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vi DISCUSSION CONCLUSION Recommendations for Further Study REFERENCES 60 61
vii LIST OF TABLES 1. Buckwheat Production (Acreage) Records 1997-2002 2. X-ray Diffraction Results for Heated and Unheated Buckwheat Starch at Various Moisture Levels 3. Differential Scanning Calorimeter Results 4. Amylose Leaching Results
viii LIST OF FIGURES 1. Diagram of a Buckwheat Groat/Achene 2. Starch Structure and Amylose and Amylopectin Formations 3. X-ray Diffraction Reading for Unheated Buckwheat Starch 4. X-ray Diffraction Reading for Heated Buckwheat Starch 5. X-ray Diffraction Reading for Unheated and Heated 13.2% Moisture Level Buckwheat Starch 6. X-ray Diffraction Reading for Unheated and Heated 26.8% Moisture Level Buckwheat Starch 7. X-ray Diffraction Reading for Unheated and Heated 32.3% Moisture Level Buckwheat Starch 8. X-ray Diffraction Reading for Unheated and Heated 40.0% Moisture Level Buckwheat Starch 9. X-ray Diffraction Reading for Unheated and Heated 44.4% Moisture Level Buckwheat Starch 10. Representative DSC Scan of 32.3% Moisture Level Buckwheat Starch 11. Representative DSC Scan of 40.0% Moisture Level Buckwheat Starch 12. Representative DSC Scan of 44.4% Moisture Level Buckwheat Starch 13. Standard Amylose Leaching Curve
1 CHAPTER I INTRODUCTION Introduction Buckwheat (Fagopyrum esculentum Moench) is a non-glutinous pseudocereal that is consumed mainly in China, Japan, and Eastern Europe, but could be profitable in the United States if new uses were found for buckwheat products (Edwardson, 1996). It has a starch composition similar to cereals, but has higher amounts of amino acids lysine, methionine, and cystine which is more typical of legumes (Qian, Rayas-Duarte, & Grant, 1998; Zheng, Sosulski, & Tyler, 1998). In order to learn more about processing buckwheat into consumer products, it is important to find out how its major components such as starch react to different processing techniques. Most processing techniques involve the use of heat and moisture. The effects of several heat and/or moisture processing techniques, such as boiling, baking in bread, and dry-heat, on buckwheat starch composition and characteristics have been studied (Skrabanja, Elmsthl, Kreft, & Bjrck, 2001; Skrabanja, Laerke, & Kreft, 1998). One area that has yet to be studied is the effect of microwave annealing and heat-moisture treatments on buckwheat starch properties. Annealing is a heat moisture process that uses treatment of starch at intermediate or excess moisture (40% moisture content and above) at a temperature below the gelatinization temperature (Jacobs & Delcour, 1998). The theory behind annealing is that it could cause changes in the molecular structures within the starch, creating structures that are more resistant to gelatinization (Stute,

4 amount of amylose that has leached out of a granule after excess heat and moisture have been supplied. Two-way analysis of variance was used to determine the influence that microwave heat-moisture and annealing treatments had on starch crystalline pattern, starch granule melting characteristics, and amylose leaching. Tests were repeated to enhance statistical significance. Data was analyzed using an SPSS 11.0 for Windows statistical analysis program.
Objectives 1. The first objective was to isolate buckwheat starch from buckwheat fancy flour (Minn-Dak Growers Ltd., Fargo, ND) and dry it to different moisture contents. 2. The second objective was to determine the temperature at which to heat the buckwheat in the microwave using a differential scanning calorimeter. 3. The third objective was to construct and conduct heat-moisture and annealing heating regimens in the microwave using the resources obtained from objectives one and two. 4. The fourth objective was to study the heat-moisture treated and annealed starch using the differential scanning calorimeter, the X-ray diffractometer, and an amylose leaching colorimetric method in order to determine whether starches resistant to
5 further heat and moisture were formed with annealing and heatmoisture treatment.
