IMPACT OF ALKALINE HYDROGEN PEROXIDE PRETREATMENT ON SORGHUM WET-MILLING AND QUALITY OF SORGHUM STARCH By Nana Baah Pepra-Ameyaw A DISSERTATION Michigan State University in partial fulfillment of the requirements Submitted to for the degree of Food Science—Doctor of Philosophy 2019 ABSTRACT IMPACT OF ALKALINE HYDROGEN PEROXIDE PRETREATMENT ON SORGHUM WET-MILLING AND QUALITY OF SORGHUM STARCH By Nana Baah Pepra-Ameyaw Sorghum is an important food security crop and a potential alternative source for industrial starch production. Sorghum starch is, however, characterized by dull off-colors, attributed to the high levels of polyphenol compounds in the grain. Under alkaline conditions, hydrogen peroxide produces perhydroxyl anions, which react with chromophore groups in colored molecules. This work evaluated the impact of alkaline hydrogen peroxide (AHP)-pretreatment on sorghum wet- milling and the quality of sorghum starch. AHP-pretreatment of sorghum was investigated to (1) determine the optimum H2O2 concentration and pH at which acceptable starch could be obtained; (2) evaluate effect of pretreatment on the recovery of wet-milling products and the quality of the wet-milled starch fraction; and (3) evaluate the hydration kinetics of the pretreated grains during soaking in water. The first study varied H2O2 concentration and pH based on a central composite design experiment to determine its effect on sorghum starch color and recovery. The change in starch color was correlated with the properties of the wet-milled starch. A response surface model determined an acceptable degree of lightness (L-value) of 90 for sorghum starch at a pH of 11.84 and 20% (w/w) H2O2 concentration. Varying pH during AHP-pretreatment had a significant effect (p < 0.05) on the starch color. A response surface model for starch recovery did not adequately fit the experimental data (Lack-of-Fit = 0.460). Changes in starch color did not influence the starch recovery and most of the starch properties analyzed, however, it was significantly correlated with the starch pH (r = 0.62), swelling power (r = -0.57) and final pasting viscosity (r = 0.62) at p > 0.01, as well as oil absorption capacity (OAC) (r = 0.48) at p > 0.05. The second study evaluated the effect of AHP-pretreatment and further steeping in water for 24 hours at 55oC on the recovery yields of wet-milling products (coarse fraction, fine fraction, starch fraction, and total solids recovery) and the properties of the wet-milled starch obtained from four sorghum cultivars. The presence or absence of tannin in the sorghum cultivars did not significantly influence (p < 0.05) the recovery yields of wet-milled fractions. AHP-pretreated grains produced significantly higher (p < 0.05) levels of starch fraction yields and total starch recoveries. AHP-pretreatment also produced brighter starches particularly from the tannin- containing cultivars, whereas, steeping in water alone reduced the brightness of starches from the tannin-free sorghum cultivars. Most starch functional properties were not significantly affected (p > 0.05) by AHP-pretreatment or steeping in water. In the third study, the hydration kinetics of AHP-pretreated sorghum was evaluated by fitting Peleg’s sorption model to experimental water absorption data with R2 values for the fitted model ranging between 0.976 and 0.996. The capacity constant (K2) was independent of the soaking temperature and pretreatment, with an average value of 0.02%-1. AHP-pretreatment resulted in a significantly higher (p < 0.05) initial rate of hydration (1.86 g/min to 5.10 g/min) compared to the control (1.56 g/min to 3.92 g/min) at 60oC. The saturation moisture content for AHP-pretreated grains (56.67%, d.b. to 82.37%, d.b.) and the control (52.02%, d.b. to 76.49%) were consistent for each grain cultivar. The entire body of work demonstrates the effectiveness of AHP-pretreatment to enhance the color of wet- milled sorghum starch without significantly changing its functionality. In memory of Kwame Pepra-Ameyaw iv ACKNOWLEDGMENTS I would like to express appreciation to my advisors Dr. Gale Strasburg and Dr. Perry K. W. Ng, who dedicated their time to directing my research and scholarship. Without their guidance, this dissertation would not have been possible. I would like to thank my committee members, Dr. Leslie Bourquin and Dr. Eric Olson, whose support and guidance was also instrumental in completing this dissertation. I am grateful to Borlaug Higher Education for Agricultural Research and Development (BHEARD) program and staff, who funded and supported me during my studies at Michigan State University. I would also like to thank Professor Karen Duca and Professor Ibok Oduro for their mentorship. I am grateful for the continued support that I receive from my friends and family, particularly, my parents Mr and Mrs. Pepra-Ameyaw, my lab mates Dr. Walid Aljarbou and Dr. Juma Myongwo, and my close friends Dr. Olaocha Nwabara, Clarence George, Dr. Clement Kubuga and Adela Myles. v TABLE OF CONTENTS LIST OF TABLES..........................................................................................................................ix LIST OF FIGURES........................................................................................................................xi CHAPTER 1....................................................................................................................................1 1. Introduction..................................................................................................................................1 1.1. Background...............................................................................................................................1 1.2. Study 1......................................................................................................................................4 1.3. Study 2......................................................................................................................................4 1.4. Study 3......................................................................................................................................5 CHAPTER 2....................................................................................................................................6 2. Literature Review.........................................................................................................................6 2.1. Sorghum Production and Uses..................................................................................................6 2.2. Sorghum Grain Structure and Composition..............................................................................8 2.2.1. Starch Content..............................................................................................................9 2.2.2. Protein Content...........................................................................................................11 2.2.3. Fat Composition.........................................................................................................12 2.2.4. Vitamins and Mineral Composition............................................................................13 2.2.5. Sorghum Polyphenols.................................................................................................13 2.2.5.1 Phenolic Acids.....................................................................................................14 2.2.5.2 Flavonoids............................................................................................................15 2.2.5.3 Condensed Tannins..............................................................................................16 2.3. Sorghum Starch Production....................................................................................................19 2.3.1. Wet Milling of Sorghum.............................................................................................20 2.3.1.1 Steeping...............................................................................................................22 2.3.1.2 Milling and Separation of Grain Components.....................................................23 2.3.1.3 Optimizing Sorghum Wet-milling For Pilot Scale...............................................25 2.3.2. Sorghum Starch Characteristics.................................................................................26 2.3.2.1 Interactions Between Sorghum Starch and Polyphenols.....................................28 2.4. Alkaline Hydrogen Peroxide...................................................................................................29 2.4.1. Mechanism of Alkaline Hydrogen Peroxide..............................................................30 2.4.1.1 Oxidation of Anthocyanins by Hydrogen Peroxide.............................................32 CHAPTER 3..................................................................................................................................35 3. Alkaline Hydrogen Peroxide (AHP)-Pretreatment of Sorghum and Effect on Starch Properties ............................................................................................................................................35 3.1. Abstract...................................................................................................................................35 3.2. Introduction.............................................................................................................................35 3.3. Materials and Methods............................................................................................................38 3.3.1. Sample Preparation.....................................................................................................38 3.3.2. Alkaline Hydrogen Peroxide Pretreatment.................................................................38 3.3.3. Wet milling of AHP-Pretreated Sorghum...................................................................39 vi 3.3.4. Characterization of Wet-Milled Starch Fractions.......................................................40 3.3.5. Experimental Design..................................................................................................41 3.3.6. Statistical Analysis......................................................................................................42 3.4. Results and Discussion...........................................................................................................43 3.4.1. Effect of AHP-pretreatment Conditions on Starch Color...........................................43 3.4.2. Effect of AHP-pretreatment on Sorghum Starch Recovery........................................47 3.4.3. Validation of response surface model.........................................................................49 3.4.4. Correlation among starch fraction properties.............................................................50 3.5. Conclusion.............................................................................................................................53 CHAPTER 4..................................................................................................................................54 4. Wet-Milling of Alkaline Hydrogen Peroxide (AHP)-Pretreated Sorghum and Characterization of the Sorghum Starch Fraction.........................................................................................54 4.1. Abstract...................................................................................................................................54 4.2. Introduction.............................................................................................................................55 4.3. Materials and Methods............................................................................................................57 4.3.1. Sorghum Samples.......................................................................................................57 4.3.1.1 Alkaline Hydrogen Peroxide Pretreatment..........................................................58 4.3.1.2 Steeping of Sorghum Grain in Water...................................................................58 4.3.1.3 Wet-Milling Procedure.........................................................................................58 4.3.2. Characterization of Starch..........................................................................................61 4.3.3. Experimental Design and Statistical Analysis............................................................61 4.4. Results and Discussion...........................................................................................................62 4.4.1. Grain Characteristics and Chemical Composition of Sorghum Cultivars..................62 4.4.2. Recovery of Wet-milling Products.............................................................................63 4.4.3. Characteristics of Starch Fraction..............................................................................67 4.4.4. Functional Properties of Starch Fraction....................................................................70 4.4.5. Pasting Properties of Wet-milled Starch.....................................................................72 4.5. Conclusion..............................................................................................................................75 CHAPTER 5..................................................................................................................................76 5. Water Absorption Characteristics of Sorghum Pretreated With Alkaline Hydrogen Peroxide (AHP).................................................................................................................................76 5.1. Abstract...................................................................................................................................76 5.2. Introduction.............................................................................................................................76 5.3. Materials and Methods............................................................................................................79 5.3.1. Sample Preparation.....................................................................................................79 5.3.2. Alkaline Hydrogen Peroxide (AHP) Pretreatment.....................................................79 5.3.3. Physical Characteristics and Chemical Composition.................................................80 5.3.4. Determination of Water Absorption During Soaking.................................................80 5.4. Results and Discussion...........................................................................................................81 5.4.1. Physical and chemical properties...............................................................................81 5.4.2. Water Absorption Characteristics...............................................................................82 5.4.3. Total Solids Loss........................................................................................................85 5.4.4. The Constant Rate of Water Absorption.....................................................................86 5.4.5. Saturation Moisture Content.......................................................................................90 vii 5.5. Conclusion..............................................................................................................................91 CHAPTER 6..................................................................................................................................92 6. Summary and Conclusions........................................................................................................92 6.1. Future Directions....................................................................................................................94 APPENDIX....................................................................................................................................96 REFERENCES.............................................................................................................................110 viii LIST OF TABLES Table 1: Chemical composition of sorghum grain...........................................................................9 Table 2: Phenolic acids, flavonoids and proanthocyanidins in sorghum grains............................18 Table 3: Comparison of wet-milling fraction yields between 100g sorghum and corn wet-milling procedures, and pilot scale wet-milling.............................................................................26 