were submerged in a graded ethanol series (25%, 50%, 75%, 95%, 100%) for 20 minutes at each gradation; then submerged in 100% ethanol for three consecutive 20 minute intervals to ensure full dehydration. Since C02 is miscible with ethanol, dough samples were critical point dried using a Balzers Critical Point Dryer (Balzers Union, FL-9496, F urstentum, Liechtenstein). Critical point drying allows ethanol removal in C02 without large surface tension forces that may distort the sample. 3.3.2 Ethanol Dehydration Without Chemical Fixation Previous research efforts included chemical fixation prior to ethanol dehydration. In this research, the same preparation procedure mentioned above, without the chemical fixation in 4% glutaraldehyde, was followed. No published record of this technique could be found. 27 3.3.3 Vacuum Desiccation Vacuum desiccation did not use chemicals or rapid freezing; rather, the sample was allowed to dry in a vacuum desiccator. Small samples (5-7 mm3), were placed on parafilm in a desiccator containing Drierite (anhydrous calcium sulfate) desiccant. Samples were dehydrated for 24 hours at room temperature in the desiccator. 3.3.4 Freeze Drying Samples were placed in scintillator vials, previously chilled in an ethanol and dry ice slush at approximately -70°C, for transfer to the freeze dryer (Labconco Corporation, Kansas City, MS). Once the dryer reached -40°C and 12x10"3 mbar, samples were freeze- dried for 24 hours. After drying, the vials were capped, wrapped in parafilm and placed in a freezer (Forma Scientific Freezer, Marietta, OH) maintained at -86°C for storage. 3 .3 .5 Cryogenic Techniques Dough samples were mounted on a special cryo stub with Tissue-Tek 11 OCT. (Lab-Tek Products, Naperville, IL) compound. The sample and holder were submerged into a liquid nitrogen slushing chamber and transferred to the JEOL JSM-35CF Cryo- SEM chamber (JEOL LTD., Tokyo, Japan) maintained at approximately -100°C. Etching heated the scanning electron microscope chamber to -65°C, allowing the water in the sample to sublime over a period of 20-30 minutes. After etching, the sample was transferred back to the EMSCOPE SP2000 (T8-84442, Ashford, Kent, England) working 28 chamber for sputter coating with gold particles. Samples were viewed on the scanning electron micrOSCOpe stage at -90°C since thermal stress caused the sample to crack at -140°C. 3.4 Data Collection 3.4.1 Scanning Electron Microscopy Images A JEOL J SM-6400V Scanning Electron Microscope (JEOL, Tokyo, Japan) scanned soft white and hard red developed, partially developed (in shear or extensional deformation), and undeve10ped dough samples using an accelerating voltage of 13 kV, 15 mm working distance, and a condenser lens setting of 10. Analog images from the scanning electron microscope are produced on a cathode-ray tube with a 2,500-line resolution capacity (Flegler et al., 1993) and converted to a digital image. The 1024 x 910 pixel size, black and white, images were magnified at 2,000 X and subjected to pixel averaging data image acquisition. While pixel averaging is slow, the resultant image is better because the beam dwells at each pixel longer and the gray level is a time averaged value. The final image was saved as a tagged image file (TIF) on a 3.5 inch floppy disk. Five SEM images were taken for each replication of a dough sample treatment. At least ten different sample replications were Observed under the microscope, resulting in fifty images per sample treatment, providing sufficient information for complete statistical analysis. 29 3.4.2 Image Analysis Visual distinction between starch and protein texture was evident in scanning electron micrographs of developed, partially developed (controlled shear and extensional deformation), and undeveloped soft white and hard red wheat dough. Image analysis software was used to compare images and quantify relative texture using edge detection and thresholding. All images subjected to numerical image analysis were from samples prepared using the technique involving chemical dehydration in ethanol and critical point drying in C02. Results were compared to rheological data of these doughs tested on the Haake RS 1 00 rheometer. Numerical digital image analysis software was used to quantify relative protein texture present in SEM images. The Optimas 4.10 (Optimas Corporation, Edmonds, WA) software program contains image processing tools which can be used to assess texture edges. The software program was loaded onto a computer interfaced with a black and white frame grabber. Before the scanning electron micrographs were analyzed, TIF images having a known amount of black and white area were used to calibrate processing tools in the software program. After calibration, micrographs in standard TIF format were loaded into the program and displayed on the frame grabber. The Sobel operator is a gradient Operator that detects gradients in gray levels and computes an angle indicating the direction and steepness of maximum gray level change. A SEM image was loaded onto the computer and displayed on the frame grabber. The 30 Sobel operator defined the edges in the image related to protein texture and changed the image to a gray level edge intensity image. Figures 3.1a and 3.2a are original SEM images that appear on the frame grabber screen. Figures 3.1b and 3.2b illustrate the gray level edge intensity detected by the Sobel operator for developed and undeveloped soft wheat dough, respectively. A pixel count routine analyzed each pixel and determined if the value was within the predefined threshold. Pixel values between 129 and 255 (inclusive) were used to compute a protein texture value percentage (PTV%): number of texture pixels .