Use of Findings Annealing and heat-moisture treatment are hydrothermal (heat and water) treatments that could have significant effects on the properties of the buckwheat starch. Microwave technology allows for faster heating of food items, decreasing the amount of time needed to process the food. The results of this experiment could help to: 1. Build knowledge of buckwheat starch behavior and its interaction with different heat/moisture processes 2. Establish new procedures for using microwave dielectric technology for annealing and heat-moisture treatments to create modified starches. 3. Encourage further study into the development of new products from buckwheat starch using the findings of this study.
Buckwheat: From Pseudocereal Food Source to Neutraceutical Buckwheat (Fagopryum esculentum) is derived from the Anglo-Saxon boc (beech) and whoet (wheat) because it resembles the beech nut (Edwardson, 1996). However, buckwheat is neither a nut nor a cereal like wheat, but rather a pseudocereal whose history dates back over 1000 years. Cereals at their most basic structure are one-seeded fruits containing a small embryonic germ and a larger, starchy endosperm surrounded by an outer aleurone layer and a hull (Hoseney, 1994). Like cereals, the seed of the buckwheat plant contains a germ, endosperm, aleurone layer, and a hull. However, buckwheat is not a part of the cereal or grain family (Gramineae) but rather comes from the same family as rhubarb (Polygonaceae) (Hoseney, 1994; Saeger & Dyck, 2001). Buckwheat can grow to be anywhere from two to five feet and produces white or pink blossoms with five petals (Saeger & Dyck, 2001). Buckwheat can be divided into groups of species: annual and multiennal (Li & Zhang, 2001). The buckwheat used for this experiment is of the annual species Fagopyrum esculentum Moench. Although it contains the same tissue components as cereals, buckwheat has different tissue features. Buckwheat is a dicotyledon as are peas and beans, while grains like wheat and corn are monocots (Starr, 2000). These different features are visible for monocots and dicots in the actual appearance of the plants as well as the way in which they grow after germination. Dicotyledons contain

7 two cotyledons or seed leaves which store and absorb food for the plant during germination and primary growth. Monocotyledons contain only a single cotyledon. The foliage of dicotyledons contains netlike vascularization whereas the foliage of a monocot contains parallel veining. The vascular structures of dicotyledons are organized in a ring-like structure in the stem whereas the vascular structures of a monocot are dispersed in the stem. The buckwheat grain consists of a triangular seed with two cotyledons running through the endosperm and surrounding it - see Figure 1 (Steadman, Burgoon, Lewis, Edwardson, & Obendorf, 2001).
Figure 1: Diagram of a Buckwheat Groat/Achene Reprinted from Journal of Cereal Science, 33, Steadman, K.J., Burgoon, M. S., Lewis, B. A., Edwardson, S. E., & Obendorf, R. L, Buckwheat seed milling fractions: description, macronutrient composition and dietary fibre, 271-278, 2001, with permission from Elsevier Science. When studying cereals, it is also important to consider their internal composition. Most grains contain 60-75% carbohydrate, 8-16% protein, and varying levels of lipid, although most contain between 2-3% (Hoseney, 1994). In a study by Zheng, Sosulski, and Tyler (1998) dehulled buckwheat groats were found to contain 75% starch, 13.9% protein, and 2.3% lipid. An estimate of the whole groat by Steadman et al. (2001) stated that groat starch contained 55% starch, 12% protein, and 4% lipid. Most of the protein and lipid were found in the bran and embryo tissue. Unlike wheat and other cereals, buckwheat does not