Table 4: Experimental responses obtained from the central composite design to determine the effect of varying H2O2 concentration and pH on sorghum starch color, yield, functional and pasting properties........................................................................................................42 Table 5: Analysis of variance results for the regression model developed from the central composite design to evaluate the effect of H2O2 concentration and pH on wet-milled starch color.........................................................................................................................45 Table 6: Analysis of variance results for the regression model developed from the central composite design to evaluate the effect of H2O2 concentration and pH on wet-milled starch recovery...................................................................................................................48 Table 7: Comparison of experimental and predicted values for starch fraction color (Huner L- value) at five randomly selected levels of H2O2 concentration and pH.............................50 Table 8: Correlation of wet-milled starch properties obtained from AHP-pretreated sorghum at varying H2O2 concentrations and pH.................................................................................52 Table 9: Grain characteristics and chemical composition of the four sorghum cultivars (Dorado, ICRISAT, Kadaaga, Kapaala)............................................................................................63 Table 10: Effect of AHP-pretreatment and steeping on the recovery yields of wet-milling products from four sorghum cultivars................................................................................66 Table 11: Effect of AHP-pretreatment and steeping on characteristics of wet-milled starch fractions from four sorghum cultivars...............................................................................69 Table 12: Effect of AHP-pretreatment and steeping on the functional properties of wet-milled starch fractions from four sorghum cultivars.....................................................................71 Table 13: Effect of AHP-pretreatment and steeping on the pasting properties of wet-milled starch fraction from four sorghum cultivars.................................................................................74 Table 14: Comparison of the physico-chemical characteristics between AHP-pretreated sorghum and untreated (control) sorghum........................................................................................82 ix Table 15: Water absorption characteristics and regression parameters following the Peleg model for AHP-pretreated Burgundy whole grain sorghum and untreated control sorghum at four soaking temperatures..................................................................................................89 Table 16: Functional and pasting characteristics of wet-milled sorghum (Naga red) starch after alkaline hydrogen peroxide pretreatment...........................................................................97 Table 17: ANOVA results for effect of grain cultivar and treatment on recovery yields of wet- milling fractions...............................................................................................................106 Table 18: ANOVA results for effect of grain cultivar and treatment on starch recovery, protein residue, amylose, and starch color...................................................................................107 Table 19: ANOVA results for effect of grain cultivar and treatments on starch functional properties..........................................................................................................................108 Table 20: ANOVA results for effect of grain cultivar and treatments on the starch pasting properties..........................................................................................................................109 x LIST OF FIGURES Figure 1: Scanning electron micrographs of (A) sorghum floury endosperm and (B) sorghum corneous endosperm. pb-protein bodies; s-starch (Emmambux and Taylor, 2013)...........10 Figure 2: Structure of (a) flavonoid ring structure with numbering, and (b) Luteolinidin reported in sorghum compared to (c) Cyanidin found in berries such as grapes and cherries.........16 Figure 3: Summary of wet milling procedure for sorghum...........................................................21 Figure 4: Reaction of hydrogen peroxide with quinone by nucleophilic addition of hydroperoxyl anion...................................................................................................................................32 Figure 5: Proposed reaction from the hydrogen peroxide mediated oxidation of anthocyanin.....34 Figure 6: Wet-milling of sorghum grain (Naga Red) pretreated with alkaline hydrogen peroxide, to obtain starch fraction.....................................................................................................40 Figure 7: Response surface plot for the effect of varying H2O2 concentration and pH on starch fraction color......................................................................................................................46 Figure 8: Procedure for wet-milling sorghum grain samples into three fractions (Coarse, fine and starch fractions)..................................................................................................................60 Figure 9: Comparison of predicted and experimental water uptake of (a) control and (b) AHP- pretreated Burgundy whole grain sorghum during hydration in water at four soaking temperatures (30, 40, 50 and 60 oC). Solid lines represent predicted values based on fitted Peleg model........................................................................................................................84 Figure 10: Total solids loss from AHP-pretreated Burgundy whole grain sorghum and control (untreated sorghum) soaked in water at four temperatures. Column bars connected by the same letter are not significantly different at p < 0.05........................................................86 Figure 11: Fitting of water absorption data during hydration of (a) the control (untreated sorghum) and (b) AHP-pretreated Burgundy whole grain sorghum, to Peleg's model......88 Figure 12: Standard curve for concentration of NH3-N using ammonium chloride standard against absorbance at 640 nm..........................................................................................100 Figure 13: Representative viscosity parameters measured on a Rapid Visco Analyzer (RVA) pasting curve with heating and cooling rate of ~6oC/min................................................105 xi CHAPTER 1 1. Introduction 1.1. Background Sorghum [Sorghum bicolor (L.) Moench] is a very important cultivated grain with potential for increased utilization as a food source. It is grown mostly in semi-arid, tropical regions of the world and serves as a dietary staple for over 500 million people (Anglani, 1998). The majority of sorghum is cultivated in Africa, which was estimated to contribute about 39% of global production between 2000 and 2010 (Bean and Ioerger, 2014). Only 35% of globally cultivated sorghum is used for human consumption, whereas the rest is mainly reserved for animal feed, alcohol production and other industrial products (Dicko et al., 2006). There are several agronomic and economic advantages to producing sorghum: for instance, in regions of the world that are hot and dry, sorghum is a desirable crop because it is drought-resistant and has the ability to grow well under extreme conditions. It is also able to withstand long periods flooding or soil water-logging (Taylor, 2003). In addition to these useful traits, sorghum also has high nitrogen utilization efficiency; thus, it requires less fertilizer compared to other cereals, making it economical for farmers to grow (Gardner et al., 1994). These beneficial attributes of sorghum make it a viable crop for global food security, especially for developing nations in Africa and Asia. In Africa, the demand for sorghum is projected to increase due to the constantly growing population and efforts by national governments to promote local foods through processing and industrial utilization (Akintayo et al., 1999). One processing potential for sorghum is the production of starch, and this is mainly because of the structural similarities between sorghum and corn seeds. Corn is currently the 1 predominant global source of starch. The potential of sorghum for starch production is similar to that of corn because the wet milling processes to isolate starch from both grains are identical and their starches have comparable physical and chemical properties (Chiremba et al., 2011; Eckhoff and Watson, 2009; Wronkowska, 2016). In the corn starch industry, wet milling is the preferred industrial process for extracting starch, mainly because it is able to produce higher yielding products with high levels of purity. Wet milling of sorghum on the other hand, was developed in the U.S.A. during World War II but this was discontinued when corn prices at the time became cheaper (Serna-Saldivar and Rooney, 1995). There is currently no record of an industrial scale sorghum wet-milling operation. The wet milling process involves three basic stages, and begins with steeping of the grain to soften the pericarp and enable more efficient separation of the grain components. In most cases the steeping solution includes chemicals that help improve yields. The second stage in wet milling involves grinding the grain into separate fractions and the final stage involves the separation of starch and protein. The potential benefits of sorghum have generated a renewed interest in sorghum starch production, and laboratory procedures have been developed to study the wet milling characteristics of different sorghum varieties (Buffo et al., 1998; Eckhoff and Watson, 2009; Xie and Seib, 2000). Sorghum starches are often associated with off-colors and dull appearance in comparison to corn starch which is white and much brighter in appearance. This discoloration of sorghum starch has been attributed to the presence of pigments in the pericarp that leach into the endosperm during weathering in the field and steeping for wet-milling (Subramanian et al., 1994). Sorghum cultivars come in a variety of different colors due to the presence of phenolic compounds in the pericarp. These compounds have also been found in the endosperm of the 2 certain varieties as well. According to Subramanian et al. (1994), dark-colored sorghum cultivars which usually contain high amounts of tannins are not suitable for starch production. This is mainly because of the dull appearance of the starch and low digestibility of the starches (Agudelo et al., 1997). Starch from tannin-free sorghum are not guaranteed to appear white and may also have off-colors as a result of polyphenols in the endosperm (Subramanian et al., 1994). To improve the color of sorghum starch, the pigments in the grain must be removed before or during the starch extraction process. Since a majority of these pigment compounds are found in the pericarp of the grain, abrasively removing this layer improves the starch color; however, this approach also results in a significant loss of starch (Chew-Guevara et al., 2016; Subramanian et al., 1994). More direct bleaching approaches have involved extracting sorghum starch in alkali solutions and treating the whole grains with sodium hypochlorite (Freeman and Watson, 1971; Mistry, 1991; Ochanda et al., 2010; Sira and Amaiz, 2004). This work investigates the use of alkaline hydrogen peroxide (AHP) pretreatment of sorghum before wet milling with the aim of improving the color and appearance of sorghum starch. Additionally, the effect of the AHP pretreatment on the quality of the isolated starch is also evaluated. Hydrogen peroxide has been used for bleaching and delignifying agricultural products since its discovery in 1818 (Brooks and Moore, 2000; Gould et al., 1989; Metzger, 2002). It has many more industrial applications as a bleaching agent and is increasingly becoming the preferred option for both industrial and domestic use (Spiro et al., 1997). Under alkaline conditions hydrogen peroxide has been suggested to be more effective at removing color (Renard et al., 1997). Treatment of sorghum with hydrogen peroxide involves degrading and solubilization of the polyphenolic compounds responsible for the color in the grain. It also presents a less toxic alternative to removing color from the grains. 3 The long-term goal of this study is to effectively wet mill and isolate starch of acceptable color, appearance and quality from sorghum grain for industrial or domestic application. In developing countries where sorghum is grown in large quantities, industrial sorghum starch production would have a positive socioeconomic impact. This work involves three studies to evaluate the use of AHP pretreatment on sorghum. 1.2. Study 1 The first study was aimed at optimizing the conditions for AHP-pretreatment of sorghum. The objective was to determine optimum H2O2 concentration and pH at which starch of acceptable color could be obtained from wet-milling sorghum. The study further evaluated the effect that varying H2O2 concentration and pH has on the functional and pasting properties of the wet-milled starch. It was hypothesized that increasing both H2O2 concentration and pH during AHP-pretreatment of sorghum will result in wet-milled starch with less off-colors. 1.3. Study 2 The second study was aimed at evaluating the effect of AHP-pretreatment and steeping in water on the recovery of wet-milled fractions and quality of wet-milled starch fraction from four sorghum cultivars. Three wet-milling fractions were evaluated: coarse, fine and starch. The starch fractions were further evaluated for their functional and pasting properties. It was hypothesized that sorghum wet-milling was enhanced by AHP-pretreatment of the grains prior to wet-milling. 4 1.4. Study 3 The third study evaluated the effect of AHP-pretreatment of sorghum on the water absorption characteristics or hydration kinetics of the grain when soaked in water. The hydration of the grain was studied using Peleg’s sorption model, which is a two-parameter equation used to predict the hydration of many food materials. Water hydration behavior of the kernels is important for wet-milling operations. The hypothesis was that AHP pretreatment of sorghum enhances its hydration in water. 5 CHAPTER 2 2. Literature Review 2.1. Sorghum Production and Uses Sorghum is a very important crop grown throughout the world, especially in Africa where it is the second most important cereal grain in terms of production. Sorghum is believed to have originated from Africa and was distributed along trade routes throughout the continent, and to the Middle East (Dicko et al., 2006). World production of sorghum is estimated at 57 million tonnes using a total of 47 million ha of area harvested globally (FAOSTAT, 2019). Sorghum production in Africa accounts for about 47% of global production, and is considered a vital grain for food security in Africa. It is cultivated in about 43 countries across the continent with the largest producers being Nigeria (6.0 million tonnes), Ethiopia (4.8 million tonnes), Sudan (3.7 million tonnes) and Niger (1.9 million tonnes). Sorghum grains are about the same size as wheat with the 1000-kernel weight averaging between 25-35 g (Emmambux and Taylor, 2013). The grains come in a variety of colors ranging from white to brown to red and even black. These colors are attributed to the presence of polyphenol pigments in the pericarp of the grain (Subramanian et al., 1994). The grain is composed of three main parts: the pericarp (seed coat), germ (embryo) and the endosperm (storage tissue). Some sorghum varieties are characterized by a highly pigmented testa which is the sub-coat layer just below the pericarp. The thickness of the testa is not uniform and some varieties have a partial testa while it appears to be absent in others (Dicko et al., 2006). Several different sorghum genotypes have been developed in Africa, resulting in thousands of hybrid varieties (Akintayo et al., 1999). The genotypes can be grouped into Milo 6 (tall, often goosenecked with compact, panicled, white or yellow grains), Feterita (compact panicles with large, pearly grains and tannin pigments hidden in the bran layer between the endosperm and the outermost white cuticle), and Kaffir and Durra (traditional African varieties with erect, cylindrical, open panicles and grain colors from purple through red and white) (Dobraszczyk and Dendy, 2001). There are no definitive lists of sorghum varieties available. However, the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) has compiled a database of over 20,000 accessions, most of which are from the developing, semi- arid tropics (Akintayo et al., 1999). Sorghum has good drought resistance and is important for areas with unpredictable rainfall. Sorghum stomata remain functional for longer periods of time under wilt conditions than that of corn (Serna-Saldivar and Rooney, 1995). Sorghum can also be grown in high-rainfall areas, although this makes it more prone to fungal attack, and is more tolerant of heat and salinity than corn. It can be grown in a wide range of soil types. (Chandrashekar and Satyanarayana, 2006). In addition to its drought tolerance, it is inherently a very efficient crop as it emerges quickly and produces rapid ground cover compared to other crops. It requires limited nutrient supply and has an efficient photosynthetic pathway with a high growth rate (Gardner et al., 1994). Sorghum has the ability to produce a second crop on the roots of the first which allows for a number of variants in practice. For instance, in the rainy season, the crop can be cut early for fodder and the secondary crop harvested for the grain under dry conditions at the end of the rainy season (Taylor et al., 2006). Some sorghum varieties exhibit a degree of resistance to damage by birds in the field. Although these varieties are capable of being eaten by birds and other pests, they are less 7 susceptible than tannin-free sorghum and other cereal grains. As a result, they are referred to as “bird proof” or “bird resistance” (Dykes and Rooney, 2006). 