>. 129 total number of pixels PTV% = x 100% [3.3] The more texture detected in SEM, the greater the protein texture value. PTV% results were compared to rheological data. 3.4.3 Rheograms Rheograms of dynamic moduli (G*, G’, G") versus frequency were obtained from oscillatory testing using the Haake R8100 rheometer. Each data set was replicated at least four times for each sample treatment (soft and hard wheat dough: developed, shear deformation, extensional deformation, undeveloped) and statistical analyses were performed to model the rheological behavior of the dough. Linear viscoelastic behavior was plotted over a frequency range of 0.1 to 100 rad/s, and the structural effect of each deformation was compared for soft white and hard red wheat dough. 31 05 ,3 8630 mac n . c a c a 0:? SEE 8E3 t8 come—05c mo 8qu A: .m .nwsov 30:3 53:5 853 $8 come—gum H Mo Jam . v . . . H . M 0.— ~nm in N1.) . o. 1.3? . .. \ . . .. . ,. . ., . a . h 1,... Quit... ..... . _. .H “$2 . .../w \. US$30 Enom on“ 3 388% @323 323 .1353 02:3 wow pogo—32cc: mo mowum .nmd .nwsou 30:3 c853 823 $8 cone—3033 .mmw PSwE ‘§.. 3:... , 0.1.x... .. 2...». 4 finfifimflk . .,.. .rfllc a CHAPTER IV RESULTS AND DISCUSSION 4.1 Scanning Electron Microscopy Sample Preparation Techniques The purpose in testing various specimen preparation methods was to determine which technique gave the best results for visual image analysis by distinguishing protein and starch. The most common preparation method for biological samples is chemical fixation and dehydration, followed by critical point drying. Micrographs of soft white winter wheat developed dough not fixed, or fixed with 4% buffered glutaraldehyde, followed by ethanol dehydration and critical point drying with C02, are shown in Figure 4.1 and Figure 4.2, respectively. Figure 4.1 shows protein continuity and definite contrast between starch and protein. The continuous protein is not evident in Figure 4.2, and the contrast between starch and protein is poor. Undeveloped dough (Figure 4.3) chemically prepared without fixation illustrates a homogeneous, undeveloped, hydrated dough that formed with the absence of mechanical energy input. It was difficult to distinguish deveIOped freeze-dried dough samples from undeveloped freeze-dried dough samples shown in Figures 4.4a and 4.4b, respectively. U) D.) .wfibv «Eon Ratio ,3 330:8 83.6432. b32805 swsou 80:3 H853 833 $8 toao_o>oQ .34 oSmE .wfibu ESQ .825 ,3 330:8 uBEPEOw Ea uuxc b33826 swzoc 30:3 .353 833 $8 coao_o>oQ .Né 0.3me \ mart: “Eon 32:6 ,3 330:8 33630: DEBS—0:0 :wsov “3:3 5:3 833 «8 come—33:3 .3. 8:3": .nwsou “00:3 02:3 0053 :8 3:205:25 0050-302": b: .33: 30:3 “8:3 823 tom come—05c 00370500.: .314 053“: 38 Although freeze drying allows sublimation of H20 occurring in the absence of surface tension, ice crystal formation may damage specimen morphology. Figure 4.5a shows a developed dough sample dehydrated in a vacuum desiccator for 24 hours, and Figure 4.5b is an undeveloped dough sample prepared in the same manner. In these images, protein envelopes the starch particles, covering them like a blanket making it difficult to distinguish starch and protein based on contrast. Furthermore, samples were not fully dehydrated which caused a low vacuum in the microscope, resulting in a grainy image texture that lacked good clarity. It was difficult to distinguish protein and starch for both deveIOped and undeveloped samples subjected to cryogenic preparation. The microscopic images produced by this method could only be Obtained with a Polaroid camera and saved as an analog image on a negative, making numerical digital image analysis difficult. Furthermore, cryogenic preparation required a significant amount of time for an individual sample. The effect of sample fracturing to expose the inner surfaces was also studied. Fracturing was done by shattering a dough sample with sharply pointed tweezers, or by cutting the dough sample with a razor blade. The tweezer method distorted the proteinaceous material surrounding the starch disconnecting the protein network. It appears that the force of fracturing, causes weakly bound proteins to shatter (Figure 4.4). This trend was found in both developed and undeveloped dough samples subjected to chemical fixation with glutaraldehyde, freeze drying, vacuum desiccation, and cryogenic 43:0: 30:3 c853 0::3 c8 come—05:5. @8000_m0:-E:=om> :3. ..So: 300:3 c853 0::3 a8 @3230: :030060:-E=30> .36 05mm— 3. 