9 contain gluten, a protein used in building volume in breads; however, this may be advantageous for people with celiac disease who are intolerant to a component of gluten and therefore must avoid items with gluten in them (Saeger & Dyck, 2001). In the study by Zheng et al. (1998) the amino acid profile of buckwheat was found to be different from grains and similar to that of other dicotyledons such as soybeans with higher amounts of lysine, methionine, cystine, arginine, and aspartic acid. Steadman et al. also found that buckwheat groats contained about 7.0 g/100 g DW total dietary fiber; of which 2.2 g/100 g DW was insoluble and 4.8 g/100 g DW was soluble. The total dietary fiber content and soluble fiber content were similar to oats. As with grains, in order for buckwheat to be used as a food product, it must first be milled. In the most basic milling process, the outer hull is removed from the seed to produce a groat. The hulls of the buckwheat can be sold for special pillows (Pomeranz, 1983). The groat can then be ground further into several fractions with varying levels of the aleurone layer remaining (Minn-Dak Growers, Ltd., 1999). Coarsely ground groats are called grits and can be used for porridges or in breads. Roasted groats (kasha) are used in Eastern European ethnic dishes (Minn-Dak Growers, Ltd., 1999; Vinning, 2001). Buckwheat flour made from the aleurone layer of the groats is called Farinetta and can be used in breads, bakery products, and pancakes (Minn-Dak Growers, Ltd., 1999). Flour made from the entire buckwheat groat (Supreme flour) can be used in breads, bakery products, extruded snacks, pancakes, and pasta. Fancy flour made from

22 Several studies have used amylose leaching to determine the stability of starches that have been heat and moisture treated. In two studies Hoover and Vasanthan (1994a; 1994b) used a modified method of Chrastil (1987) to determine the extent of amylose leaching of cereal, legume, and tuber starches which had been heat-moisture treated and annealed. They found that annealing starches caused a marked decrease in amylose leaching, particularly in starches with high amounts of amylose such as lentils and oats. They also found that heatmoisture treatment caused a marked decrease in amylose leaching but more so in tuber and legume starches than cereal starches. Other components of a cereal/legume/tuber also interact with starch, affecting the susceptibility of the granule to gelatinization. These interactions are visible on DSC endotherms (readings). In a study by Szczodrak and Pomeranz (1992) starch-lipid interaction in high-amylose (43-49%) barley caused an increase in initial DSC readings from 58-85C to 89-110C. Complexation of amylose starch with lipids was also found to prevent amylose-amylose interaction. Liu, Arntfield, Holley, and Aime (1997) found similar findings with pea starch. Lipids are able to complex with amylose by hiding within the helical complexes formed by amylose. Starch granules are also known to interact with proteins. In a study by Eliasson and Tjerneld (1990) wheat proteins were found to adsorb unto wheat starch granules, potato starch, and maize starch. Adsorption increased with initial increased starch temperature due possibly to formation of starch gels or changes in the nature of the granule surface. Fardet, Abecassis, Hoebler, Baldwin,
23 Bulon, Brot, and Barry (1999) in their study of protein and starch interactions in pasta products found that starch became entrapped in protein networks which rendered them less accessible to water. Fornal, Smietana, Soral-Smietana, Fornal, and Szpendowski (1985) in their research of buckwheat starch granule interaction with proteins and lipids in an extrusion process with milk proteins found that the protein and lipids did interact with the starch. Addition of milk protein and extrusion temperature increased the degree of gelatinization and decreased the swelling power of the starch granule. Starch-lipid formations did take place but were greatest at lower extrusion temperatures (100C). In order to fully understand a starch, it is important to study its interaction with common processing factors such as heat and moisture. Heat and moisture treatments can have effects on characteristics that relate to digestion and stability under adverse storage conditions. It is also important to look at the manner in which heat and moisture treatments are elicited as more efficient processes, such as microwave technology, are being used to process foods in less time than conventional oven heating methods.