2.2. Sorghum Grain Structure and Composition Sorghum has a macromolecular composition similar to that of corn and wheat. The sorghum grain or caryopse is covered by glumes which are described by Dicko et al. (2006) as the part of the panicle that holds the caryopse after pollination. The grain, which is round and bluntly pointed, comes in varying size (4-8 mm), shape and color. The variety in grain color is a unique trait of the grain that affects sorghum grain quality. The grains are made up of an outer seed coat also known as the testa or pericarp layer, an oil-rich germ (embryo) and a starchy endosperm which is the storage tissue of the grain. The endosperm of sorghum contains between 65.7 to 70.8%, db starch, which accounts for a majority of the total grain starch, although some starch granules can also be found trapped in the grain pericarp (Beta et al., 2001b). Some sorghum genotypes have highly pigmented testa which is a genetic trait. The thickness of the testa also varies among different genotypes. In fact, some genotypes have a partial testa, whereas others do not possess a testa at all. 8 Table 1: Chemical composition of sorghum grain Macro-components (g/100 g, f.m.) Amino Acids (mg/g) Vitamins Minerals (mg/100 g, d.m.) (mg/100 g, d.m.) 21 RE Ca Cl 0.35 0.14 Cu I 2.8 0.5 Fe 0.007 Mg Vit. A Thiamine Riboflavin Niacin Pyridoxine Biotin Pantothenate 1.0 Vit. C P <0.001 K Na Zn 832 245 87 Lysine 126 Phenylalanine 306 189 Threonine 63 Tryptophan Valine 313 167 94 Carbohydrates Starch Amylose Amylopectin Non starch Low MW carbohydrates Proteins α-Kafirins β-Kafirins γ-Kafirins Other Proteins Fat Ash Moisture Sources: (Dicko et al., 2006; FAO, 1995). f.m. - fresh matter; d.m. - dry matter; RE – Retinol equivalent. Leucine 65-80 60-75 Isoleucine 12-22 Methionine 45-55 2-7 2-4 7-15 4-8 0.2-0.5 Tyrosine 0.7-1.6 Cystine 2-5 1.5-6 1-4 8-12 21 57 1.8 0.029 5.7 140 368 220 19 2.5 2.2.1. Starch Content Starch is the primary source of stored energy in sorghum and is a glucan comprising two main types of macromolecules: amylose and amylopectin. Amylose is a linear polymer of D- glucose connected by α-1,4-links and exists as a helix. Native sorghum starch contains around 24% amylose; heterowaxy and waxy starches contain significantly less amylose. Amylopectin on the other hand is more abundant in the starch and is a branched polymer with α-1,6 branches at every 20 to 25 glucose residues. 9 Sorghum starch granules also have two morphologies within the grain Figure 1. The starch granules in the center of the endosperm are spherical, less dense and floury in comparison to the starch granules in the outer portion of the endosperm which is polygonal and often dented. This is due the tight packing of the granules and the dents result from pressing against protein bodies (Emmambux and Taylor, 2013). Figure 1: Scanning electron micrographs of (A) sorghum floury endosperm and (B) sorghum corneous endosperm. pb-protein bodies; s-starch (Emmambux and Taylor, 2013). Whole grain sorghum is estimated to contain an average energy value of 356 kcal/100g (Dicko et al., 2006). Sorghum also contains resistant starch which makes it less digestible, especially by infants (Anglani, 1998). Sorghum-based foods exhibit a low glycemic index and increased satiety due to its slow digestibility (Barros et al., 2012). This makes sorghum desirable for applications in which food is prepared for diabetics and for people on diets. The pericarp and endosperm cell walls of sorghum contain non-starch polysaccharides (NSP) which are mainly composed of arabinoxylans and other β-glucans (Dicko et al., 2006). These macromolecules are mostly insoluble in water and form viscous and sticky solutions, which causes some problems with processing particularly in the brewing industry. During 10 brewing, they result in poor wort and beer filtration rates as well as the occurrence of haze (Dicko et al., 2006). 2.2.2. Protein Content Sorghum contains approximately 7-15% protein which is categorized into water-soluble albumins, salt-soluble globulins, aqueous-alcohol-soluble kafirins (prolamins), and cross-linked kafirins and glutelins (Bean et al., 2006). Kafirins are the major storage protein of sorghum and make up about 50 to 70% of the total protein (Belton et al., 2006). Hamaker et al., (1995) reported that kafirins make up about 70% of whole grain flour protein and about 80% of decorticated flour protein. Due to the high proportion of kafirin storage proteins in sorghum, they further suggested a simpler classification of sorghum proteins into kafirin and non-kafirin groups. Further discussion of sorghum proteins in this review will focus on kafirins which impact the functionality of sorghum flour or starch. Sorghum proteins have a high degree of sequence homology with corn proteins (DeRose et al., 1989). However, in comparison, sorghum proteins contain higher proportions of cross-linked kafirins which undergo intermolecular disulfide-crosslinking. Kafirin proteins are classified further into alpha-, beta-, and gamma-kafirins based on the difference in molecular weight, extractability, structure, and cross-reactivity with analogous corn proteins (Shull et al., 1991). Alpha-kafirins are the predominant storage protein and make up 80% of total kafirins followed by gamma-kafirins and beta-kafirins at 15% and 5% respectively. The influence of kafirins on the sorghum functionality depends mainly on their cysteine contents and their placement within the protein body. Beta- and gamma kafirins have high cysteine contents (5 and 7 mol%), compared to alpha-kafirins which contain relatively low levels of 11 cysteine. The alpha-kafirins are often found encapsulated within a disulfide-bound polymeric network made up of gamma- and beta-kafirin protein bodies (Shull et al., 1992). This network of protein bodies is coated by other non-kafirin protein in a matrix that surround the starch granules in the endosperm (Figure 1). The structures formed by sorghum proteins with themselves and other grain constituents have a direct impact on the functional properties of sorghum during processing as well as the digestibility of sorghum products. Kafirins are protease-resistant and thus are considered to be of poor nutritional quality (Duodu et al., 2002). Protein digestibility of sorghum may decrease upon cooking (Duodu et al., 2002) but fermentation prior to cooking may increase its digestibility (Elkhalifa et al., 2004). The low digestibility of sorghum proteins results from protein-protein, protein-carbohydrate, protein- polyphenol, and carbohydrate-polyphenol interactions (Duodu et al., 2003; Emmambux and Taylor, 2003; Wong et al., 2009; Zhu, 2015). With regards to the production of starch from sorghum, wet-milling processes are often optimized to limit the amount of protein in the starch fraction. A protein-rich fraction can be isolated from the wet-milling process as gluten feed and have several applications including use as feed or syrups. Typically, the protein in wet-milled sorghum starch ranges between 0.31% – 0.64% (Buffo et al., 1998). 2.2.3. Fat Composition The fat in sorghum is located mainly in the germ which is rich in polyunsaturated fatty acids (Awadalkareem et al., 2008). The crude fat content of sorghum is typically higher than that of wheat and rice but lower than that in corn (Rooney and Waniska, 2000). The amount of oil in sorghum is not enough to be considered as a source for industrial oil production; however, it may 12 still have some nutritional benefits due to the distribution of fatty acids within the grain. Mehmood et al. (2008) analyzed the fatty acid composition of seed oil from different sorghum varieties by subjecting solvent extracted oils to GC and GC-MS. They found total oil content up to 8.2%, w/w with significantly more polyunsaturated fatty acids compared to monounsaturated fatty acids. The fatty acid composition in sorghum oil is made up of oleic acid (31.12-48.99%), palmitoleic acid (0.43-0.56%), linoleic acids (27.59-50.73%), linolenic acid (1.71-3.89%), stearic acid (1.09-2.59%) and palmitic acid (11.73-20.18%) (Mehmood et al., 2008). 2.2.4. Vitamins and Mineral Composition Sorghum is a good source of vitamins and minerals. It contains the B vitamins thiamine, riboflavin, pyridoxine, etc., and liposoluble vitamins A, D, E and K (Dicko et al., 2006). It has also been shown to contain minerals such as phosphorus, potassium, iron and zinc (Awadalkareem et al., 2008). The ash content of sorghum is a crude estimation of the mineral content and typically ranges between 1-4 g/100g f.m (Table 1). Most of the mineral composition is located within the pericarp of the grain and is significantly lower in wet-milled starch fractions. High amounts of ash or inorganic content in wet-milled starch could indicate the presence of bran or germ components in the extracted starch. 2.2.5. Sorghum Polyphenols Sorghum is unique among other cereal grains because of the amount and variety of polyphenolic compounds that it contains. Sorghum grains come in a variety of colors mainly due to the presence of anthocyanins which are polyphenol compounds. Other types of polyphenols found in sorghum are condensed tannins which are present in the testa of many sorghum lines. In 13 the mature plant, red pigmentation is formed from the presence of polyphenols in response to damage, stress, or infection (Butler, 1982). The presence of polyphenols in the sorghum grains has been suggested to protect the grains against insect damage and birds (Bullard, 1979). It also protects the grain against preharvest germination (Beta et al., 2000). The polyphenols may also protect the sorghum plant against fungi, bacteria and viruses that may cause diseases (Lee et al., 2014; Waniska et al., 2001). Despite the agronomic benefits of sorghum polyphenols, their presence in sorghum products has been reported as being anti-nutritional. Studies on animal feed with high levels of tannins result in low rates of growth in animals (Ali and Mahmood, 2013; Al- Mamary et al., 2001; Bagliacca et al., 1997; Jambunathan and Mertz, 1973). Despite the claims of anti-nutritive properties related to sorghum feed products, there is also a case for possible health conferring effects based the results of antioxidant evaluation of the sorghum anthocyanidins and other grain polyphenols (Awika et al., 2005, 2004; Awika and Rooney, 2004). Butler (1982) suggested different ways of limiting the anti-nutritional effects of tannin in sorghum that included extraction with alkali, supplementation with methionine, and dehulling or removing the grain pericarp. Although the conflicting effect of sorghum polyphenols is beneficial to producers but a disadvantage to consumers, it is however, possible to take advantage of the anti-nutritional properties of sorghum polyphenols by developing slowly digestible commercial food products for people with specific nutritional requirements. 2.2.5.1 Phenolic Acids Phenolic acids in sorghum grains are located in the pericarp, testa, aleurone layer, and endosperm. They can be classified as hydroxybenzoic or hydroxycinnamic acids, and are listed in Table 2. The phenolic acids may also be free or bound. The free phenolic acids can be extracted in methanol and are found in the outer pericarp, testa, and aleurone layers. Bound 14 phenolic acids on the other hand can be extracted in dilute HCl (2M) at high temperature and are found in the cell walls of the grains (Hahn et al., 1984, 1983). 2.2.5.2 Flavonoids Sorghum contains a variety of flavonoids, some of which are listed in Table 2. Anthocyanins which form a major class of flavonoids are mostly responsible for the colors observed in the grains. They are found in many colored plants; however, sorghum anthocyanins do not contain a hydroxyl group in the C-3 position depicted in Figure 2 which compares the structure of luteolinidin reported in sorghum, and cyanidin found in red berries such as grapes and cherries. This difference in structure makes sorghum anthocyanins more stable at high pH compared to other anthocyanins (Awika et al., 2005). Apigeninidin, which is yellow, and luteolinidin, which is orange, are the two most common sorghum 3-deoxyanthocyanidins. Sorghum grains with black pericarps have the highest levels of 3-deoxyanthocyanins concentrated in the bran. Flavan-4-ols are common in sorghum with red pericarps and play an important role in the ability of the grains to resist mold growth (Audilakshmi et al., 1999). 15 7 6 8 A 5 HO OH 3' B 6' 4' 5' 2' 1' 2 3 O C 4 (a) OH HO + O OH OH OH (c) HO HO + O (b) Figure 2: Structure of (a) flavonoid ring structure with numbering, and (b) Luteolinidin reported in sorghum compared to (c) Cyanidin found in berries such as grapes and cherries. 2.2.5.3 Condensed Tannins Condensed tannins, also known as proanthocyanidins or procyanidins, are high molecular weight polyphenols present in sorghum grains. The amount of condensed tannins in sorghum varies depending on genotypes. Sorghum grains that contain no tannins are classified as type I sorghum, whereas Type II and Type II sorghum contain tannin levels between 0.02-0.19 mg/100 mg and 0.4-3.5 mg/100 mg, respectively (Dykes and Rooney, 2006). Condensed tannins in sorghum are able to bind to proteins, carbohydrates, and minerals, resulting in reduction in nutritional value (Awadelkareem et al., 2009). During malting, condensed tannins also bind to amylases and limit their availability for starch degradation (Elmaki et al., 1999). These negative effects can however be limited by decortication, fermentation, germination, and chemical 16 treatment with alkali, HCl, formaldehyde, etc. (Adetunji et al., 2015; Agudelo et al., 1997; Ahmed et al., 1996). While other undesirable effects resulting from the presence of condensed tannins in sorghum have been reported, such as astringency in sorghum food products and off-colors in wet-milled starch (Anglani, 1998), some agronomical benefits have also been suggested. Mold resistance in sorghum grains have been correlated with high levels condensed tannins (Waniska et al., 1989). Tannins also provide a degree of resistance to damage by predatory birds in the field, and such high tannin cultivars are often referred to as “bird resistant” or “bird proof” (Dykes and Rooney, 2006). The presence of tannins does not completely prevent bird damage, since these birds are able to eat high tannin sorghum when no other food is available. 