40 preparation techniques. Slicing samples with a razor blade at freezing temperatures also produced unacceptable results because the samples appeared sliced and lacked contrast. The contrast in SEM images created by chemical dehydration and critical point drying produced the best visual distinction between starch and protein in wheat dough samples. Dough preparation was the least time consuming for freeze drying and vacuum desiccation methods; however, visual distinction between developed and undeveloped dough was difficult. Furthermore, vacuum desiccated samples were not fully dehydrated and produced grainy microscopic images. Cryogenic preparation is advantageous because dough samples are observed as water sublimes; however, this method is the most laborious and only produces analog images. 4.2 Development of the Protein Matrix in Dough Scanning electron micrographs of soft white and hard red wheat dough revealed significant visual distinctions between developed and undeveloped dough. Figure 4.6 is a micrograph of soft wheat dough developed in the farinograph. The figure shows a weblike protein structure surrounding starch particles. Figure 4.7 illustrates soft wheat undeve10ped dough which was prepared in the absence of mechanical energy. The figure shows large round starch particles and minimal protein development. Figure 4.8 is developed hard wheat dough and Figure 4.9 is undeveloped hard wheat dough. Similarly, the developed wheat dough micrograph shows an abundance of starch particles surrounded by a developed protein network while the micrograph for undeve10ped dough Figure 4.6. Developed soft white winter wheat dough. Figure 4.7. Undeveloped soft white winter wheat dough. .:w=o: “00:3 c033 :0: Eu: Ego—035 .wé 05mm”— .:w=o: “00:3 00:23 :0: :3: :0Qo_0>0:~5 6.: 05mm“: 45 shows starch particles with a minimal protein network. Figures A.l to A8 are additional images of developed and undeveloped, soft and hard wheat dough. It was not as easy to visually distinguish scanning electron micrographs of soft wheat dough from hard wheat dough. Generally, hard wheat dough contains more protein than soft wheat dough; however, this generalization could not be verified from simple visual observation of micrographs. 4.2.1 Dough Development in Shear Flow Images of soft and hard wheat dough subjected to 785% shear deformation were also taken in the scanning electron microscope. Figure 4.10 is a micrograph of soft wheat dough and Figure 4.11 is of hard wheat dough. In both images, protein forms a weblike network with the starch particles. Since samples were prepared perpendicular to the shear field, the protein network appears to form in the direction of the shear force. Figures A9 to A.12 are additional images of soft and hard wheat dough subjected to shear deformation. 4.2.2 Dough Development in Extensional (Biaxial) Flow Soft and hard wheat dough was partially developed in extensional deformation using squeezing flow displacement. An image of extensionally developed soft wheat dough is found in Figure 4.12 and hard wheat dough in Figure 4.13. Figures A.13 to A.16 are additional images of biaxially strained soft and hard wheat dough. It was not easy to distinguish soft wheat dough from hard wheat dough; however, protein 00:08:80: 0.00:0 8 380.33 :wso: 30:3 c883 0::3 80m .0: .v 05mm: .co:0::0.:0: :00:m 8 8:00.38 :mso: 30:3 853 :0: :33 .:.v 05$: 2;, .,h .580880: 3:830:08 8 80080320 :mso: «00:3 :853 8:3 tom .2 .v 05%: .co:0:::80: _0=o:m=0:x0 8 380.38 :wzo: 80:3 :853 :0: :8: .m _ .v 0::wE 50 development characterized by squeezing flow was evident. Protein development from extensional deformation appears to cover more surface area compared to developed and undeveloped wheat dough. The protein does not have a “stringy” appearance and covers the starch particles like a blanket. 4.2.3 Dynamic Rheological Properties after Shear and Extensional (Biaxial) Deformation Dynamic rheological measurements were used to determine the structural characteristics of soft and hard wheat dough. Developed and undeveloped samples, as well as samples subjected to shear and extensional deformation, were tested on the Haake RS100 rheometer. Dynamic moduli (G*, G’, G”) described the linear viscoelastic behavior of dough at constant shear stress of 50 Pa over a frequency range of 0.1 to 100 rad/s. At least four data sets were taken of soft and hard wheat dough at each deformation, and data from these replicates were pooled because there was no statistical difference between curves. Dynamic rheological behavior of wheat dough followed a power law model: (34:40b [4.1] where the constants, a and b, were determined from linear regression analysis after a logarithmic transformation of the raw data. Dynamic variables G’ and G" also showed power law behavior with respect to frequency. Tables 4.1-4.3 contain values of a and b from regression analysis for G*, G' and G”, respectively. 