Microwave Technology Unlike ovens which rely on conduction (transfer of energy from metal or food molecule to food molecule) and convection methods (transfer of energy from liquid or air to food molecule) to heat food, microwaves heat food using dielectric energy (Fellows, 2000). Dielectric energy affects food components that contain
24 positive and negative poles (dipoles), particularly water, and a common component of food. Microwaves are able to create an environment where a moving electrical field is created, which causes the dipolar molecules to continually turn back and forth, creating frictional heat. Heating depends on distribution of water and other dipolar molecules such as salt. Unlike with conduction and convection methods the surface of the food is less warm than within the food due to evaporative loss of water. The temperature just below the surface, however, is much warmer and from there heat is conducted to the center of the food (Buffler, 1992). Microwave ovens use a magnetron which produces electrons that are sent through a waveguide and scattered in the heating chamber where they contact food items (Fellows, 2000). Magnetrons provide bursts of energy at variable powers (load) for variable lengths of time (time base) and create fields that move from top to bottom, side to side, and front to back in the heating compartment (Buffler, 1992). To prevent microwaves from concentrating in only a few areas of the food, creating hot or cold spots, most microwave systems are equipped with stirrers or turntables to produce an even exposure of the food item to the microwaves. Sensors are also used, though they may be inaccurate up to 8F (3C). Microwaves are best used for thawing, tempering, dehydrating, and baking, but not blanching or pasteurization (Fellows, 2000). Microwave ovens, like any other heating equipment, work on the concept of power, rate at which work is done or, in other words, the rate at which energy is expended or utilized (Buffler, 1992). Many factors may affect the
25 power supplied to the food including the load and time base. These factors are influenced by the temperature and the power level at which the microwave is set. The shape of the food may also have an effect on the power supplied to it as items that are flat or square experience more corner heating than oval or circular foods. Individual food dielectric constants also play a part in the amount of power absorbed by the food (Miller, Gordon, & Davis, 1991). Dielectric constants look at the interaction between the material being heated and the microwave energy (K'), as well as its ability to dissipate energy as heat (K''). These constants are affected by the charge of the components of the food, the environment in which it is in, and the presence of water in the food. Several researchers have investigated the effect of microwave energy on starch properties. Khan, Johnson, and Robinson (1979) studied the effect of water content and heating time in a microwave oven on the degradation of wheat starch flour. They found that water had a direct relationship with sugar production in that sugar production increased with increased starch hydration. Heating time also had a direct effect up to a point with total soluble sugar increasing except at high water and heating time where total soluble sugar was reduced due to sugar destruction. Glucose concentrations also increased with increased amounts of water and heating time. Sumnu, Ndife, & Bayindirli (1999) studied the effect of water, sugar, and protein on starch gelatinization in wheat starch that was microwaved. They found that wheat starch gelatinized even before applying heat at a 2:1 (w/w, water:starch) concentration. Of the three components, sugar had

26 the most significant effect on starch gelatinization and significantly interacted with protein and water to prohibit gelatinization. Zylema, Grider, Gordon, and Davis (1985) compared the effect of microwave dielectric heating and conduction/convection heating in an oil bath on heating rate (up to 65C and 85C), microstructure, and swelling of wheat starch systems with 1:2 to 1:8 starch-to-water ratios. They found that heating time did not vary between the two types of heating but microwave heating did result in more uniform gelatinization at both the 65C and 85C temperatures in 1:2, 1:4, and 1:8 starch systems. In microwave 1:1 and oil bath 1:1-1:4 systems chalky regions were formed where the granules were not as swollen as in the gelled regions. In microwave 1:4 and 1:8 and oil bath 1:8 starch systems watery regions also formed in which granules swelled similar to those found in the gelled regions. Water concentration was found to play a great role in helping to distribute heat transfer by increasing microwave coupling with the starch and helping to conduct heat throughout the starch. The effect of convection and microwave heat methods on wheat granule swelling was also studied by Goebel, Grider, Davis, & Gordon (1984). Varying levels of water:starch concentrations from 1:1 (w/w) starch:water to 5:95 were heated to 75C using the 177F convection and low/medium microwave mode of a convection/microwave oven. The researchers found that heating in both applications was uneven, forming distinct regions that were described as gel, chalky, watery gel, chalky gel, soft gel, paste, watery paste, and chalky paste. Studying the different regions under a scanning electron microscope and
27 light/polarized light microscope the researchers found that with increased water ratios there was higher swelling, and, looking at both convection and microwave modes, starch from chalky regions of samples heated using the convection mode had higher starch swelling than those heated using the microwave mode. Except for the 1:4 water: starch ratio level, little difference was noted between samples heated at low and medium microwave mode. Differences between convection and microwave mode heated samples decreased as water:starch ratio increased. The researchers stated that the advanced swelling in higher water:starch ratio samples was probably due to the longer heating period that these samples had as noted by the longer periods of time it took higher water:starch ratio samples to heat to 75C. Yiu, Weisz, and Wood (1991) compared microwave heating of regular and quick-cooking oats to that of conventional boiled oats. Both samples were hydrated to a 1:8 starch-to-water ratio and were kept at temperatures between 9095C for 1 minute and 20 minutes. The researchers found, when studying the starch samples from the different cooking techniques that oat starch granules remained intact even after 20 minutes heating while those of boiled oatmeal fragmented. However, this was attributed to the boiled oatmeal being stirred more. Although microwave technology usage with heat and moisture treatment of some types of starch had been explored, the effect of microwave heat/moisture treatment had yet to be studied with buckwheat starch. Buckwheat starch with its

33 time. After the microwaving was completed, samples were immediately capped, wrapped with parafilm wax to prevent moisture loss or gain, and placed in a 25C water bath to prevent further heating. Once cooled, granules were separated by applying a mortar and pestle to the contents of the centrifuge tubes. Visible gelatinized starch granules were removed.