17 Table 2: Phenolic acids, flavonoids and proanthocyanidins in sorghum grains Phenolic acids Hydroxybenzoic acids: Flavonoids and Proanthocyanidins Flavan-4-ols Flavonols Anthocyanins Gallic Apigeninidin Luteoforol Protocatechuic P-Hydroxybenzoic Apigeninidin-5-glucoside Apiforol Luteolinidin Flavones Kaempferol 3-rutinoside- 7-glucuronide Dihydroflavonols Taxifolin Gentisic 5-Methoxyluteolinidin Apigenin Taxifolin 7-glucoside Luteolin Flavanones Eriodictyol 7-glucoside 7-Methoxyapigeninidin 5-glucoside Salicylic Vanillic Syringic Hydroxycinnamic acids Luteolinidin 5-glucoside Eriodictyol 5-glucoside Ferulic Caffeic P-Coumaric Cinnamic Sinapic Sources: (Dykes et al., 2009; Dykes and Rooney, 2006) 5-Methoxyapigeninidin Naringenin 7-Methoxyluteolinidin 18 Proanthocyanidin monomers/dimers Catechin Procyanidin B-1 Proanthocyanidin polymers Epicatechin- (epicatechin)n-catechin Prodelphinidin Proapigeninidin Proluteolinidin 2.3. Sorghum Starch Production Starch is extracted from sorghum in the same way as corn using the wet milling process. This method results in highly purified starch fractions in comparison to dry milling. There are also several valuable co-products obtained from this method which are discussed in the next section. Industrial sorghum starch production began in the United States during World War II when corn production was insufficient for all its end uses. However, by 1948, only one factory was reported to produce starch and glucose from sorghum, and this factory was closed shortly afterward because sorghum prices began to exceed those of corn and it was no longer cost- effective to produce starch from sorghum (Taylor and Dewar, 2001). The need to address food security challenges in developing countries has made it necessary to reevaluate the value of sorghum and its potential as a source of starch. Several studies have examined optimal conditions for sorghum wet milling through laboratory-scale studies (Buffo et al., 1998; Wang et al., 2000; Xie et al., 2006; Xie and Seib, 2002). For instance, Buffo et al. (1998) evaluated the relationship between grain characteristics of 24 sorghum hybrids and yields and composition of their wet-milled products. They found significant correlations between the proximate and physical characteristics of sorghum, and the yields and protein contents of laboratory wet-milled products and recovery. There are still ongoing studies attempting to extract starch from sorghum with the same color and quality characteristics as corn starch which is the industry standard. These studies are based on methods used in the corn industry which involve treatment of the grains with sulfur dioxide and lactic acid to facilitate effective separation of the grain components and improve the color of the extracted starch. 19 2.3.1. Wet Milling of Sorghum Wet-milling is industrially used for the extraction of starch from corn which is the predominant source of starch. It is also applied to other cereals such as wheat, sorghum, barley, oats or rice. The conventional wet-milling process involves several steps that involve grain handling, and steeping, separation and recovery of the germ, fiber, proteins, and starch (Wronkowska, 2016). Wet-milling is often preferred to dry milling for starch extraction because it results in isolation of higher amounts of starch that require minimal further processing to enable its application as food additives or adhesives. It is also possible to fractionate different components that may find wider application in both food and nonfood products. The most common by-product from wet-milling is gluten feed and gluten meal which are high in protein and used for animal feed (Ambula et al., 2003; Bagliacca et al., 1997; Larrain et al., 2009; Tandiang et al., 2014). Industrial wet-milling was developed in the United States in during World War II, and a sorghum wet-milling plant was built in Texas by the Corn Products Company in 1948 which processed up to 20,000 bushels of sorghum per day. The wet-milling plant was closed in 1975 mainly because of the competing low cost of corn (Serna-Saldivar and Mezo-Villanueva, 2003). There are reports of a low capacity (150t/day) wet-milling plant in Sudan (Wang et al., 2000). Recovery of starch from sorghum is more complicated than corn because the pericarp of sorghum is fragile and during wet-milling, small pericarp particles interfere with starch separation and result in dull colored starch as well. Laboratory-scale wet-milling has been developed to assess the millability of cereals and estimate the quantity and quality of the recoverable components as well as the 20 difficulty in separating the different components (Jackson and Shandera, 1995). Laboratory wet- milling process involves sample preparation, steeping, first and second grind, germ and fiber separation and finally a starch-protein separation step as shown in Figure 3. Preparing the grains for laboratory wet milling begins with cleaning the grains. They are hand-picked, hand-sieved, or mechanically cleaned to remove damaged kernels and foreign material before steeping. The choice of cleaning methods usually depends on the objective of the milling test. If the objective is to compare wet milling among different grain varieties, it is important to ensure that the samples have comparable levels of damaged kernels and foreign materials (Singh and Eckhoff, 1996). The subsequent steps in the wet milling process are discussed below. Figure 3: Summary of wet milling procedure for sorghum. 21 2.3.1.1 Steeping The first critical step in wet-milling is steeping which involves soaking the whole grains in aqueous solutions for a period of time under controlled conditions of temperature. This step is crucial for ensuring effective milling operation and a maximum yield of quality starch (Jackson and Shandera, 1995). Industrially, steeping is done to soften the kernel, reduce deterioriation of the grain by undesirable microorganisms, and assist in the recovery of pure starch. Several studies employed sulfur dioxide during steeping mainly to disrupt the protein matrix by breaking disulfide bonds and preventing reformation of these bonds by forming soluble S-sulfoproteins (Buffo et al., 1997). As steeping progresses, reducing sugars that leach out of the grain are fermented by Lactobacillus species which have a high tolerance for acids, sulfur dioxide, and elevated temperature (Dailey, 2002). In practice, lactic acid which is the main fermentation product is added to lower the pH and restrict the growth of microorganisms and also soften the kernel components (Buffo et al., 1997). The amount of steep water relative to the amount of grain is also important, especially when it contains chemicals that need to be absorbed by the grains. Ratios of the volume of steep water to the amount of sample used in wet-milling studies vary among different studies. This was demonstrated with corn wet-milling by Krochta et al. (1981), who reported an increase in mill- starch (contains starch, germ and fiber) yields from an average of 67.5% to 70.3% when the steep water to grain ratio was increased from 1:1 to 2:1. Most laboratory wet-milling protocols use steeping times between 24-96 hours to achieve conditions comparable to industrial wet-milling which use average steeping times between 28-30 22 hours (Buffo et al., 1997; Singh and Eckhoff, 1996; Wronkowska, 2016). Long steeping times for laboratory wet-milling are impractical for maintaining daily milling regimens when processing large quantities of samples (Buffo et al., 1997). Maintaining consistent steeping time for laboratory wet-milling could provide a means of identifying suitable millable samples and help to discriminate between different hybrids or cultivars but may not be sufficient to optimize yields of fractions. Longer steep times could mask differences by compensating for poor processing conditions. Long steeping times could also be costly and inconvenient, and there are merits to developing wet-milling protocols with shorter steeping times. Enzymes have also been used to speed up the steeping process and enhance wet-milling (Moheno-Pérez et al., 1997). Serna- Saldivar and Mezo-Villanueva (2003) found a proportional increase in starch recovery using a cell-wall-degrading enzyme complex. 2.3.1.2 Milling and Separation of Grain Components The main aim of the milling process is to separate the various grain components. These operations vary slightly among different studies but generally follow the same basic procedures with similar results (Beta et al., 2001a; Eckhoff et al., 1996; Xie and Seib, 2002, 2000; Zheng et al., 1997). There two milling steps followed separation of a starch and protein slurry. The first milling process during wet-milling is considered the degermination step. This process involves detaching the germ with minimal damage to it and other kernel components. Damaging the germ makes it more difficult to isolate starch, because the damaged germ releases oil into the wet-mill slurry which is absorbed by the grain protein. The oil/protein mixture may also coat the milling equipment, resulting in increased maintenance costs and reduced yields (Buffo et al., 1998). The first grinding step in laboratory wet-milling is often done using a 23 commercial Waring blender with blunt blades (Buffo et al., 1997; Xie and Seib, 2002). In most cases, the blades in the blender are reversed and the blender operated at reduced speed in order to avoid damaging the germ (Jackson and Shandera, 1995). The germ can be removed by skimming off the slurry after adjusting the specific density or sieving the slurry (Eckhoff et al., 1996). The resulting slurry obtained after separating the germ and bran fraction is finely and thoroughly milled to release the remaining starch from the endosperm cellular structure. This usually involves the use of a plate mill (Quaker City mill). The mill plates are conditioned by grinding them down for several hours before first use (Eckhoff et al., 1996; Xie and Seib, 2000). The resulting slurry from fine grinding is separated to obtain the fine fraction by shaking over a sieve or mesh ranging between 63 to 74 microns. Shaking the slurry over the sieve provides for mechanical washing of the fiber. This process can be time-consuming, Eckhoff et al. (1996) spent 4 hours separating the fine fractions for each wet-milling iteration. Industrially, pressure screens are employed for this process. The slurry obtained after the fine fraction separation is often referred to as mill starch and consists of starch and protein (gluten) particles. These can be separated based on their density differences. For commercial operations, centrifuges are used for this purpose and the resulting starch fraction is washed using a battery of hydroclones. Under laboratory settings, the starch is separated by tabling. This involves adjusting the specific density of the mill starch slurry and pumping it onto a sloped wooden troughs or aluminum channels called starch tables. The starch would settle on the table, leaving the protein suspended the water that is run off the table (Singh and Eckhoff, 1996). 24 2.3.1.3 Optimizing Sorghum Wet-milling For Pilot Scale Studies on sorghum wet-milling date back to the 1950’s and the procedures used are based on well established corn wet-milling processes. The lack of sorghum wet-milling plants make it difficult to compare laboratory based experiments to industrial scale productions, however due to the structural similarities between sorghum and corn, there is justification for comparing laboratory scale sorghum wet-milling to pilot scale corn wet-milling. Laboratory and pilot scale wet-milling procedures differ in the amount of sample and the equipment used in fractionation and separation (Singh and Eckhoff, 1996). While laboratory procedures use sample sizes between 50 g to 2 kg in addition to smaller equipment, pilot scale productions use between 10 kg to 70 metric tonnes of grain samples and small-scale industrial equipment (Eckhoff et al., 1996). Singh and Eckhoff (1996) reviewed laboratory and pilot plant-scale corn wet-milling and both procedures were labor and time-intensive which limited the number of experiments that could be run in a given time. This was inadequate for screening large varieties of samples for wet-milling characteristics in terms of mass balance and yields of fractions. To address this issue, Eckhoff et al. (1996) developed and evaluated a 100 g procedure that could be performed routinely within a shorter time and with consistent precision. They obtained an average total solids recovery of 98.3%, which was the same as that recovered from a 1 kg procedure. Xie and Seib (2000) adopted the 100 g wet-milling procedure for sorghum and obtained average total solids of 98.9%. They further isolated six fractions from the sorghum grains: germ and bran (8.3%), fine (4.8%), protein (8.5%), starch (69.4%), process water solubles (2.0%) and steep liquor solids (5.6%). A comparison of the wet-milling fraction in Table 3, shows similar yields between sorghum and corn wet-milling. 25 Table 3: Comparison of wet-milling fraction yields between 100g sorghum and corn wet-milling procedures, and pilot scale wet-milling Corn 100 g wet- milling Corn 1 kg wet- milling Pilot Scale Corn wet-milling (Eckhoff et al., 1996) 5.9 9.8 8.6 67.4 2.3 4.3 98.3 (Singh and Eckhoff, 1996) 10.5 21.8 7.6 58.8 5.1* - 103.8 Sorghum 100 g wet-milling (Xie and Seib, Source (Eckhoff et al., 2000) 8.3 4.8 8.5 69.4 2.0 Germ and Bran Fine Fiber Protein Starch Process Water Solubles Steep Water Solubles Total Recovered Solids *Sum of process water and steep water fractions. 98.9 5.6 1996) 5.2 10.2 8.8 67.3 2.5 4.3 98.3 2.3.2. Sorghum Starch Characteristics Dry unmodified sorghum starch appears pink compared to corn starch which is a white powder. For most industrial applications of starch such as the production of aspirin in the pharmaceutical industry, absolute white starch is most preferred. This is sometimes achieved with bleaching. The most important determinant of starch application is however, related to its pasting properties during cooking (Abegunde et al., 2013; Adil Gani et al., 2010; Chew-Guevara et al., 2016; Sandhu and Singh, 2007). Finished starch obtained after wet-milling can be further dried and sold directly as unmodified starch. They can also be modified by chemical or physical treatments. These 26 modifications can permanently alter the properties of the starch or be designed to preserve the granule structure after which the reagents used can be washed out. Dried, gelatinized starch is also a commodity that can be used in a number of food and non-food products. Finished starches can also be hydrolyzed completely into D-glucose, which can be used in the fermentation industry for the production of ethanol and other products (Eckhoff and Watson, 2009). Sorghum starch granules have two morphologies depending on their location in the grain. The starch granules from the outer, dense, corneous endosperm are polygonal in shape and often dented. Their shape results from tight packing, and the denting is due to the nature of the protein bodies. On the other hand, the starch granules from the inner, less dense, floury endosperm are more spherical and without dents (Emmambux and Taylor, 2013). Sorghum starch granules are very similar to that of corn although corn starch granules may be larger. The starch granules have pores on their surface that create channels to their central cavity (Serna-Saldivar and Rooney, 1995). The rheology of starches is mostly defined by its pasting, viscosity and gelling properties. These properties are exhibited during wet heat processing of the starches in excess water. Most of the rheological information can be obtained from the pasting properties which are determined using equipment such as the Brabender amylograph and the Rapid Visco Analyzer (RVA) (Crosbie and Ross, 2007). Pasting properties are affected by heating and cooling rate, holding temperatures, and solid content. Amylose is important in forming a three-dimensional network during cooling and as a result, amylose and amylopectin ratios affect pasting properties of starches. Waxy and heterowaxy sorghum starches are characterized by lower amylose contents and have higher peak viscosity (Sang et al., 2008). Sang et al. (2008) suggested that amylose 27 prevents swelling of starches during pasting by forming a barrier around the granules, as such low amylose starches also had low setback viscosities. 2.3.2.1 Interactions Between Sorghum Starch and Polyphenols Sorghum is unique among cereals in that it contains a variety of polyphenolic compounds such as condensed tannins (proanthocyanidins), 3-deoxyanthocyanidins, and other flavonoids. The polyphenol compounds in sorghum are responsible for the variety of colors in some sorghum varieties and contribute to the pink off-colors observed in finished sorghum starch. These polyphenols have been shown to protect the seeds against insects, birds, and preharvest germination (Bullard, 1979; Hoshino and Duncan, 1982, 1981). They may also protect the grain against some fungal pathogens (Chandrashekar and Satyanarayana, 2006). Condensed tannins are the major polyphenol present in sorghum and have high antioxidant activity (Beninger and Hosfield, 2003), but they also limit nutrient digestibility by interacting with proteins and digestive enzymes (Agudelo et al., 1997; Butler et al., 1984; Duodu et al., 2003). On studying the nutritional effect of polyphenols on sorghum quality, Butler (1982) suggested that sorghum polyphenols are generally beneficial on the field but may be harmful in the diet. There is more evidence that despite the agronomic advantages of sorghum polyphenols, they also have significant nutritional disadvantages. Their presence contributes to the low digestibility of sorghum starch by binding and forming complexes with proteins and digestive enzymes, making them unavailable for absorption (Barros et al., 2012; Dunn et al., 2015; Zhu, 2015). 