51 Table 4.1. Power law constants for G*, determined from linear regression analysis. Soft White Winter Hard Red Winter Wheat Dough Wheat Dough development a b R2 a b R2 developed 8905 0.27 0.90 20320 0.23 0.97 extensional deformation shear deformation undeveloped 6415 0.27 0.72 2263 0.40 0.83 1326 0.42 0.88 10490 0.23 0.94 5758 0.32 0.98 5305 0.27 0.87 52 Table 4.2 Power law constants for G', determined from linear regression analysis. Soft White Winter Hard Red Winter Wheat Dough Wheat Dough development a b R2 a b R2 developed 7806 0.28 0.89 18682 0.23 0.97 extensional deformation shear deformation undeveloped 5484 0.28 0.71 1706 0.40 0.84 973 0.40 0.84 9584 0.24 0.94 4581 0.34 0.97 4749 0.26 0.86 53 Table 4.3 Power law constants for G", determined from linear regression analysis. Soft White Winter Hard Red Winter Wheat Dough Wheat Dough development a b R2 a b R2 developed 4246 0.27 0.91 7909 0.22 0.95 extensional deformation shear deformation undeveloped 3299 0.25 0.71 1476 0.38 0.81 889 0.44 N 0.91 3245 0.29 0.97 4214 0.22 0.93 2358 0.28 0.90 54 Data analysis showed G’ (storage modulus) to be consistently greater than G” (loss modulus), indicating dough to behave more as a solid than a liquid over the entire frequency range studied. Moduli were greatest for developed dough, followed by dough subjected to extensional deformation and shear deformation. As expected, undeveloped dough had the smallest moduli. Figures 4.14 and 4.15 are rheograms showing the structural characteristics of soft and hard wheat dough, respectively. Error bars to indicate the 95% confidence interval were very small and could not be clearly displayed in the figures. Figures 4.14 and 4.15 clearly indicate independent structural development of wheat dough in which the type Of deformation or development are unrelated. An F -test further attested that the four models in Figures 4.14 and 4.15 do not correlate with each other. Figures 4.14 and 4.15 show greater structural development for extensional deformation than shear deformation even though the strain percentage was 80.5% and 1570% for extensional strain and shear strain, respectively. The difference in structure indicates greater molecular aligmnent and disulfide bonding in extensional flow fields compared to shear flow fields. Campos (1996) also tested structure development for soft white and hard red, developed and undeveloped wheat dough; however, moduli in the current study were lower. The difference was expected because enzymatic activity increased over time as indicated by the falling number test, and decreased viscoelasticity. Image analysis techniques identified a protein texture value allowing for comparison with structural development determined from rheological measurements. 55 :96: 80:3 0:53 02:; :80 802002: :50 005020 00:0 605080 >__mco_0c0:x0 .:0ao_0>0: :o 530:0: 390.300 .36 050E A0200: 3:03:09“. coo? cow or F to :0ao_0>0::: -- o , :0590 50:0 --o .0590 >__mco_0c0:x0 .0: -0 :0ao_0>0: -. I- ..-.b.\0 r t 1 l ..... , . -. _ \.\-0 Q..O . ...O 0-.- ---o .0...- .--e ox... .\0 -ox. .... .-0. ..\o\ 0- ext 0 o I \Q-- .0- ¢\ 4 . \ .\ \\0\ u\‘\\‘ I. . to - -O .07. -I\,I \O\ \.0 -,.-C. \.- o \o o a I\I t. 0.\0 -0 §\ ...I\ Q o.-- th\¢- I. a -.l \6 -\\‘-\ 11.}. . Q \ \‘Nh ..\\.\ .. C . t- t a I--I 4 .-I- I-- It- I 5.0 00? : 000? 0000? ooooo F (ea) .9 ‘snlnpow xeldwoo 56 :96: 00:3 .853 :0: :0: 00020085 90 605080 00:0 005000 20:00:80 000200: :0 530:0: 00905000 .0 3V 050E 0:000 >o:0:00:n_ (ea) .9 ‘snlnpow xeldwoo 000v 00: or F 0.0 5.0 n... .1: . r : :FL phi :.|r l,r_lll:._Ll.-..1-_ rlL .- Fill. :..rt...... . ... -. .. ...-1:. .-rlrLl. - .. 1r .. . : .. r 00F :0ao_0>0::: o _ 005050 00:010-- :0:_0:0 >__0:o_0:0:x0- «1 :0Qo_0>0: --I: 1-, .- l- . -. .-l: 000v to .---orb...- \-®\0\ \ h - - \-§\ o\Q\m.\ 0? . a- \110 3- o-,-o\o\ ltd--0, I\I 0000 _. \o\?.. -O\ 4-- - ---I- ~0\\ ‘\t\‘\\ .1 . 30H“ Oxbx 4 --h-LT- I-ItI; \ - - - It “US-Ia I\,l-I\I\ x!- -I I\I\I\I If- - . 00000 P 57 Table 4.4 gives the average protein texture value percentage (PTV%) and standard deviation determined from image analysis of soft and hard wheat dough. A comparison of the means and standard deviations in Table 4.4 statistically indicates that both shear and extensional deformation treatments resulted in similar protein development compared to developed dough. However, rheological data indicate that the type of deformation results in different levels Of structural development. Reasons the results from image analysis do not correspond with rheological data include daily contrast and brightness variability in the microscope, and micrOSCOpe contamination by volatile materials. F urtherrnore, visual observation of extensionally strained samples do not reveal distinct edges in the protein webbing, rather the protein appears flat. The Sobel operator would not detect these edges and subsequently, the protein texture value calculated for the image would be lower. Figures 4.16 (a-d) and 4.17 (a-d) allow a visual comparison of the effect of deformation type on soft and hard wheat dough, respectively. Images of deveIOped wheat dough have a protein web in which edges are clearly detected and undeve10ped dough images show less protein texture. Images of extensionally strained dough show protein development in the x and y direction whereas, protein development is primarily in the x direction in shear strained dough samples. Protein development in shear strained samples is clearly detectable by the Sobel operator. 