X-ray Diffraction Evaluation of Starch Crystalline Structure X-ray diffraction is a method used to characterize the crystalline structure of a material (Pomeranz and Meloan, 2000). X-rays consist of high energy waves created when a high concentration of electrons hits a heavy target, causing the electrons to penetrate the atoms of the target and give off high energy waves. These waves then penetrate a sample such as a starch granule where they are diffracted by crystalline layers. The spacing of the crystalline layers may be examined by the distance (d) between the wavelengths that are diffracted. The intensity of the d-spacing peaks relates to the concentration of the crystalline phase within the starch granule (Cullity, 1978). X-ray diffraction was performed on a Scintag PAD-X Advanced Diffraction System X1 (Thermo ARL, Waltham, MA). A small amount of buckwheat starch powder was placed in a plastic x-ray sample holder and flattened with a piece of glass in order to entirely fill the holder and to make the sample level with the edges of the holder to reduce scanning errors. The buckwheat was scanned through the 2 range of 0-40 using MDI Data Scan 3.2 software (Livermore, CA). The angles used were similar to those described in
34 Hoover and Vasanthan (1994a) and are typical for x-ray diffraction starch analyses. D-spacing and intensities were examined for the samples using MDI Jade 6.5 software (Livermore, CA) which contained a manual cursor function that gave d-spacing and intensity data at selected points. For this procedure a starch sample with 13.247 0.041% moisture and a sample with 26.809 0.331% moisture were created in order to have a more complete view of the effect of moisture level and heat treatment on x-ray diffraction analyses of buckwheat starch. Preparations were similar to previous air temperature drying with 30 grams of buckwheat starch from the lowest and highest moisture level starches placed in evaporation dishes at ambient temperature for approximately 24 hours. New moisture levels were determined as previously described.

40 different graphs were presented by the x-ray diffraction machine. Table 2 reports the d-spacing angles at which the crystalline layer in the starch refracted the x-ray and intensities for the two major peaks found on each graph. Most graphs peaked at 3.8 and 5.0 with intensities increasing with less moisture for unheated samples (Figure 3), but less so with heated samples (Figure 4). Within each moisture level changes in intensity were not seen with heating except for starch samples with moisture levels 40.0% and 44.4% (Figures 8 and 9).
Figure 3. X-ray Diffraction Reading for Unheated Buckwheat Starch Moisture levels: a - 13.2%, b - 26.8%, c 32.3%, d 40.0%, e. 44.4% (2)
Figure 4. X-ray Diffraction Reading for Heated Buckwheat Starch Moisture levels: a 13.2%, b 26.8%, c 32.3%, d 40.0%, e. 44.4%
Figure 5. X-ray Diffraction Reading for Unheated and Heated 13.2% Moisture Level Buckwheat Starch
Figure 6. X-ray Diffraction Reading for Unheated and Heated 26.8% Moisture Level Buckwheat Starch
Figure 7. X-ray Diffraction Reading for Unheated and Heated 32.3% Moisture Level Buckwheat Starch
Figure 8. X-ray Diffraction Reading for Unheated and Heated 40.0% Moisture Level Buckwheat Starch
Figure 9. X-ray Diffraction Reading for Unheated and Heated 44.4% Moisture Level Buckwheat Starch
Table 2. X-ray Diffraction Results for Heated and Unheated Buckwheat Starch at Various Moisture Levels Second Second Moisture Microwave First Peak First Peak Peak Peak DIntensity D-space Level (%) Time Intensity space () (Counts) () (Minutes) (Counts)* 6** 0* 6 3.5.* Reflects the average of two or more readings taken for starches with this treatment. Most starches were only run one time for each treatment group. **Only the one, unheated 44.4% moisture level starch graph with readable peaks was recorded in this table.