28 Besides the nutritional quality, sorghum polyphenols also influence some physical properties of starch. In sorghum varieties with high polyphenol contents, the starch in the grain is able to absorb and retain the phenolic compounds after extraction (Davis and Hoseney, 1979). Evaluation of pasting properties for high polyphenol contents show low peak time and high peak viscosities in comparison with starch that does not contain any polyphenols (Beta et al., 2001a). Boudries et al. (2009) studied the functional properties of pure starches from sorghum grown in the Sahara of Algeria and found high peak viscosities for red sorghum starch that contained polyphenols compared to white sorghum starch. It followed that polyphenols bind with starch molecules to produce pastes with high viscosities at low peak times. The exact mechanism of this interaction is speculative and the phenolic-starch property relationship depends on the type of starch, chemical composition, the structure of the specific phenolic compounds and the pH of the system (Zhu, 2015). Zhu (2015) reviewed the interactions between starch and phenolic compounds, and attributed the changes in physicochemical and nutritional properties of such foods to non-covalent bonding between starch and polyphenols. Other studies suggested the formation of inclusion complexes between starch and polyphenols to have an impact on starch rheology (Beta and Corke, 2004; Bordenave et al., 2014). 2.4. Alkaline Hydrogen Peroxide The use of hydrogen peroxide (H2O2) for bleaching purposes is an established industrial process and has been studied extensively since the discovery of hydrogen peroxide in 1818 (Hardman et al., 1985). In the present study, application of hydrogen peroxide in alkaline medium is proposed as a process to enhance the color of sorghum starch obtained after wet- milling. There is an increase in the use of hydrogen peroxide for bleaching operations in both 29 industrial and domestic settings. Hydrogen peroxide is environmentally friendly and reduces the use of other chemicals used in bleaching operations such as ClO2, Cl2 and NaOCl. This is because hydrogen peroxide breaks down into water and oxygen making it less toxic that most of the alternatives (Salem et al., 2000). Hydrogen peroxide is commercially available in different concentrations and for general applications as bleach, it is often diluted to lower its concentrations (Kandathil, 1980). It is widely used in industries such as the textile and wood pulp industries, and is also employed in the delignification of some agricultural products (Gould, 1985; Zeronian and Inglesby, 1995). In fact, during the bleaching of wood pulp, the process starts with delignification and then ends with eliminating the chromophores that are responsible for the color (Dannacher and Schlenker, 1996). 2.4.1. Mechanism of Alkaline Hydrogen Peroxide To determine how hydrogen peroxide can be used to improve the quality of sorghum starch, it is necessary to understand its chemistry and mechanism of action. The mechanism by which hydrogen peroxide decolorizes compounds has been studied to some extent as many industries interested in using it, require optimum bleaching effects without significant damage to the colored material or products. Hydrogen peroxide is a weak acid and ionizes in water to form hydrogen and perhydroxyl ions (Equation 1). (Equation 1) 30 Several studies have suggested that the perhydroxyl ion is the main species responsible for decolorizing colored chromophores (Zeronian and Inglesby, 1995). Brooks and Moore (2000) postulated that hydroperoxide anion formation was key to H2O2 bleaching and the degree of bleaching was directly related to the concentration of the anion in the bleaching solution. They concluded that there were two competing reactions during H2O2 bleaching: the reaction with the colored bodies and the decomposition of H2O2 to form oxygen. Other studies have suggested that decolorization also occurs by the oxidation of chromophores by radicals which are formed from decomposition of the perhydroxyl ion (Equation 2) and alternatively, further decomposition of H2O2 by the perhydroxyl ion (Equation 3). (Equation 2) (Equation 3) As increasing amounts of perhydroxyl ion are indicative of peroxide decomposition, neutral solutions of hydrogen peroxide are relatively stable due to minimal amounts of perhydroxyl ions at pH 7. In most bleaching operations, the peroxide is activated by adding a base to the bath solution. Under these alkaline conditions the decomposition of hydrogen peroxide is shifted to the right as shown in Equation 4. (Equation 4) Increasing pH results in an increase in the amount of perhydroxyl ions formed from H2O2 dissociation (Thompson et al., 1992). Although the bleaching action of hydrogen peroxide may be enhanced at high pH, it also becomes unstable and decomposes readily. Decomposition of hydrogen peroxide is further catalyzed by metal ions such as copper and iron which is often 31 undesirable in most bleaching industries because it causes damage to fibrous material (Salem et al., 2000). Bleaching occurs when the colored compounds are solubilized and removed, or when unsaturated or conjugated chromophores are altered or destroyed (Zeronian and Inglesby, 1995). There is a general consensus that formation of free radicals and a nucleophilic attack by perhydroxyl anions causes a reaction with colored chromophore groups. The perhydroxyl ions demobilize the electrons in the conjugated double bonds present in chromophores, thereby decolorizing it (Spiro et al., 1997). This reaction between perhydroxyl anions formed from the decomposition of hydrogen peroxide and colored organic molecules is illustrated with the de- conjugation of a quinone structure in Figure 4. The reaction proceeds with a nucleophilic addition of the perhydroxyl anion to the enone structure which results in a disruption of the aromatic ring structure (Hosoya, 1998). Figure 4: Reaction of hydrogen peroxide with quinone by nucleophilic addition of hydroperoxyl anion. 2.4.1.1 Oxidation of Anthocyanins by Hydrogen Peroxide Anthocyanins are widely distributed in the plant kingdom and are attributed to the variety of colors ranging from red, violet, and blue, observed in plants. Their increasing levels in sorghum grains with darker pericarps, suggests their role in the color of sorghum starch. While 32 some carotenoid pigments may also contribute to the dark color of sorghum starches, this review will focus mainly on the color associated with anthocyanins which is more predominant in sorghum. The predominant anthocyanidin in sorghum is 3-deoxyanthocyanins which show a red color (Awika et al., 2004). The color of anthocyanins depends on the number of hydroxyl groups on the B-ring (Figure 2). It also depends on the pH, presence of co-pigments such as flavones and flavonols, and metal ions. The anthocyanin color observed in sorghum and other food crops results from subtle balances between all the factors mentioned above. Altering any of these factors or the structure of anthocyanins could affect its colors. For instance, at low pH (pH<3), the anthocyanins are red and more stable but become colorless under mildly acidic conditions between pH 3-6 (Tanaka et al., 2008). At high pH above 6, it becomes blue and unstable as the quinonoidal base forms. Anthocyanins are degraded in the presence of hydrogen peroxide and contribute more to peroxide scavenging more than other phenolic compounds (Bi et al., 2014; Özkan, 2002; Özkan et al., 2005, 2002). Satake and Yanase, (2018) proposed a mechanism for hydrogen peroxide mediated anthocyanin oxidation based on the products they obtained from the reactions. They found that oxidation occurred at the 3-position from the electron donation by a hydroxyl group which was followed by the cleavage of the C2-C3 bond (Figure 5). 33 OH OH OH OH HO O HO O OH A OH O O OH B Figure 5: Proposed reaction from the hydrogen peroxide mediated oxidation of anthocyanin. 34 CHAPTER 3 Alkaline Hydrogen Peroxide (AHP)-Pretreatment of Sorghum and Effect on Starch 3. Properties 3.1. Abstract Alkaline hydrogen peroxide (AHP)-pretreatment of sorghum was evaluated for its effect on the color and recovery of wet-milled sorghum starch, which often appears dull with off-colors due to the presence of polyphenols. A central composite response surface design was used to determine the optimum H2O2 concentration and pH at which acceptable starch color and recovery could be obtained. The characteristics and functional properties of the starch were also evaluated. AHP- pretreatment increased the degree of lightness for the sorghum starch. Acceptable starch color was obtained with L-value greater than 90 at optimum conditions of pH 11.84 and 20% (w/w) H2O2 concentration. The pH used during AHP-pretreatment was the most significant factor in determining the color of sorghum starch. Varying concentration and pH did not significantly affect the starch recovery based on the response surface (RSM) model. Changes in starch color did not influence the starch recovery and a majority of the starch properties analyzed; however, color was significantly correlated with the starch pH (r = 0.62), swelling power (r = -0.57) and final pasting viscosity (r = 0.62) at p > 0.01, as well as oil absorption capacity (OAC) (r = 0.48) at p > 0.05. AHP-pretreatment of sorghum increases the degree of lightness and removes the off- colors present in sorghum starch with minimal changes in the functional properties of the starch. 3.2. Introduction Sorghum is a drought-tolerant crop and an important food source for food security (Belton and Taylor, 2004). The grains contain between 57% to 79% starch which could be 35 extracted and used for various industrial applications (Sira and Amaiz, 2004). The demand for starch in general continues to increase significantly due to its versatility, however, this demand is met with a limited number of sources. Corn remains the predominant global source of starch, whereas sorghum starch production is almost non-existent, despite similarities between sorghum and corn starches (DeRose et al., 1989). Production of starch from sorghum is done in the same way as corn starch; however, sorghum starch appears darker compared to corn starch which is usually brighter or whiter in appearance. The off-colors present in sorghum starch limits its market desirability and industrial application (Subramanian et al., 1994). Sorghum grains contain a variety of polyphenolic compounds which contribute to the range of colored pigments present in different sorghum varieties. The presence of these pigments in the grain pericarp and the leaves of sorghum result in the off-colors observed after starch extraction. These pigments leach into the endosperm during weathering in the field or during steeping before wet milling (Norris, 1971). Darker colored sorghum grains produce darker colored starch; however, white sorghum grains also produce starches with off-colors due to the presence of carotenoid pigments in the endosperm and tannins in the testa (Beta et al., 2001a). Laboratory wet-milling of sorghum grain has been extensively studied with emphasis on improving production yields by optimizing processing conditions (Buffo et al., 1998; Wang et al., 2000; Xie and Seib, 2000; Yang and Seib, 1995). Only a few studies have addressed the issue of discoloration in sorghum starch, especially from dark-colored sorghum varieties. Strategies to improve sorghum starch color have included decortication or dehulling of the kernels, solvent extraction or bleaching of the whole grains. Decortication or dehulling the kernels is based on the assumption that the outer grain layer, which can be removed abrasively by mechanical force, 36 contains most of the colored material (Chew-Guevara et al., 2016; Mwasaru et al., 1988). Although this approach has been successful in producing brighter sorghum starches, it also results in significant loss of solids and low starch yields (Chew-Guevara et al., 2016). Subramanian et al. (1994) found that extraction of dull starch with methanol resulted in brighter starch which suggested that certain alcohol-soluble compounds were responsible for dull appearance of sorghum starch. (Sira and Amaiz, 2004) isolated starch from red sorghum grains using a bleaching reagent containing an alkaline mixture of sodium hypochlorite and potassium hydroxide. They obtained sorghum starch with improved color and appearance. Toxicity of the extracted starches was a concern for the authors and there is a need to find a safer alternative treatment of the grains. The use of hydrogen peroxide under alkaline conditions is an established industrial process used to bleach agricultural products and other cellulosic materials. It is safer and less toxic compared to sodium hypochlorite and has been tested extensively on various materials (Brooks and Moore, 2000). Treatment of agricultural by-products with hydrogen peroxide under alkaline conditions produces nontoxic carbohydrate sources for use in ruminant feed as microbial feedstocks, and as sources of dietary fiber for humans and other monogastrics (Gould, 1989). Preliminary experiments established that alkaline hydrogen peroxide was capable of removing color from sorghum whole grains; however, it is necessary to determine if the loss of color from the kernels corresponds with an improvement of starch color after wet-milling. The objective of the present study was to investigate AHP-pretreatment of sorghum and determine optimum combinations of H2O2 concentration and pH at which sorghum starch with acceptable color could be obtained. The effect of varying concentration and pH was also determined on the recovery of starch, starch properties, and the functional characteristics of the wet-milled sorghum starch. 37 3.3. Materials and Methods 3.3.1. Sample Preparation Red sorghum (Naga Red, 2018) grains were obtained from Garu in the Northern Region of Ghana. The samples were chosen based on the color of the grain pericarp which appeared dark red, as well as their availability and extensive use in the region. The grains were cleaned by sifting and winnowing to remove particulate matter and broken kernels. After cleaning, all the grain samples were vacuum-packed and stored at 4oC. All reagents and chemicals used were analytical grade. 3.3.2. Alkaline Hydrogen Peroxide Pretreatment Sorghum samples were pretreated with alkaline hydrogen peroxide prior to wet-milling for starch based on procedures described by Brooks and Moore (2000) with some modifications. Approximately 50 g of each sample was pretreated in 100 ml of solution containing different combinations of hydrogen peroxide concentration ranging from 2% (w/w) to 20% (w/w) and pH (9 – 12). The sorghum samples were weighed into a flask containing the required amount of buffer adjusted to the required pH with 0.1 M NaOH and equilibrated to 60o C on a hot plate with a magnetic stirrer. The required amount of H2O2 was measured into the solution to make up the required concentration with a total volume of 100 ml. The temperature of the mixture was maintained at 60oC while stirring continuously for 60 min with the magnetic stirrer. The pretreated samples were then filtered off and washed with distilled water. 38 3.3.3. Wet milling of AHP-Pretreated Sorghum The pretreated sorghum samples were wet-milled to extract starch fractions using the procedure in Figure 6 (Yang and Seib, 1996). Each batch of pretreated sample was ground along with 150 ml of distilled water for 2 min at low speed and then for 4 min at high speed using a commercial blender (CG-Sierra 500, Crompton Greaves Appliance Division, Mumbai, India). The slurry with a temperature of approximately 40o C was poured onto a nylon mesh screen (64 μmm opening), and washed with 5 x 100 ml of distilled water. The resulting throughs were centrifuged at 2800 x g in a temperature-controlled centrifuge set at 4oC (Thermo Scientific Heraeus Megafuge 1.0 Centrifuge) for 15 min, and the supernatant was discarded. The sediment was slurried in 100 ml of distilled water and centrifuged again. After 3 iterations, the mucilaginous protein-rich top layer of the sediment was scraped off with a spatula and the bottom fraction was dried overnight at 40o C. The dried fractions were weighed and assayed for moisture (method 44-19.01, AACC, 2000) and total starch content (McCleary et al., 1994). The amount of starch recovered was determined as the ratio of the amount of starch determined in the starch fraction to the total amount of starch in the whole grain sorghum 39 Figure 6: Wet-milling of sorghum grain (Naga Red) pretreated with alkaline hydrogen peroxide, to obtain starch fraction. 