58 Table 4.4 Protein texture values from numerical digital image analysis. Soft White Winter Hard Red Winter Wheat Dough Wheat Dough development PTV% standard PTV% standard deviation deviation developed 35.3 5.7 - 31.9 5.7 extensional deformation 28.0 7.0 32.8 7.0 shear deformation 32.3 4.6 32.6 5.7 undeveloped 23 .4 3.8 ' 18.8 6.6 00:08:80: :00:m .: 00:08:80: 3:80:0me .0 .:0080>0:=D .: .:0080>0Q .0 .:m:o: 000:3 0:53 02:3 80m .08.: 05?": 00:08:80: :00:w .: 00:08:80: :Eofifiim .0 .:0mo_0>0::5 .: .:0qo_0>0n_ .0 .:m=o::00:3 02:3 :0: :33 .2: 0SmE CHAPTER V SUMMARY AND CONCLUSIONS The undeveloped dough concept of Campos et al. (1996) achieved a uniform distribution of water in a wheat dough system by combining flour and ice particles. The advantage of an undeveloped dough matrix is that the contribution of shear and extensional flow fields can be separately analyzed in subsequent controlled deformation experiments. In traditional instruments such as the farinograph and mixograph, shear and extensional deformation are combined, but the relative contribution Of each flow field is undefined. In this study, the development of soft white and hard red winter wheat dough was controlled. Four development treatments were analyzed: fully deveIOped, controlled shear deformation, controlled extensional (biaxial) deformation, undeveloped. Shear and extensional deformation were controlled between parallel plate sensors of a Haake R8100 Controlled Stress Rheometer. Once samples were partially developed, either the dynamic rheological behavior was characterized, or the sample was prepared for imaging in a scanning electron microscope. 61 62 Curves describing viscoelastic behavior of wheat dough were fit to a power law model using linear regression analysis. Rheological testing revealed that G’ was consistently greater than G” over the frequency range of 0.1 to 100 rad/s, indicating that the behavior of wheat dough was more solid-like than liquid-like. Rheograms further depicted greater viscoelasticity for developed doughs, followed by samples subjected to extensional deformation and shear deformation, respectively. Undeveloped samples showed the lowest moduli, reflecting a low level of structural development. A statistical comparison of regression lines clearly indicated significant differences between the developed, shear strained, extensionally strained and undeveloped dough models. Scanning electron microsc0py is a powerful method for visualizing wheat dough samples and investigating structural development related to rheological data. Dough samples were submerged in a graded ethanol series, critical point dried, and sputter coated with evaporated gold particles to create a conductive surface. Although four other preparation methods were investigated, this preparation technique provided best visual distinction between starch and protein for numerical digital image analyses. Numerical digital image analysis was used to quantitatively determine protein texture values in dough images. A Sobel operator was used as an edge detector. After the Sobel operator passed through the image, processing tools computed the percentage of protein texture related pixels within a predefined threshold. Statistical analysis of the protein percentages showed there was no significant difference in protein texture between 63 developed, shear strained, and extensionally strained wheat dough. There was however, a difference between developed wheat dough and undeveloped wheat dough. Although it was difficult to select and generate consistent images in the microsc0pe, numerical image analysis is an excellent tool for investigating the physical structure of wheat dough. Based on the results of this study, the following conclusions are drawn: 1. Shear and extensional deformation can be controlled using undeveloped dough as the starting point for induced deformation. Shear deformation was controlled using serrated parallel plates attached to the Haake R8100 rheometer. Samples were sheared until a breaking strain was obtained. Biaxial deformation was also controlled using the rheometer by placing samples between parallel plates and squeezing them to a predetermined extensional strain. Both techniques to control deformation, and further investigate the influence Of deformation on dough development, were successful. 2. Scanning electron micrographs provide meaningful information about the nature of dough deformation, and can be used to investigate the structural development of a wheat dough samples. The best method (one that minimizes artifacts and maintains sample integrity) to prepare samples for microscopic analysis involves soaking samples in sequential graded ethanol solutions, critical point drying, and sputter coating with gold. Images prepared in this way revealed a clear network of starch and protein in wheat dough samples. q 3. 