Differential Scanning Calorimeter Results Data analysis for differential scanning calorimeter (DSC) readings are shown in Table 3. All data analyses were set at an alpha level of 0.05. In onset temperature a two-way analysis of variance (ANOVA) indicated that moisture level did have a significant effect on mean onset melting temperature F(2, 51) = 6.053, p< 0.01 with a large effect size (Eta = 0.212). According to least significant difference (LSD) analysis, the 44.4% moisture level starch had a significantly higher mean onset temperature than the 32.3% moisture level starch (p < 0.01) while there was no significant difference between the 44.4% and 40.0% moisture level starches (p = 0.072) and the 32% and 40% moisture level starch (p = 0.147). Application of microwave heating did not have a significant effect on

Figure 10. Representative DSC Scan of 32.3% Moisture Level Buckwheat Starch
Figure 11. Representative DSC Scan of 40.0% Moisture Level Buckwheat Starch
Figure 12. Representative DSC Scan of 44.4% Moisture Level Buckwheat Starch
Amylose Leaching Results In order to determine the amylose leaching percentage 0-100% amylose standards were prepared and tested with the same procedure as the treated samples. The resulting graph is shown in Figure 13. Since there was a large deviation from 40-60%, these data points were eliminated. The resulting graph gave an equation of y = 0.573x which was used to determine the percent of amylose that leached out of the starch granules during the test using the absorbance readings from the starch-iodine test.
Figure 13. Standard Amylose Leaching Curve In order to analyze the amylose leaching results two-way ANOVA and independent sample T-tests were performed at an alpha level of 0.05. Results are shown in Table 4. The two-way ANOVA indicated that microwave heating had a significant effect on mean amylose leaching readings F(1, 54) = 10.873, p < 0.01 with a large effect size (Eta = 0.185) and that the interaction between moisture level and microwave heating also had a significant effect on mean amylose readings F(2, 54) = 4.288, p < 0.05 with a large effect size (Eta = 0.152). However, moisture level alone did not have a significant effect on mean amylose readings F(2, 54) = 1.480, p = 0.238 with a medium effect size (Eta = 0.058). In
52 other words, moisture level alone did not affect mean amylose leaching, however it did have a combined effect with microwave heating. Levenes test of equality of error variances showed that there was no significant difference in variances among the different treatment groups F (5, 48) = 1.314, p = 0.274. Since LSD could not be performed to determine the significance of the difference between the different treatment groups, independent sample t-tests were performed. The results of the t-tests indicated that the mean amylose leaching reading for the unheated 44.4% moisture level starch was significantly higher than the heated 44.4% moisture level starch, p < 0.001, and that the unheated 40.0% moisture level starch and all of the 32.3% moisture level starch were significantly higher than the heated 44.4% moisture level starch, p < 0.01. The unheated 44.4% moisture level starch had significantly higher mean amylose leaching than the heated 40.0% moisture level starch, p < 0.01. Differences among the other treatments were not significant at the selected alpha level. This means that mean amylose leaching was lowest for the heated 44.4% moisture level starch, followed by the heated 40.0% moisture level starch, the unheated 40.0% moisture level starch and both treatments of 32.3% moisture level starch, and finally the unheated 44.4% moisture level starch.