3.3.4. Characterization of Wet-Milled Starch Fractions1 Wet milled starch fractions were evaluated for color using a chromameter (Konica Minolta CR-410, Osaka, Japan). The instrument was calibrated with a white standard (L= 92.32, a= -0.36, b= 4.00). The L value from the Hunter Lab color space was used to determine the brightness of the starch fractions on a scale of 0 to 100, where 100 was indicative of white and 0 was for black. The amount of inorganic or mineral residue in the starch fractions was determined as the ash content using AACC procedures (AACC, 2000 Method 08-01). The pH of the starch fraction was determined using the method described by Benesi et al. (2004). Water and oil absorption capacity (WAC) of the starch fractions were determined at ambient temperature using 1 Details of experimental methods are shown in the appendix 40 the methods outlined by Awolu (2017). The swelling power and water solubility index of each wet-milled starch fraction were determined according to procedures described by Boudries et al., (2009). The pasting properties of the each wet-milled sorghum starch fraction was determined using a Rapid Visco Analyzer (Perten RVA 4500) with procedures described by Crosbie and Ross (2007). Each starch fraction was accurately measured and suspended in distilled water (14% moisture basis). A 23-min heating and cooling cycle was programmed to hold the suspensions (3 g sample/24 g water) at 50 °C for 1 min, then heating to 95 °C in 7.5 min, holding at 95 °C for 5 min before cooling back to 50 °C in 7.5 min and holding at 50 °C for 1 min. The starch viscosity parameters measured were pasting temperature, peak viscosity, peak time, breakdown viscosity, final viscosity and setback viscosity. 3.3.5. Experimental Design A central composite design (CCD) was used to study the effect of hydrogen peroxide concentration and pH on the color and characteristics of the wet-milled starch fractions. The design combined two-level fractional factorials with center points and axial points. The center points were set to the mid-range for all the factor values and the axial points were determined such that all but one factor value was set at the cube-face center. The two factors considered were H2O2 concentration with levels 2% (w/w) and 20% (w/w), and pH with levels 9 and 12. The center points for the experiment were replicated and determined as 11.0% (w/w) H2O2 concentration and pH 10.5. Axial points for H2O2 concentration ranged from 0% (w/w) to 23.7% (w/w) and 8.4 to 12.6 for pH. The chosen variables for optimization were starch color and starch recovery. All experimental runs and results of the CCD were listed in Table 4. 41 Table 4: Experimental responses obtained from the central composite design to determine the effect of varying H2O2 concentration and pH on sorghum starch color, yield, functional and pasting properties Run H2O2 concentration (%(w/w)) 1 2 3 4 5 6 7 8 9 10 20.0 11.0 2.0 0.0 23.7 20.0 2.0 11.0 11.0 11.0 pH 12.0 10.5 12.0 10.5 10.5 9.0 9.0 12.6 10.5 8.4 Starch Color (Hunter L-value) Starch Recovery (%, d.b.) 90.49 ±1.71 86.94 ±8.44 80.93 ±0.28 79.52 ±0.01 82.49 ±2.74 83.04 ±2.98 82.54 ±3.57 90.04 ±6.49 82.91 ±1.58 78.87 ±6.34 71.89 ±9.79 68.40 ±3.98 69.30 ±5.86 66.40 ±4.74 48.96 ±4.13 73.51 ±10.44 61.03 ±13.50 60.08 ±9.21 62.76 ±3.91 88.99 ±3.65 Response values are reported as mean ± standard deviation 3.3.6. Statistical Analysis Each experimental run was carried out in duplicate and thee mean value reported. The experimental design was generated using the statistical software package JMP (Version 7. SAS Institute Inc., Cary, NC). All statistical analyses were performed using Minitab 17 statistical software (Minitab Inc, 2013). The significance of the response surface model was determined with ANOVA and the Lack-of-fit test was used to determine the adequacy of the model to fit the experimental data. Correlations between the starch fraction characteristics were also determined 42 using Person correlation coefficient. All statistical analyses were considered significant at p < 0.05. 3.4. Results and Discussion 3.4.1. Effect of AHP-pretreatment Conditions on Starch Color The color of starch is an important quality characteristic for wet-milling of sorghum, with the ultimate goal of obtaining a pure white starch. The central composite design (CCD) was used to investigate the optimal values, and interaction of H2O2 concentration and pH on the sorghum starch color obtained after AHP-pretreatment. Starch color was measured in terms of the degree of lightness indicated by the Hunter L-value. The experimental data for starch color in Table 4, were subjected to quadratic multiple regression and the polynomial equation that was obtained (Equation 5), indicated the quantitative effect of concentration and pH and the interactions on the wet-milled starch color. Starch color = 132.9 – 1.97 (Concentration) - 9.5 (pH) – 0.0150 (Concentration)2 + 0.42 (pH)2 + 0.24 (Concentration*pH) (Equation 5) The results of the regression and ANOVA on the CCD model are presented in Table 5. The regression model for sorghum starch color was statistically significant based on the p-value (<0.05). The p-value for the lack-of-fit test was not significant (p = 0.460 > 0.05), indicating that the regression model was adequate and could be used to fit the experimental data. The 43 determination coefficient (R2) showed that the model explained 76.13% of variability in results for sorghum starch color obtained from the experiment. Significance tests for the model coefficients showed that varying the H2O2 concentration within the experimental limits did not have a significant effect (p = 0.220 > 0.05) on sorghum starch color. On the other hand, varying the pH of pretreatment medium had a significant effect on sorghum starch color (p <0.05). Both squared and interaction terms in the model were not statistically significant (p > 0.05). 44 Table 5: Analysis of variance results for the regression model developed from the central composite design to evaluate the effect of H2O2 concentration and pH on wet-milled starch color Remarks Significant F-Value P-Valuea Prob > F 0.032 0.027 0.009 0.220 0.006 0.415 0.436 0.463 0.052 0.052 4.25 7.32 8.94 1.77 14.11 0.98 0.67 0.59 5.19 5.19 2.41 0.460 Not significant Mean square 44.44 76.54 93.4 18.49 147.45 10.28 7.02 6.2 54.2 54.2 10.45 11.28 4.68 Sum of squares 266.64 76.54 186.81 18.49 147.45 20.57 7.02 6.2 54.2 54.2 83.62 78.94 4.68 350.27 df 6 1 2 1 1 2 1 1 1 1 8 7 1 14 Source Model Blocks Linear Concentration pH Square Concentration2 pH2 2-Way interaction Concentration*pH Error Lack-of-Fit Pure Error Total 76.13% R2 Adjusted R2 58.22% a P < 0.05 denotes significant difference The response surface plot was obtained based on the second order polynomial equation (Figure 7). An increase in pH was followed by a corresponding increase in the brightness of the starch fraction indicated by the Hunter L-value. The influence of pH on the starch color occurred at all concentrations of H2O2 but significantly increased at higher concentration. Wang et al. (2000) suggested that L-values greater than 90, which characterized the degree of lightness for sorghum starch were satisfactory. 45 Figure 7: Response surface plot for the effect of varying H2O2 concentration and pH on starch fraction color. The results show that an increase in the degree of lightness for sorghum starch obtained after AHP-pretreatment was dependent on the pH level used. The formation of perhydroxyl anions from hydrogen peroxide under alkaline conditions was proposed as the primary mechanism for removing color (Gould, 1985; Hosoya, 1998; Spiro et al., 1997; Thompson et al., 1992; Zeronian and Inglesby, 1995). It is likely that increase in the degree of sorghum starch lightness resulting from a corresponding pH increase was due to higher perhydroxyl anion concentration at high pH during AHP-pretreatment. 46 3.4.2. Effect of AHP-pretreatment on Sorghum Starch Recovery The recovery of starch from AHP-pretreated sorghum was determined as a ratio of the amount of assayable starch in the wet-milled starch fraction to the amount of assayable starch in the whole grain prior to wet-milling. The experimental data obtained for starch recovery are also shown in Table 4. Equation 6 shows the polynomial equation determined from regression of the experimental data for starch recovery. Starch recovery = 432 + 1.68 (Concentration) – 69.2 (pH) – 0.0823 (Concentration)2 + 3.12 (pH)2 + 0.045 (Concentration*pH) (Equation 6) Results for the regression and ANOVA on the starch recovery data are shown in Table 6. The regression model was not statistically significant (p = 0.256 > 0.05) and did not fit the experimental data as indicated by a significant (p < 0.05) lack-of-fit test. Based on the results, varying the AHP-pretreatment conditions (H2O2 concentration and pH) within the experimental limits used in the study did not significantly influence the recovery of sorghum starch after wet- milling. Due to the inability of the model to adequately fit the experimental data, a response surface plot was not presented. 47 Table 6: Analysis of variance results for the regression model developed from the central composite design to evaluate the effect of H2O2 concentration and pH on wet-milled starch recovery Source Model df 6 Sum of squares Mean square F-Value P-Valuea Prob > F Remarks Not significant 0.256 0.516 0.389 0.948 0.185 0.085 0.233 0.137 0.906 0.906 1239.50 206.58 1.63 0.46 1.07 0.00 2.11 3.40 1.66 2.74 0.01 0.01 58.68 58.68 270.70 135.35 0.58 0.58 267.40 267.40 863.32 431.66 211.34 211.34 347.80 347.81 1.89 1.89 1.89 1.89 1015.99 127.00 1015.99 145.14 88593.31 <0.01 2255.49 <0.01 1 2 1 1 2 1 1 1 1 8 7 1 14 Blocks Linear Concentration pH Square Concentration2 pH2 2-Way interaction Concentration*pH Error Lack-of-Fit Pure Error Total R2 Adjusted R2 a P < 0.05 denotes significant difference 54.95% 21.17% 0.003 Significant Although the response surface model did not fit the experimental data for starch recovery, the values obtained were comparable to levels reported in similar studies (Wang et al., 2000; Xie et al., 2006; Xie and Seib, 2002; Yang and Seib, 1995). Without steeping, an average starch recovery of 78.0% was reported by Xie and Seib (2002). In practice, grains to be wet-milled are 48 steeped in aqueous solutions for long periods of time in order to facilitate separation of grain constituents and improve starch recovery. 3.4.3. Validation of response surface model The optimum conditions for H2O2 concentration and pH during AHP-pretreatment of the sorghum grain were determined with the goal of obtaining degree of lightness (starch color) equal to or exceeding an L value of 90. Since varying H2O2 concentration within the experimental limits did not have a significant effect on starch color, the variable setting for concentration was held at 20% (w/w). To obtain a predicted L-value of 90 using a H2O2 concentration of 20% (w/w) according to the model, an average pH of 11.84 would be used during AHP-pretreatment of the sorghum grains. The ability of the fitted model to predict starch color was validated by randomly selecting combinations of H2O2 concentration and pH, and comparing the experimental results for starch color to the predicted values (Table 7). The starch color obtained from four out of the five runs fell within acceptable boundaries at the 95% confidence interval. The confidence interval represented a range of values which were likely to contain the mean L-value for the selected combinations of H2O2 concentration and pH. The prediction interval on the other hand, represented the range that was likely to contain single future responses for the selected combinations with added uncertainty. The optimum L value obtained at the optimum condition in the study was higher than the color of sorghum starches obtained by Sira and Amaiz (2004), who obtained an average L-value of 78.4 after treating the sorghum grains with a combination of sodium hypochlorite and potassium hydroxide. 49 Table 7: Comparison of experimental and predicted values for starch fraction color (Huner L- value) at five randomly selected levels of H2O2 concentration and pH H2O2 Run Concentration pH Starch color (Hunter L-value) Confidence limits* Prediction limits* 1 2 3 4 5 10.50 10.50 12.00 10.50 9.00 (%w/w) 11.00 0.00 2.00 0.00 20.00 Expa 92.91 79.52 81.12 79.51 80.93 Pred 83.07 78.81 80.35 78.81 78.75 *95% confidence and prediction limits aExp-Expeirmental results, Pred-Predicted fit, SE-Predicted standard error High 87.42 85.26 87.16 85.26 83.99 SE 1.89 2.80 2.95 2.80 2.27 Low 78.71 72.37 73.54 72.37 73.51 Low 74.43 68.96 70.26 68.96 69.64 High 91.70 88.67 90.45 88.67 87.87 3.4.4. Correlation among starch fraction properties Correlations between the color, recovery and other properties of the wet-milled starch fraction were determined and correlation coefficients shown in Table 8. Changes in starch color as a result of varying H2O2 concentration and pH used during AHP-pretreatment was not associated with a majority of the starch characteristics and functional properties determined. This included the ash content, WAC, WSI, peak time, pasting temperature, peak pasting viscosity, breakdown viscosity and setback viscosity. Significant correlations were, however, determined between the starch color and the starch pH (r = 0.62), swelling power (r = -0.57) and final pasting viscosity (r = 0.62) at p > 0.01, and with oil absorption capacity (OAC) (r = 0.48) at p > 0.05. The starch pH increased as the degree of lightness for the starch also increased, mainly due to the influence of pH on starch color during AHP-pretreatment. Positive correlations were found between the starch pH, final pasting viscosity (r = 0.54 at p < 0.05) and setback viscosity (r = 50 0.68 at p < 0.01), but a majority of the starch properties were not associated with the changes in starch pH. Starch granules in aqueous suspensions, when heated, take up water and swell, ultimately releasing some of their soluble constituents. The results showed that starch swelling power determined at 90oC decreased as the starch color became lighter, suggesting that AHP- pretreatment restricted swelling of the starch granules. Decreasing swelling power also significantly correlated (p < 0.05) with higher peak time (-0.52) and pasting temperature (-0.54). Swelling power was also associated with increasing OAC (r = -0.50 at p< 0.05). 51 Table 8: Correlation of wet-milled starch properties obtained from AHP-pretreated sorghum at varying H2O2 concentrations and pH 1. Starch recovery 2. Ash content 1 -0.26 0.38 2 3 4 5 6 7 8 9 10 11 12 13 0.24 3. Starch pH 0.62** -0.13 0.35 0.07 0.14 -0.01 0.10 -0.13 -0.01 0.16 0.11 4. WAC 5. OAC 6. Swelling power 7. WSI 8. Peak time 9. Pasting temp 10. Peak viscosity 11. Breakdown viscosity 12. Final viscosity 13. Setback viscosity ‘***’: p < 0.001, ‘**’: p < 0.01, ‘*’: p < 0.05. WAC–Water absorption capacity; OAC–Oil absorption capacity; WSI–Water solubility index. 0.02 0.48* -0.57** 0.09 -0.37 -0.30 -0.16 -0.50* -0.30 -0.12 -0.15 0.03 0.05 0.15 0.17 -0.38 -0.08 0.05 0.30 0.20 0.44* -0.18 0.25 0.17 0.29 0.24 -0.38 -0.16 -0.30 -0.13 -0.14 -0.11 -0.11 0.32 -0.33 -0.36 0.62** -0.34 0.18 0.54* -0.21 0.13 0.01 0.30 0.68** -0.28 -0.16 0.18 0.20 -0.52* 0.01 0.26 -0.54* 0.07 0.82*** 0.20 0.26 -0.64** -0.70*** 0.44 0.11 -0.88*** -0.77*** 0.81*** 0.82*** -0.18 0.20 -0.44* 0.12 0.00 -0.82*** -0.59** 0.66** 0.81*** 0.68** 0.48* -0.43 52 3.5. Conclusion AHP-pretreatment of sorghum is presented as alternative approach to obtaining sorghum starch with acceptable color and quality. The presence of polyphenols in sorghum results in starches with off-colors which limits their use as a source of industrial starch. AHP-pretreatment of the sorghum grains prior to wet-milling improves the starch color by increasing the degree of lightness. Hydrogen peroxide under alkaline conditions produces perhydroxyl anions which may have been responsible for the loss of color from the starch observed after sorghum wet-milling. Optimum conditions for AHP-pretreatment required to maintain acceptable starch color with L- values of 90 were obtained at pH 11.84 and 20% (w/w) H2O2 concentration, based on response surface methods. The pH of the medium during pretreatment was most significant in determining the color of the wet-milled starch. Starch recovery on the other hand, was not influenced by varying the H2O2 concentration and pH. Changes in starch color resulting from varying the conditions during AHP-pretreatment did not influence most of the starch characteristics and functional properties studied. While AHP-pretreatment is effective at enhancing the color of wet- milled sorghum starch, more research is needed to evaluate its application in pilot scale sorghum wet-milling. 