64 Numerical digital image analysis is a unique tool to determine image texture related to protein development; however, it cannot serve as the only analytical tool in a controlled deformation experiment. The electron micrOSCOpe is a sensitive instrument that may cause grainy and dark images, due to contamination. Poor images make edge detection difficult using the Sobel operator and subsequently, to calculate the percentage of foreground pixels. Further investigations in this area are necessary. The oscillatory behavior of developed, shear strained, extensionally strained, and undeveloped wheat dough showed the rheological properties of each were statistically different. Fully developed dough exhibited the greatest moduli followed by extensionally strained and shear strained dough samples, respectively. UndeveIOped dough exhibited the lowest moduli because protein development was smaller compared to the other deformation treatments. CHAPTER VI RECOMMENDATIONS FOR FUTURE RESEARCH The following recommendations are for further research and potential industry applications: 1. To better distinguish starch and protein, scanning electron micrographs should be compared to laser scanning confocal micrographs of samples containing a protein specific stain. This would clearly distinguish starch from protein and ensure more accurate protein texture values in numerical image analysis. 2. Apply information regarding the structural characteristics of a partially strained dough to optimize engineering design. The protein development of dough as influenced by mixing or sheeting, could be investigated by taking samples at one consistent location to determine Optimal protein development, specific to a process or product. 3. Compare baked product prOperties of shear and extensional deformed dough samples. 65 66 . Compare protein development in wheat dough subjected to the same amount of shear and extensional strain. . Compare wheat dough samples subjected to a combination of shear and extensional deformation. Samples could be deformed in an extensional flow field and then deformed in a shear flow field. Compare wheat dough samples strained to various levels of shear and extensional deformation. Study the effects of temperature on partial dough development. Once an Optimum temperature is established, prepare products of controlled deformation to determine baking quality. . Investigate shear stress effects on samples subjected to shear deformation, as well as the effect of squeezing flow rate on samples subjected to extensional deformation. . Develop more sophisticated image analysis algorithms allowing better distinction between large round starch particles and protein webbing. APPENDICES APPENDIX A 67 Figure A. 1. Developed soft white winter wheat dough. .:w=o: 00:3 :0:E3 0:33 :00 302009 .N.< Rama Figure A.3. Undeveloped soft white winter wheat dough. Figure A.4. Undeveloped soft white winter wheat dough. .:w=o: 00:3 02:3 :0: ::0: :0mo_0>0D .m.< 0:33.: .:m=o: 00:3 02:3 :0: ::0: :0020>0Q .0.< Emmi .:w=o: 00:3 .883 :0: ::0: :0mo_0>0:=D .04 05%.: Figure A.8. Undeveloped hard red winter wheat dough. 00:08:80: 00:0 8 “0080.330 :mao: 000:3 0:53 8:3 com .04 0::mE .:o:0E:80: 50:0 8 :080 .330 :wso: 00:3 0:83 02:3 80m .0: .< 0::mE J’fiv-‘l‘ 2. ' ' A < ”A I .. _V"*‘1,. y r 4.; - ‘ o‘ , a: "ké..-__~R.*).iz' ‘9. {VI-V i2 \ ‘r 5‘ A.4 Figure A.11. Hard red winter wheat dough subjected to shear deformation. Figure A. 12. Hard red winter wheat dough subjected to shear deformation. Figure A. 1 3. Soft white winter wheat dough subjected to extensional deformation. Figure A. 14. Soft white winter wheat dough subjected to extensional deformation. .coumgomov Esau—~85 8 @300 33m 53% “8:3 “853 c8 Bum .m H .< oSwE dons—58% 3:230pr 8 880.33 :mzoc 80:3 BEE um: BE .2 .< oezmfi It P1 ..., A .1..." .. N. APPENDIX B 84 85 Table B.1 Protein texture values from numerical digital image analysis of developed soft white winter wheat dough. sample total PTV PTV% 26 403456 144392 35.79 pixels pixels 27 403456 1 34054 33.23 1 W‘ 26 403456 135320 33.54 2 403456 139669 34.67 29 403456 165970 46.09 3 403456 121 153 30.03 30 403456 162505 40.26 4 403456 1 17627 29.15 31 403456 139832 34.66 5 403456 1 12164 27.60 32 403456 163366 40.50 6 403456 167579 41.54 33 403456 152965 37.91 7 403456 144660 35.66 34 403456 131659 32.63 6 403456 165976 41.14 35 403456 166366 41.74 9 403456 107596 26.67 36 403456 166426 41.75 10 403456 124453 30.65 37 403456 143637 35.60 11 403456 172144 42.67 36 403456 131901 32.69 12 403456 156510 36.79 39 403456 166743 41.33 13 403456 126792 31.43 40 403456 139052 34.47 14 403456 125437 31.09 41 403456 135552 33.60 15 403456 135266 33.53 42 403456 120322 29.62 16 403456 116356 29.34 43 403456 134265 33.26 17 403456 106023 26.77 44 403456 124451 30.85 16 403456 126630 31.93 45 403456 127474 31.60 19 403456 133216 33.02 46 403456 161670 45.03 20 403456 147153 36.47 47 403456 155532 36.55 21 403456 115473 28.62 46 403456 131972 32.71 22 403456 142027 35.20 49 403456 226302 56.59 23 403456 156032 36.67 50 403456 156001 39.16 24 403456 1 19660 29.66 A VERAGE 35.34 25 403456 145929 36.17 STAND. DEV. 5.68 86 Table B.2 Protein texture values from numerical digital image analysis of soft white winter wheat dough subjected to shear deformation. total PTV PTV% 41 403456 123801 30.69 pixels pixels 42 403456 128195 31.77 . 