53 Table 4. Amylose Leaching Results Moisture Level (%) Microwave Time Amylose Leaching (%) (Minutes) 32.14.25 6.29bc 32.14.35 6.82bc 40.13.66 5.95bc 40.9.43 5.21ab 44.16.89 3.44c 44.6.57 3.51a All data is given as mean and standard deviation. Subscripts within the same column denote significant difference among data of at least p < 0.01. n = 9
54 CHAPTER V DISCUSSION In examining the results for this experiment it is important to note that, for some buckwheat starch characteristics, heat treatment or the interaction of heat treatment and moisture level had a significant effect, while for other characteristics moisture level alone had a significant effect. Three main moisture levels 32.3%, 40.0%, and 44.4% - and two heating options microwave heated or unheated at below the gelatinization temperature - were used to create microwave heat-moisture (32.3%, heated) and annealed (40.0%, 44.4%, heated) samples. These factors, moisture and heat, created six treatment groups which were applied to the buckwheat starch and then used to examine buckwheat starch characteristics. The three tests used in this experiment examined a characteristic which has to do with amylose interactions in the starch granule and characteristics which have to do with the crystalline region of the starch granule (concentration and stability). Results from these tests showed that buckwheat granule structures can be stabilized in some ways using microwave and moisture heat treatment to make it more resistant to breaking apart from further addition of heat and water. X-ray diffraction results were found to be similar to previous x-ray diffraction readings of buckwheat starch (Zheng, Sosulski, & Tyler, 1998). The starch did have a cereal A-type crystallinity with two major d-spacing peaks at 5.0 (~17.7) and 3.8 (~23.4) and one smaller peak that was not recorded but was visible as a shoulder at about 5.7 (~15.4). This did not change with percent moisture or heat treatment (see Figures 3 and 4). In general the intensity of the x-
55 ray diffraction readings increased as moisture level decreased. X-ray intensity also increased with microwave annealing treatment of buckwheat starch with moisture levels of 40.0% and 44.4% (see Figures 8 and 9). Hoover and Vasanthan (1994a; 1994b) found that heat-moisture and annealing treatment of cereal did increase peak intensities without changing dspacing. Stute (1992) found that heat-moisture treatment of B-type crystalline structures caused a change in crystalline structure to A-type and C-type whereas annealing did not cause any crystalline changes. Contrary to some of these experiments heat-moisture treatment did not result in significant changes to intensity (see Figures 5-7) while annealing did (see Figures 8 and 9). Percent moisture, particularly of unheated starch (see Figure 3), also influenced x-ray diffraction readings which could be expected since less water would mean lower swelling in amorphous regions, decreasing concentration of amorphous regions and increasing concentration of crystalline regions (Cullity, 1978). A possible explanation for the increased intensity with annealing is that the excess moisture coupled with heat may have been able to more evenly spread the amylose throughout the starch granule, allowing interaction of the amylose and amylopectin branches in the crystalline regions which would account for higher intensity readings between heated and unheated starch at higher percent moisture levels and comparable readings among several heated starches as seen in Figure 4. As suggested in Hoover and Vasanthan (1994b) interaction between amylose and amylopectin chains may also have occurred at the two moisture levels, which would also account for increased concentration of the crystalline regions. Loss of

56 moisture due to heating was not considered a major factor for increased x-ray diffraction readings since percent moisture level analyses of heat treated starches found little percent moisture loss (32.261% pre-treatment, 30.745% posttreatment; 40.017% pre-treatment, 38.954% post-treatment; 44.379% pretreatment, 43.335% post-treatment). More tests, however, would need to be run to confirm these findings. Temperature of fusion and heat of fusion results using the differential scanning calorimeter (DSC) for this experiment were higher than previous experiments which involved the use of heat and moisture treatment of cereal and buckwheat starches (Hoover & Vasanthan, 1994a; Hoover & Vasanthan, 1994b; Li, Lin, & Corke, 1997; Qian, Rayas-Duarte, & Grant, 1998). This is expected per the results of Donovans experiment (1979) because, unlike the other experiments, this experiment did not involve the addition of water to the DSC samples prior to testing. With intermediate to low moisture levels higher endotherms could be expected since, according to Donovans research (1979), DSC readings at lower moisture levels were due to the melting of the majority of the crystalline structure versus the small amount of crystalline structure stripping that takes place at the lower (66C) endotherm when excess moisture is available. In preliminary tests with buckwheat starch that had higher moisture levels and with some of the 44.4% starch samples some endotherms in the 66C area were visible. The lowest peak temperature for any of these readings was 67.64C. Heat treatment temperature was set at 65.6C (150F) in order to supply enough