53 CHAPTER 4 Wet-Milling of Alkaline Hydrogen Peroxide (AHP)-Pretreated Sorghum and 4. Characterization of the Sorghum Starch Fraction 4.1. Abstract Alkaline hydrogen peroxide (AHP)-pretreatment of sorghum was evaluated as an alternative approach to improving the quality of wet-milled sorghum starch, which typically appears dull as a result of colored polyphenols in the grain. A factorial experiment was carried out to study the effect of AHP-pretreatment and further steeping in water for 24 hours at 55oC on the wet-milling recovery yields and the properties of the wet-milled starch fraction from four sorghum cultivars. The presence or absence of tannins were identified in the sorghum cultivars but had no effect on the yields of wet-milled fractions and the properties of the wet-milled starch. AHP-pretreated grain produced significantly higher (p < 0.05) starch fraction yields and total starch recovery. It also produced brighter starches particularly from the tannin-containing cultivars which produced darker starches without pretreatment. Steeping in water alone reduced the brightness of starches from the tannin-free sorghum cultivars. The water solubility index of the starches increased significantly (p < 0.05) after AHP-pretreatment; however, the majority of starch functional properties were not significantly affected (p > 0.05) by AHP-pretreatment or steeping in water. This study demonstrates that acceptable wet-milled starch can be obtained from tannin- containing sorghum cultivars generally considered unsuitable for wet-milling due to their colored pericarps and high polyphenol content. 54 4.2. Introduction Global sorghum production in 2017 was estimated at 58 million tonnes and ranked fifth among other cereal crops in terms of global production (FAOSTAT, 2019). Africa contributed to 47% of global sorghum production, and approximately 32% of the global production was grown in the United States. The grain is food staple in many developing countries but is used mainly for animal feed in the United States (Anglani, 1998). The structure and composition of sorghum is similar to that of corn, and is made up of the outer bran (8%), oil-rich germ (10%), and a starchy endosperm (82%), which contains ~73.8% starch (Serna-Saldivar and Rooney, 1995). Recent interests in sorghum as a viable food security crop and its low cost in comparison to corn has led to investigation of sorghum wet-milling as an alternative source of industrially produced starch (Eckhoff and Watson, 2009). Wet-milling of sorghum is done in the same way as corn, and involves a combination physical, chemical and biochemical operations designed to separate the grain components (Buffo et al., 1998; Xie et al., 2006; Xie and Seib, 2002; Yang and Seib, 1995). Recovery of starch from sorghum wet-milling is reported to be much lower than corn, with an average yield of 30% compared corn starch yields which range between 80% to 90% (Wronkowska, 2016). It is more difficult to recover starch from sorghum because of the fragile nature of the pericarp, which breaks up into small particles that impede the separation of starch from protein during wet- milling, and also influences the color of the starch (Beta et al., 2001a). Sorghum starch appears darker than corn starch, and this is a major challenge for sorghum starch production. These colors are mainly associated with presence of polyphenols in the grain pericarp and endosperm. 55 Subramanian et al. (1994) previously suggested that highly pigmented sorghum was unsuitable for wet-milling and there was a need to improve the color of sorghum starch. Several studies have investigated the wet-milling of sorghum with emphasis on increasing starch yield and recovery, but few efforts have addressed improvement of starch color from pigmented grains (Buffo et al., 1997; Sira and Amaiz, 2004; Wang et al., 2000; Xie et al., 2006; Xie and Seib, 2000). Buffo et al. (1998) evaluated wet-milling of 24 sorghum hybrids and found that grain characteristics such as the thousand kernel weight and chemical composition had significant correlations with different wet-milling attributes. Regardless of the type of grain, steeping them in aqueous solutions before wet-milling is important in facilitating the separation of grain components. Steeping solutions usually include optimal combinations of sulfur dioxide and lactic acid; however, plain water has also been considered with comparable recovery yields of wet-milled products (Buffo et al., 1997; Wang et al., 2000). Adequate steeping of sorghum grains for wet-milling requires between 24 hours to 96 hours; however, acceptable starch yields up to 80% were also obtained using shorter steeping and no steeping at all (Xie et al., 2006; Xie and Seib, 2002). The use of enzymes in steeping solutions has also been considered and Serna- Saldivar and Mezo-Villanueva (2003) found that increasing concentration of cell-wall-degrading enzymes during steeping resulted in a proportional increase in starch recovery. There is still a need to address the challenges regarding the color and appearance of wet- milled sorghum starch. Yang and Seib (1995) found washing the starch with sodium hydroxide solution improved starch color. Sira and Amaiz (2004) using a mixture of sodium hypochlorite and potassium hydroxide, obtained acceptable color for sorghum starch wet-milled from highly pigmented sorghum. These treatments present issues of toxicity resulting from reagent residue 56 and as such, a need to investigate less toxic treatment options. Alkaline hydrogen peroxide (AHP) treatment is an established industrial process used for bleaching textiles, paper, and agricultural byproducts and is considered less toxic, because hydrogen peroxide readily breaks down into water and oxygen (Brooks and Moore, 2000; Renard et al., 1997; Zeronian and Inglesby, 1995). While AHP-pretreatment is increasingly being preferred as an alternative for chlorine-based bleaches, it has not yet been considered for sorghum wet-milling, where white starch is valued over darker sorghum starch. The present study investigated the effect of pretreating sorghum with AHP and steeping in plain water on the recovery of wet-milled products, as well as the color and quality of wet-milled sorghum starch. 4.3. Materials and Methods 4.3.1. Sorghum Samples Four sorghum cultivars were obtained from the Savannah Agricultural Research Institute in Northern Ghana. These were Dorado, Kapaala, ICRISAT, and Kadaaga. Dorado and Kapaala were both white in color compared to Kadaaga which appeared red and ICRISAT which had brown stripes. All the samples were cleaned by sifting and winnowing to remove foreign materials and broken kernels. The sorghum samples were characterized by determining the presence of tannins using the rapid “bleach” test described by (Waniska et al., 1992). Other parameters determined were the thousand kernel weight (TKW) and the proximate composition of the whole grains, which included the moisture content, crude protein, crude fat, ash and total starch content (AACC, 2000; AOAC, 1990). 57 4.3.1.1 Alkaline Hydrogen Peroxide Pretreatment AHP-pretreatment was done by soaking 100 g of sample in 200 ml of a solution containing 20% (w/w) H2O2 adjusted to pH 12 with 0.1 M NaOH. The mixture was placed in a water bath at 60o C and shaken at 10-min intervals for a period of 60 min. The grains were then filtered and washed with 500 ml of distilled water. 4.3.1.2 Steeping of Sorghum Grain in Water Samples of AHP-pretreated and untreated sorghum were further steeped by immersing 100 g of sample in 200 ml of distilled water maintained at 55oC for 24 hours in a thermostatically controlled water bath. After steeping, the samples were removed and washed with 500 ml of distilled water. 4.3.1.3 Wet-Milling Procedure The sorghum samples were wet-milled based on the procedures described by Xie and Seib (2002) as shown in Figure 8. Accurately weighed samples (100 g, dry basis) along with 150 ml of distilled water were transferred into a grinding mill (Quaker City, Model No. 4E, Straub Co., Warminster, PA) and ground into a slurry. The slurry was then sieved through a nylon mesh (420 μmm opening). The overs were washed with 500 ml of distilled water, dried overnight at 40o C and weighed as the coarse fraction. The throughs on the other hand were allowed to settle for 30 min and decanted. The bottom layer was ground into a fine slurry in the plate mill, and rinsed off with the previously decanted upper layer. The resulting slurry was sieved through a nylon mesh (74 μmm opening) and washed with 200 ml of distilled water. The overs were dried at 40oC overnight and weighed as the fine fraction. The throughs were centrifuged at 2800 x g for 15 min 58 and the supernatant decanted and discarded. The sediment was re-slurried in 100 ml of distilled water and centrifuged again. After decanting the supernatant, the mucilaginous protein-rich top layer was carefully scraped off with a spatula. The sediment was dried overnight at 40oC and weighed as the starch fraction. All the fractions obtained were assayed for moisture and Total Starch contents (McCleary et al., 1994). Coarse, fine and starch fraction yields were determined as the amount of dry product obtained, per dry weight of wet-milled grain. Starch recovery was determined as the percentage of assayable starch in the starch fraction relative to the total starch determined in the whole grain. 59 Figure 8: Procedure for wet-milling sorghum grain samples into three fractions (Coarse, fine and starch fractions) 60 4.3.2. Characterization of Starch2 The wet-milled sorghum starch fraction was further characterized by determining the amount of crude protein in the starch based on the Total Kjeldahl Nitrogen (AOAC, 1990). Amylose content of the starch fraction was determined colorimetrically based on amylose-iodine binding as described by Beta et al. (2001a). Starch color was also evaluated by measuring the L- value based on the Hunter Lab color space using a chromameter (Konica Minolta CR-410, Osaka, Japan). Functional characteristics of the starch were evaluated by determining the water and oil absorption capacities, swelling power and water solubility index according to methods described by Awolu (2017) and Boudries et al. (2009). Pasting properties were determined using a Rapid Visco Analyzer (Perten RVA 4500) as described by Crosbie and Ross (2007). 4.3.3. Experimental Design and Statistical Analysis A three-factor experiment was conducted to evaluate the effect of grain cultivar, AHP- pretreatment, and 24-hour steeping on the wet-milling characteristics and the properties of wet- milled starch. All the experiments were replicated and the effect of the experimental factors was determined by ANOVA. Significant differences between levels of the experimental factors were determined using the least significant difference test at 95% confidence. 2 Details of experimental procedures are reported in the Appendix 61 4.4. Results and Discussion 4.4.1. Grain Characteristics and Chemical Composition of Sorghum Cultivars The characteristics and chemical composition of the sorghum cultivars used in the study were determined and reported in Table 9. Based on the rapid “bleach” test described by Waniska et al. (1992), cultivars Dorado and Kapaala, which were both characterized by white seed colors were determined as Type I (tannin-free) cultivars. The ICRISAT and Kadaaga cultivars both had pigmentation in their pericarps and were found to contain tannins. They were classified as Type II/III. The physical and chemical properties for all four sorghum cultivars were within the range of values recommended for wet-milling (Pérez-Carrillo and Serna-Saldívar, 2006). Typically, sorghum is reported to contain approximately 12.3% protein, 3.6% fat and 1.65% ash (Wang et al., 2000). The tannin-free cultivars, Dorado and Kapaala had significantly higher thousand kernel weights (TKW) of 32.86 g and 41.16 g, respectively, compared to the tannin-containing cultivars, ICRISAT (26.02 g) and Kadaaga (23.47 g). The tannin-free cultivars also contained higher amounts of protein. The amount of starch and fat content among the sorghum cultivars did not differ significantly (p <0.05). 62 Table 9: Grain characteristics and chemical composition of the four sorghum cultivars (Dorado, ICRISAT, Kapaala, Kadaaga) Dorado White Kapaala White Kadaaga Red ICRISAT White/Brown stripes Type II/ III 26.02 ± 0.26 c 9.89 ± 0.11 c 2.46 ± 0.20 a 1.38 ± 0.002 a 72.16 ± 6.06 a Type I 41.16 ± 0.73 a 11.20 ± 0.02 a Type I 32.86 ± 1.44 b 10.63 ± 0.03 b Pericarp description Grain type† TKW‡ (g) Protein (N x 6.25),% Fat (%) Ash (%) Starch (%) Results are shown as mean ± standard error. Values with different letters for each parameter are significantly different (p < 0.05) † Type I – Tannin-free; Type II – Tannin-containing ‡Thousand Kernel Weight Type II/ III 23.47 ± 0.79 c 10.28 ± 0.24 bc 2.46 ± 0.22 a 1.34 ± 0.005 b 65.31 ± 2.64 a 2.53 ± 0.64 a 1.24 ± 0.001 c 66.46 ± 3.08 a 2.02 ± 0.15 a 1.25 ± 0.001 c 69.05 ± 4.92 a 4.4.2. Recovery of Wet-milling Products The main wet-milling products evaluated were the coarse, fine and starch fractions. Table 10 shows the yields of these wet-milled fractions excluding the soluble losses which were not quantified. The total solids recovered was indicative of the consistency of the wet-milling procedure, where the objective was to recover the maximum amount of solids from the grains after wet-milling. The total solids recovered in this study ranged from 68.97% for untreated ICRISAT grains also steeped in water for 24 hours to 98.99% recovered from AHP-pretreated Kapaala grains without further steeping in water. On average, AHP-pretreated grains produced significantly higher (p< 0.05) amounts of total solids compared to untreated grains, irrespective of the grain cultivar or further steeping in water. The amount of coarse and fine fractions 63 quantified as a proportion of the total amount of wet-milled grain, ranged between 12.99% - 45.97% and 14.00% - 34.99%, respectively, while the starch fraction yields ranged from 19.99% to 52.98% for all the sorghum cultivars both pretreated and untreated. The effect of grain cultivar or pretreatments on the coarse fraction could not be established due to significant interactions (p >0.05) between the grain cultivars and different treatments. The average fine fraction yield from the Kadaaga cultivar on the other hand, was found to be significantly higher (p < 0.05) than that recovered from the ICRISAT and Dorado cultivars but not significantly different from Kapaala. AHP-pretreatment and steeping in water did not have a significant effect (p > 0.05) on the fine fraction yields obtained. Starch fraction yields obtained from AHP-pretreated grains were significantly higher (p < 0.05) than that obtained from untreated grains. Differences in sorghum cultivar and steeping in water did not have a significant effect (p > 0.05) on the starch fraction yields. Su et al. (2015) demonstrated that AHP-pretreatment was capable of breaking down plant cell wall and this could have accounted for the comparatively higher starch fraction yields obtained from AHP-pretreated grains. In comparison to the results reported in similar wet- milling studies, lower levels of total solids and starch fraction yields were obtained in this study. Such differences could have occurred as a result of the variations in the laboratory wet-milling setup. This discrepancy was also noted by Jackson and Shandera (1995). Xie and Seib (2000) obtained total recoveries of 98.9% from sorghum using the same wet-milling procedure described by Eckhoff et al. (1996), who obtained 98.3% total solids for corn. Both of these studies improved the separation of grain components by steeping the grain samples in aqueous solutions containing varying amounts of sulfur dioxide and lactic acid for periods between 24 64 and 96 hours. Since the starch fraction is the most economically viable product from wet-milling, it is important to obtain high starch fraction yields. Typically for corn, starch fraction yields reported from laboratory wet-milling range between 58.4% and 68.5%, while an average of 58.5% is obtained from pilot scale wet-milling (Singh and Eckhoff, 1996). 