43 403456 109415 27.12 403456 142062 35.21 44 403456 113107 28.03 403456 121699 30.16 45 403456 111095 27.54 403456 156292 38.74 46 403456 132852 32.93 403456 132406 32.82 47 403456 128374 31.82 403456 141616 35.10 48 403456 141002 34.95 403456 126182 31.28 49 403456 100956 25.02 403456 158224 39.22 50 403456 124345 30.82 403456 131468 32.59 51 403456 112214 27.81 403456 99076 24.56 52 403456 122807 30.44 403456 133236 33.02 53 403456 123339 30.57 403456 146851 36.40 54 403456 143740 35.63 403456 143733 35.63 55 403456 134957 33.45 403456 126369 31.32 56 403456 83553 20.71 403456 127848 31.69 57 403456 110707 27.44 403456 108493 26.89 58 403456 133187 33.01 403456 158228 39.22 59 403456 107335 26.60 403456 152979 37.92 60 403456 124674 30.90 403456 147694 36.61 61 403456 98177 24.33 403456 136136 33.74 62 403456 115558 28.64 403456 165908 41.12 63 403456 113194 28.06 403456 1 13063 28.02 64 403456 130242 32.28 403456 174353 43.21 65 403456 148405 36.78 403456 179822 44. 57 66 403456 138217 34.26 403456 151628 37.58 67 403456 165220 40.95 403456 134296 33.29 68 403456 160395 39.76 403456 139031 34.46 69 403456 113041 28.02 403456 111265 27.58 70 403456 127012 31.48 403456 113180 28.05 71 403456 133697 33.14 403456 124862 30.95 72 403456 115358 28.59 403456 146825 36.39 73 403456 132843 32.93 403456 151936 37.66 74 403456 148900 36.91 403456 121865 30.21 75 403456 112534 27.89 403456 105036 26.03 76 403456 129654 32.14 403456 117936 29.23 77 403456 142976 35.44 403456 134951 33.45 78 403456 119359 29.58 403456 140120 34.73 79 403456 118655 29.41 403456 138151 34.24 80 403456 124348 30.82 403456 135406 33.56 81 403456 135142 33.50 403456 104153 25.82 82 403456 128441 31.84 Table B.2 (Cont’d.) 83 403456 84 403456 85 403456 86 403456 87 403456 88 403456 89 403456 90 403456 91 403456 92 403456 93 403456 94 403456 95 403456 96 403456 97 403456 98 403456 136630 120828 100920 116799 105841 115169 138948 145195 118412 130242 100816 152583 173769 151287 136642 125167 .AUEHEAGE? STAND. DEV. 1K186 29£K5 25£H 28£K5 2Ei23 281fi5 3444 1N399 291%5 3228 24£K9 13182 43Lfl' 13150 II387 3102 .3234 4464 87 88 Table B.3 Protein texture values from numerical digital image analysis of soft white winter wheat dough subjected to extensional deformation. sample total PTV PTV% 26 403456 129277 32.04 pixels pixels 27 403456 86022 21 .32 1 4m 26 403456 122670 30.40 2 403456 65665 16.28 29 403456 109449 27.13 3 403456 48314 1 1.98 30 403456 128166 31.77 4 403456 81605 20.23 31 403456 116615 28.90 5 403456 97521 24.17 32 403456 109736 27.20 6 403456 125722 31.16 33 403456 110907 27.49 7 403456 111174 27.56 34 403456 117495 29.12 8 403456 134882 33.43 35 403456 97815 24.24 9 403456 1 18930 29.48 36 403456 108739 26.95 10 403456 145726 36.12 37 403456 129087 32.00 1 1 403456 133704 33.14 38 403456 134380 33.31 12 403456 143437 35.55 39 403456 129369 32.07 13 403456 125338 31.07 40 403456 134500 33.34 14 403456 155491 38.54 41 403456 99584 24.68 15 403456 90495 22.43 42 403456 120772 29.93 16 403456 86928 21.55 43 403456 98890 24.51 17 403456 78860 19.55 44 403456 91123 22.59 18 403456 100418 24.89 45 403456 87410 21.67 19 403456 148069 36.70 46 403456 150898 37.40 20 403456 95615 23.70 47 403456 108570 26.91 21 403456 84639 20.98 48 403456 132682 32.89 22 403456 71556 17.74 49 403456 223384 55.37 23 403456 97519 24.17 50 403456 127492 31.60 24 403456 1 10551 27.40 A VERA GE 28.00 25 403456 93746 23.24 STAND. DEV. 7.00 89 Table B.4 Protein texture values from numerical digital image analysis of undeveloped soft white winter wheat dough. sample total PTV PTV% 26 403456 76041 18.85 pixels pixels 27 403456 95217 23.60 1 W‘ 28 403456 104788 25.97 2 403456 95453 23.66 29 403456 128852 31.94 3 403456 91517 22.68 30 403456 92532 22.93 4 403456 80765 20.02 31 403456 86989 21.56 5 403456 73658 18.26 32 403456 103796 25.73 6 403456 106772 26.46 33 403456 80407 19.93 7 403456 90049 22. 32 34 403456 1 05044 26.04 8 403456 95025 23.55 35 403456 71191 17.65 9 403456 94308 23.38 36 403456 136535 33.84 10 403456 95942 23.78 37 403456 131620 32.62 1 1 403456 1 13000 28.01 38 403456 108568 26.91 12 403456 104627 25.93 39 403456 82930 20.55 13 403456 98809 24.49 40 403456 125763 31.17 14 403456 89976 22.30 41 403456 82301 20.40 15 403456 95574 23.69 42 403456 89935 22.29 16 403456 95838 23.75 43 403456 85644 21.23 17 403456 98640 24.45 44 403456 74064 18.36 18 403456 85733 21.25 45 403456 69967 17. 34 19 403456 64807 16.06 46 403456 97593 24.19 20 403456 86147 21.35 47 403456 102667 25.45 21 403456 92070 22.82 48 403456 107446 26.63 22 403456 80840 20.04 49 403456 94357 23.39 23 403456 78393 19.43 50 403456 83510 20.70 24 403456 107395 26.62 A VERA GE 23.37 25 403456 87433 21.67 STAND. DEV. 3.83 Table B.5 Protein texture values from numerical digital image analysis of developed hard red winter wheat dough. 90 sample NNNNNN—L—l—LA—A—l—L—A-A-h cn-b 0360-4 cauacn \toacn.h 0353.4 c,“>CD-fl O>cn.> 0)hJ.a total PTV PTV% pixels pixels 403456—T3813? 