57 heat to cause changes in the crystals without causing gelatinization which did partially occur in some samples as was noted in the results section. DSC endothermic changes did occur, but, as stated in the results, were attributed to moisture level changes, particularly between the 32.3% and 44.4% moisture levels. The shift in higher endothermic parameters is contrary to Donovan (1979) and other researchers who have studied the effect of moisture content on DSC parameters (Rolee & LeMeste, 1999) and found DSC parameters such as onset and peak temperature to decrease with increasing moisture content. Change in enthalpy was more consistent with the findings of Donovan (1979) and Rolee and LeMeste (1999) where peaks became smaller with decreased moisture content. Although hard to conclude due to the great amount of variance in especially peak temperatures, buckwheat starch with its higher water binding capacity and higher amylose content may actually form stronger internal bonds between amylose and itself or amylose and amylopectin at higher moisture levels which would contribute to increased resistance to melting. Amylose leaching results focused on the interaction of amylose with itself and other starch granule components. The results of this experiment found that amylose leaching was not significantly affected solely by moisture level as were DSC endotherm readings; rather the amylose leaching was affected more by the use of microwave heat treatment, and the combination of moisture and microwave treatment. This was most significant especially with the 40.0% and 44.4% moisture level annealed starch. Although the unheated 44.4% moisture level starch had the highest mean amount of amylose leaching, it was not significantly

58 different from the other unheated starches. The most significant finding from this test was that annealed starches had significantly lower amylose leaching. This finding is consistent with annealing treatments of different starches by Hoover and Vasanthan (1994b) but not heat-moisture treatment of different starches by Hoover and Vasanthan (1994a). The findings are also consistent with the restrictive swelling properties of buckwheat starch found by Qian, Rayas-Duarte, and Grant (1998). Lower swelling relates to lower amylose leaching in that granules that are more resistant to swelling are more resistant to leaching of their components. Higher amounts of amylose, coupled with the effects of annealing conditions, could help to form strong internal bonds between amylose and itself and amylose and other starch granules components which would make the granules more resistant to changes caused by the further addition of heat and moisture. Overall significant changes were observed in amylose leaching and DSC endotherm parameters. Visible changes were observed in x-ray diffraction readings in heated buckwheat starch at high moisture levels and in unheated buckwheat starch at low moisture levels. The addition of moisture and in some cases heat helped to form starch granules that were resistant to the breakdown of crystalline structures and the leaching of amylose in the presence of supplemental heat and moisture. Most of these changes were attributed to changes in interactions between amylose and other components throughout the starch granule.
59 CHAPTER VI CONCLUSION The purpose of this experiment was to explore the effect of microwave heat-moisture treatment and annealing on buckwheat starch properties. The
hypothesis was that both treatments would make the buckwheat starch granules more resistant to destruction by further heat and moisture application. This
hypothesis was tested by isolating buckwheat starch from flour, preparing five moisture levels, and setting up three different tests which looked at the resistance of the buckwheat starch granule to melting from additional heat, the leaching of amylose, a component of starch, with application of heat and water; and the crystalline structure of the starch before and after heat treatment at the different moisture levels. High moisture levels were found to have a significant effect on melting parameters whereas annealing treatment was found to have a significant effect on amylose leaching. There were no changes in d-space angles in x-ray diffraction; however, intensities did increase with lower moisture level and annealing. These findings were attributed to interactions between amylose and other starch components throughout the starch granule.

65 Zheng, G. H., Sosulski, F. W., & Tyler, R. T. (1998). Wet-milling, composition and functional properties of starch and protein isolated from buckwheat groats. Food Research International. 30, 493-502. Zylema, B. J., Grider, J. A., Gordon, J., & Davis, E. A. (1985). Model wheat starch systems heated by microwave irradiation and conduction with equalized heating times. Cereal Chemistry. 62, 447-453.



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