34:24 403456 143215 35.50 403456 1 52999 37.92 403456 141388 35.04 403456 157483 39.03 403456 1 18539 29.38 403456 128313 31.80 403456 130482 32.34 403456 151 147 37.46 403456 171 128 42.42 403456 128592 31.87 403456 97980 24.29 403456 1 19763 29.68 403456 101348 25.12 403456 1 14997 28.50 403456 136236 33.77 403456 1 10278 27.33 403456 118435 29.36 403456 128019 31.73 403456 105884 26.24 403456 96898 24.02 403456 92944 23.04 403456 124935 30.97 403456 1 14629 28.41 403456 122002 30.24 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 45 46 47 48 49 403456 91277 403456 83344 403456 116794 403456 129992 403456 156137 403456 139989 403456 109067 403456 108492 403456 151064 403456 111161 403456 109814 403456 93750 403456 152104 403456 137811 403456 137730 403456 116675 403456 143129 403456 136372 403456 149426 403456 148541 403456 122821 403456 166149 403456 189729 403456 157922 .AVEHZAGHE JSIAAMIIJEVC 22.62 20.66 28.95 32.22 38.70 34.70 27.03 26.89 37.44 27.55 27.22 23.24 37.70 34.16 34.14 28.92 35.48 33.80 37.04 36.82 30.44 41.18 47.03 39.14 31.89 5.71 91 Table B.6 Protein texture values from numerical digital image analysis of hard red winter wheat dough subjected to shear deformation. sample total PTV PTV% 26 403456 109009 27.02 pixels pixels 27 403456 134519 33.34 1 403456 146524 3631" 26 403456 1 13406 26.1 1 2 403456 1 10260 27.33 29 403456 1 19294 29.57 3 403456 154156 38.21 30 403456 184823 45.81 4 403456 154108 38.20 31 403456 157956 39.15 5 403456 154842 38.38 32 403456 150587 37.32 6 403456 147242 36.50 33 403456 18881 1 46.80 7 403456 129641 32.13 34 403456 142912 35.42 8 403456 130404 32.32 35 403456 109382 27.1 1 9 403456 1 13159 28.05 36 403456 120585 29.89 10 403456 156843 38.87 37 403456 1 19140 29.53 1 1 403456 92728 22.98 38 403456 1 14959 28.49 12 403456 97160 24.08 39 403456 99145 24.57 13 403456 132897 32.94 40 403456 129947 32.21 14 403456 127329 31.56 41 403456 92728 22.98 15 403456 144951 35.93 42 403456 97160 24.08 16 403456 141596 35.10 43 403456 132897 32.94 17 403456 122635 30.40 44 403456 127329 31.56 18 403456 133091 32.99 45 403456 144951 35.93 19 403456 90793 22.50 46 403456 141596 35.10 20 403456 131216 32.52 47 403456 122635 30.40 21 403456 168055 41.65 48 403456 133091 32.99 22 403456 164451 40.76 49 403456 90793 22.50 23 403456 152258 37.74 50 403456 131216 32.52 24 403456 140429 34.81 A VERAGE 32. 62 25 403456 132221 32.77 STAND. DEV. 5.74 92 Table B.7 Protein texture values from numerical digital image analysis of hard red winter wheat dough subjected to extensional deformation. sample total PTV PTV% 26 403456 1 13302 28.08 pixels pixels 27 403456 1 15606 28.65 1 7103256 133451 3308" 28 403456 106845 26.48 2 403456 151252 37.49 29 403456 123174 30.53 3 403456 129732 32.16 30 403456 128801 31.92 4 403456 142214 35.25 31 403456 180868 44.83 5 403456 144676 35.86 32 403456 161654 40.07 6 403456 131427 32.58 33 403456 137868 34.17 7 403456 153798 38.12 34 403456 187442 46.46 8 403456 137604 34.1 1 35 403456 176604 43.77 9 403456 135925 33.69 36 403456 1 19893 29.72 10 403456 131 158 32.51 37 403456 128074 31.74 1 1 403456 13051 1 32.35 38 403456 123961 30.72 12 403456 1 17649 29.16 39 403456 118039 29.26 1 3 403456 1 19887 29.72 40 403456 124029 30.74 14 403456 129875 32.19 41 403456 158693 39.33 15 403456 137830 34.16 42 403456 128519 31.85 16 403456 132660 32.88 43 403456 144064 35.71 17 403456 131870 32.69 44 403456 147304 36.51 18 403456 136743 33.89 45 403456 134639 33.37 19 403456 135460 33.57 46 403456 71848 17.81 20 403456 192075 47.61 47 403456 98900 24.51 21 403456 124533 30.87 , 48 403456 56701 14.05 22 403456 146641 36.35 49 403456 59527 14.75 23 403456 163126 40.43 50 403456 63966 15.85 24 403456 164000 40.65 A VERA GE 32.82 25 403456 155328 38.50 STAND. DEV. 7.04 93 Table B.8 Protein texture values from numerical digital image analysis of undeve10ped hard red winter wheat dough. sample total PTV PTV% 26 403456 60908 15.10 pixels pixels 27 403456 59667 14.79 1 403456 4622T 11.46 28 403456 56609 14.03 2 403456 67586 16.75 29 403456 56683 14.05 3 403456 1 14363 28.35 30 403456 64051 15.88 4 403456 64216 15.92 31 403456 63696 15.79 5 403456 76103 18.86 32 403456 1 13421 28.1 1 6 403456 1 19868 29.71 33 403456 60122 14.90 7 403456 122554 30. 38 34 403456 74615 18.49 8 403456 84255 20.88 35 403456 58479 14.49 9 403456 101249 25.10 36 403456 95685 23.72 1 0 403456 99534 24.67 37 403456 56220 13.93 1 1 403456 166554 41.28 38 403456 78019 19.34 12 403456 124209 30.79 39 403456 106783 26.47 1 3 403456 101660 25.20 40 403456 90574 22.45 14 403456 92553 22.94 41 403456 45018 1 1.16 15 403456 56791 14.08 42 403456 59716 14.80 16 403456 73254 18.16 43 403456 43599 10.81 17 403456 24076 5.97 44 403456 44227 10.96 18 403456 99564 24.68 45 403456 63967 15.85 19 403456 51092 12.66 46 403456 77766 19.27 20 403456 73204 18.14 47 403456 82870 20.54 21 403456 56262 13.95 48 403456 84229 20.88 22 403456 79009 19. 58 49 403456 61782 15.31 23 403456 53755 13.32 50 403456 62581 15.51 24 403456 60724 15.05 A VERAGE 18. 76 25 403456 54391 13.48 STAND. 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