65 Table 10: Effect of AHP-pretreatment and steeping on the recovery yields of wet-milling products from four sorghum cultivars Dorado ICRISAT Kadaaga Kapaala No steep 24 hr steep No steep 24 hr steep No steep 24 hr steep No steep 24 hr steep c c ab ab 18.99 ±1.00 21.00 ±1.00 24.98 ±0.99 26.00 ±2.00 c c ab ab 15.00 ±3.00 16.99 ±1.00 21.00 ±9.00 14.00 ±10.00 14.99 ±9.00 15.00 ±5.00 32.99 ±9.00 c c a 14.00 ±2.00 16.00 ±0.00 34.99 ±6.99 c c a 24.00 ±2.00 bc 17.00 ±5.00 45.97 ±3.99 a 13.99 ±0.00 29.99 ±6.00 ab 29.99 ±4.00 ab 19.99 ±2.00 29.00 ±3.00 ab ab 22.00 ±2.00 28.98 ±3.00 c c ab ab abc abc Coarse Fraction (%) AHP* 12.99 ±2.99 c 17.00 ±7.00 NT 34.98 ±6.99 ab 14.00 ±2.00 Fine Fraction (%) AHP NT 15.99 ±1.99 14.99 ±7.00 Starch Fraction (%) b b a 22.99 ±5.01 28.99 ±3.00 c c ab b ab bc AHP NT 52.98 ±7.01 44.98 ±10.98 ab 39.97 ±0.01 26.98 ±3.00 bc 35.99 ±16.00 abc 28.00 ±0.00 abc abc 47.98 ±17.99 37.98 ±0.01 40.98 ±10.99 30.99 ±1.00 abc abc 34.99 ±3.00 37.99 ±7.99 Total Solids Recovery (%) AHP NT 81.96 ±2.02 76.95 ±2.99 bc bc 84.97 ±1.03 78.98 ±11.00 ab bc 83.94 ±0.02 abc 83.98 ±5.98 abc 88.96 ±7.00 74.99 ±1.00 bc 68.97 ±11.02 c 75.98 ±2.00 ab bc 83.98 ±5.99 83.97 ±3.99 abc abc abc abc 46.00 ±4.00 ab 43.99 ±2.00 43.98 ±0.01 19.99 ±0.00 98.99 ±1.00 c a 82.98 ±4.99 abc 85.94 ±1.99 ab 86.95 ±3.02 c *AHP- AHP-pretreatment; NT- No treatment. Values are means ± standard error of two replications. Means within each category with different letters are significantly different at p < 0.05. 66 4.4.3. Characteristics of Starch Fraction The wet-milled starch fraction obtained in its crude form, was a combination of pure starch and other grain components which was mainly protein residue. To account for the total starch recovered, the amount of assayable starch in the wet-milled starch fraction was determined as a percentage of the initial amount of starch determined in the whole grain samples. Total starch recovery ranged between 30.06% to 74.68% on a dry weight basis. The four cultivars had similar total starch recoveries in general (Table 11); however, AHP-pretreatment of the grains resulted in significantly higher (p < 0.05) total starch recovery. There were no significant differences (p > 0.05) resulting from steeping the grains in water alone. The amounts of protein residue in the starch fractions were similar for all the grain cultivars and the different treatments and ranged between 1.32% and 3.01%. During wet-milling, adequate separation of protein from starch is essential in ensuring high quality wet-milled starch. Steeping of the grains in sulfur dioxide prior to wet-milling has been suggested as aiding in this separation by breaking the disulfide bonds between the protein bodies (Buffo et al., 1997). The use of water alone during steeping showed no evidence of improving protein-starch separation. The results also did not indicate a significant change (p > 0.05) in the amylose content of the starch fractions which ranged between 24.53% and 36.00%. The color of wet-milled sorghum starch is one the most important characteristics that determines the quality of the starch. The color values for the wet-milled starches are presented in Table 11. Without additional treatment, the tannin-containing cultivars ICRISAT and Kadaaga, which also had pigmented testa and pericarps, produced starches with darker colors at L-values of 85.08 and 84.61, respectively, compared to starches from the tannin-free cultivars Dorado 67 (94.95) and Kapaala (94.40). The study showed that AHP-pretreated sorghum produced starch fractions with significantly (p < 0.05) higher L-values compared to the untreated sorghum. Starches from the tannin-containing cultivars which had been pretreated with AHP appeared whiter with higher L-values (ICRISAT – 99.04; Kadaaga – 95.96) compared to starches obtained from the tannin-free cultivars also pretreated with AHP (Dorado – 93.42; Kapaala – 93.83). This significant increase in the degree of lightness was likely due to interactions between AHP and the polyphenols responsible for the color in sorghum. Since these colored polyphenols were not present in the tannin-free cultivars, a similar effect in starch color was not observed in that case. Steeping the grains in water alone did not improve the starch color, and rather resulted in a significantly (p < 0.05) lower degree of lightness, especially for the white-seeded and tannin-free cultivars. The L-value for Dorado decreased from 94.95 to 82.50, while Kapaala decreased from 94.40 to 85.59. The off-white color in starch obtained from tannin-free sorghum has been attributed to the presence of noncarotenoid pigments present in the grain endosperm (Subramanian et al., 1994). 68 Protein Residue (%) AHP NT 1.32 3.01 ±0.07 ±1.01 Amylose Content (%) AHP 27.22 ±5.92 NT 29.88 ±3.09 Starch Color (L-value) b a a a 2.10 3.01 ±0.51 ±0.24 36.00 ±4.66 24.53 ±0.88 ab a a a 1.67 1.88 ±0.61 ±0.37 33.52 ±0.32 26.55 ±2.05 b ab a a 1.45 1.58 ±0.61 ±0.64 27.81 ±2.57 25.64 ±3.11 AHP 93.42 ±2.26 a-f 86.39 ±0.09 c-g 99.04 ±0.39 a 98.54 ±2.07 NT 94.95 ±0.73 abc 82.50 ±6.74 g 85.08 ±2.53 efg 79.09 ±2.62 b b a a a g 2.30 1.67 ±0.10 ±0.10 27.40 ±6.52 25.27 ±3.49 95.96 ±2.79 84.61 ±1.42 ab b a a ab fg ab d ab ab a a 68.50 ±1.25 70.19 ±0.49 abc abc 1.53 2.03 ±0.12 ±0.20 b ab 30.98 ±3.95 26.20 ±1.75 a a 1.72 1.50 ±0.14 ±0.35 27.32 ±5.49 31.32 ±0.01 b b a a 2.22 2.47 ±0.07 ±0.23 25.37 ±5.62 33.10 ±5.41 Table 11: Effect of AHP-pretreatment and steeping on characteristics of wet-milled starch fractions from four sorghum cultivars Dorado ICRISAT Kadaaga Kapaala No steep 24 hr steep No steep 24 hr steep No steep 24 hr steep No steep 24 hr steep Total Starch Recovery (%) AHP* 74.68 ±9.44 NT 37.31 ±4.27 a cd 69.56 ±16.24 51.91 ±24.48 abc a-d 55.89 ±4.30 a-d 65.99 ±23.10 39.91 ±0.31 bcd 53.09 ±1.35 abc a-d 62.65 ±15.60 44.90 ±2.96 a-d a-d 53.51 ±5.36 60.07 ±12.01 a-d a-d 72.63 ±8.52 30.06 ±0.81 94.24 ±4.60 87.69 ±1.54 a-e b-g 93.83 ±1.68 94.40 ±2.49 a-f a-d 95.16 ±4.61 85.59 ±4.64 abc d-g *AHP- AHP-pretreatment; NT- No treatment. Values are means ± standard error of two replications. Means within each category with different letters are significantly different at p < 0.05. 69 4.4.4. Functional Properties of Starch Fraction The functional properties of the wet-milled starches were determined and reported in Table 12. The water and oil absorption capacities ranged between 1.09 g/g – 1.70g/g and 0.98 g/g – 1.69 g/g, respectively. The swelling power of the starch fractions at 90oC was also determined and ranged between 8.88 g/g and 11.89 g/g for all the sorghum samples. Water solubility index (WSI) of the starch fractions ranged between 2.04% and 19.46%. Analysis of the results showed no significant difference (p < 0.05) between the functional properties for all the sorghum cultivars. With the exception of the water solubility index, AHP-pretreatment and steeping did not have a significant effect (p > 0.05) on the functional properties of the wet-milled sorghum starch. The water solubility index of the starches increased significantly after AHP-pretreatment of the grains, which was likely due to the ability of hydrogen peroxide under alkaline conditions to breakdown the carbohydrate molecules (Yu et al., 2015; Zeronian and Inglesby, 1995). 70 Table 12: Effect of AHP-pretreatment and steeping on the functional properties of wet-milled starch fractions from four sorghum cultivars Dorado ICRISAT Kadaaga Kapaala No steep 24 hr steep No steep 24 hr steep No steep 24 hr steep No steep 24 hr steep Water Absorption Capacity (g/g) AHP* NT 1.10 1.70 ±0.10 ±0.10 c a 1.49 1.09 ±0.11 abc 1.59 ±0.20 ab 1.20 ±0.10 c 1.49 ±0.10 abc 1.39 ±0.20 ±0.00 bc 1.40 abc 1.49 ±0.20 ±0.10 abc abc 1.19 1.19 ±0.01 ±0.20 Oil Absorption Capacity (g/g) AHP NT 1.07 1.44 ±0.11 ±0.05 ab ab 1.08 1.29 ±0.11 ±0.10 ab ab 1.36 1.47 ±0.03 ±0.11 ab ab 1.45 1.29 ±0.32 ±0.10 ab ab 1.47 1.36 ±0.09 ±0.39 ab ab 1.36 1.64 ±0.17 ±0.10 bc bc ab sb 1.19 1.59 1.38 1.37 ±0.20 ±0.20 ±0.39 ±0.02 bc ab ab ab 1.18 1.09 0.98 1.69 ±0.01 ±0.10 bc c ±0.18 ±0.49 b a Swelling Power (g/g) AHP NT 11.48 ±0.01 9.65 ±0.19 abc a-d 8.88 ±0.82 11.89 ±0.87 Water Solubility Index (%) AHP NT 14.48 ±7.27 4.71 ±1.28 ab b 11.36 ±1.50 10.75 ±7.78 d a ab ab 10.87 ±1.76 9.83 ±0.15 a-d a-d 11.25 ±1.51 a-d 10.38 ±0.55 9.46 ±0.23 bcd 9.18 ±0.10 a-d cd 9.49 ±0.04 bcd 11.80 ±1.01 ab 10.55 ±0.86 10.20 ±0.02 a-d 9.89 ±0.37 a-d 9.68 ±1.01 a-d a-d 11.61 ±6.76 2.05 ±0.06 ab b 11.64 ±1.52 14.63 ±10.92 ab ab 5.54 4.27 ±0.23 ±1.56 b b 5.05 3.38 ±1.15 ±0.21 b b 15.17 ±6.35 5.37 ±0.78 ab b 19.46 ±3.47 4.05 ±0.55 a b *AHP- AHP-pretreatment; NT- No treatment. Values are means ± standard error of two replications. Means within each category with different letters are significantly different at p < 0.05. 71 4.4.5. Pasting Properties of Wet-milled Starch The pasting properties of the starch fractions encompass the events occurring after gelatinization when further swelling of the granules and subsequent leaching of polysaccharides results in an increase in viscosity and formation of an amylopectin gel network (Waterschoot et al., 2015). The results reported in Table 13 show the pasting properties of the wet-milled starch fractions using the rapid visco analyzer (RVA). The RVA pasting temperatures for all the wet- milled starches ranged between 77.95oC and 89.33oC. This was followed by peak viscosities (PV) recorded that were between 84.71 RVU and 148.75 RVU and occurred at peak times between 8.57 min and 10.63 min. The breakdown viscosity (BV), determined as the extent of granule disruption after the peak viscosity ranged between 7.17 RVU and 27.00 RVU, while the setback viscosity (SV) which indicated the re-association of the granule constituents after pasting was recorded between 30.71 RVU and 67.25 RVU. The final viscosities (FVs) for all the wet- milled sorghum starches were between 104.00 RVU and 193.08 RVU. Analysis of the pasting properties showed significant interaction effects (p < 0.05) between the grain cultivars and the grain treatments for all the pasting parameters determined except for BV which was significantly higher (p < 0.05) for starches obtained from steeped grains. Despite the interaction effects between the study factors, tannin-containing cultivars ICRISAT and Kadaaga recorded higher PV values compared to tannin-free cultivars, likely due to higher levels of polyphenolic compounds. The results corresponded with observations by Beta et al. (2001) who reported increased PV associated with sorghum polyphenols. Boudries et al. (2009) also found higher PVs for red sorghum starches compared to white sorghum starches. While the interaction between sorghum polyphenols and sorghum starch is little understood, 72 these interactions could account for the differences in peak viscosity. AHP-pretreatment and steeping in water further increased the peak viscosity of the wet-milled starches. 73 Table 13: Effect of AHP-pretreatment and steeping on the pasting properties of wet-milled starch fraction from four sorghum cultivars Dorado ICRISAT Kadaaga Kapaala No steep 24 hr steep No steep 24 hr steep No steep 24 hr steep No steep 24 hr steep bcd 9.60 8.57 ±0.73 ±0.10 bc d 9.23 9.30 ±0.30 bcd 10.63 ±0.03 a ±0.17 bcd 9.87 ±0.20 ab 9.00 9.10 ±0.20 cd ±0.43 bcd Peak Time (min) AHP* NT 9.20 9.83 ±0.33 bcd 9.27 ±0.23 abc 9.10 ±0.13 ±0.10 bcd bcd 9.30 9.67 ±0.10 bcd 9.17 ±0.07 bc 8.67 ±0.10 ±0.33 Pasting Temperature (oC) AHP NT 79.53 87.38 ±1.18 ±0.63 c a 79.73 81.53 ±0.98 c ±2.38 bc 78.93 85.28 ±0.63 c ±0.17 ab 80.33 78.38 ±0.42 ±0.78 Peak Viscosity (RVU) d c c 79.55 77.95 ±1.20 ±0.40 c c 79.88 82.28 ±0.83 c 81.33 ±0.63 bc 79.10 ±1.20 ±2.38 bc 89.33 ±1.02 a 81.78 ±4.58 c bc AHP 136.79 ±5.54 a-d 144.63 ±6.79 a-d 153.13 ±2.71 NT 91.71 ±0.46 f 126.21 ±3.71 de 115.83 ±4.17 a e 143.04 ±5.88 146.83 ±2.33 a-d abc 139.46 ±6.63 129.42 ±0.50 a-d cde 143.33 ±7.75 a-d 134.25 ±6.58 a-e 148.75 ±2.42 ab 130.00 ±2.25 b-e 84.71 ±6.54 f 134.71 ±16.88 a-e Breakdown Viscosity (RVU) AHP 15.75 ±1.50 a-e 16.25 NT 7.17 ±2.42 e 16.67 ±1.33 ±3.25 a-e a-e 13.29 ±0.54 b-e 14.00 9.04 ±0.38 de 26.21 ±0.92 ±7.46 b-e 16.42 ±7.25 a-e 18.25 ±6.33 a-e 15.96 ±3.21 a-e 22.92 ±4.83 ab a 27.00 ±1.67 a 19.42 ±0.42 a-d 11.42 ±0.92 cde 22.25 ±5.00 abc Final Viscosity (RVU) AHP 178.58 ±9.00 NT 117.33 ±0.75 ab g 182.38 ±0.96 146.00 ±4.83 ab ef 179.13 ±2.29 ab 182.75 ±1.67 ab 162.92 ±4.00 138.92 ±6.08 f 174.33 ±8.83 abc 150.96 ±2.96 b-e def 170.21 ±7.38 bcd 179.54 ±6.13 ab 193.08 ±2.92 a 152.88 ±1.79 def 104.00 ±8.00 g 156.42 ±18.58 c-f Setback Viscosity (RVU) AHP NT 57.54 32.79 ±4.96 abc 54.00 ±7.17 a-d 39.29 ±0.96 def 53.71 ±5.12 ±2.13 ef 36.46 ±4.37 ef 32.12 ±1.54 f 53.71 ±13.96 a-d a-d 39.88 48.54 ±4.63 ±0.79 def b-e 45.12 42.29 ±5.96 ±0.87 b-f c-f 61.25 ±2.75 ab 67.25 ±5.33 a 30.71 ±2.38 f 43.96 ±6.71 c-f *AHP- AHP-pretreatment; NT- No treatment. Values are means ± standard error of two replications. Means within each category with different letters are significantly different at p < 0.05. 74 4.5. Conclusion Alkaline hydrogen peroxide (AHP)-pretreatment of sorghum grains is proposed as a means of improving the color and quality of wet-milled sorghum starch. The recovery and quality of wet-milled products in general is affected by pretreatment of the grains prior to wet- milling. The four sorghum cultivars studied had similar wet-milled recovery yields and starch fraction quality irrespective of the presence or absence of tannins in the grain. AHP-pretreatment of the grains increased the starch fraction yields and the total starch recovery in all the sorghum grains. The degree of lightness of the wet-milled starch also increased after AHP-pretreatment particularly for the tannin-containing cultivars, which typically produced darker starches without pretreatment. Steeping the grain in water alone did not lighten the starch color, but instead resulted in comparatively darker starches even from the tannin-free cultivars. A majority of the starch functional properties were not influenced by pretreating the sorghum grains, however an increase in the water solubility index of the starch resulted after AHP-pretreatment. The study demonstrates that AHP-pretreatment allows for wet-milling tannin-containing sorghum with colored pericarps to produce starch fraction of acceptable quality, without significantly affecting the starch functionality. Further steeping in water does not appear to affect the effectiveness of AHP-pretreatment; however, further study is needed to evaluate how additional steeping reagents may influence the impact of AHP-pretreatment. 75 CHAPTER 5 Water Absorption Characteristics of Sorghum Pretreated With Alkaline Hydrogen 5. Peroxide (AHP) 5.1. Abstract Pretreatment of sorghum grains with alkaline hydrogen peroxide was evaluated for its influence on water absorption of the grains during soaking in water at four temperatures ranging from 30oC to 60oC. The amount of water absorbed by AHP-pretreated sorghum grains at the different soaking temperatures was recorded at 10-min intervals over a period of 130 min, and compared to the water absorption for a control group that was untreated. Peleg’s two-parameter sorption equation was fitted to the experimental water absorption data, which enabled determination of a rate constant (K1) and a capacity constant (K2). R2 values for the fitted model ranged between 0.976 and 0.996. The rate constant was inversely related to the soaking temperature and suggested an increasing rate of hydration following an increase in temperature. The capacity constant was relatively stable over the temperature range, with an average value of 0.02%-1 and was independent of both the soaking temperature and pretreatment. The initial rate of hydration at 60oC based on Peleg’s model was higher for the AHP-pretreated sorghum (1.86 g/min to 5.10 g/min), compared to the control (1.56 g/min to 3.92 g/min). The saturation moisture content did not vary significantly (p > 0.05) for AHP-pretreated sorghum (56.67%, d.b to 82.37%, d.b.) and the control (52.02%, d.b. to 76.49%) across different soaking temperatures. 5.2. Introduction Sorghum is a particularly important crop that is well adapted to semi-arid and sub- tropical climates (Belton and Taylor, 2004). In addition to being drought resistant, it is an 76 important source of carbohydrates and a viable crop for food security (Dicko et al., 2006). There is an interest in promoting sorghum for industrial starch production as an alternative to corn starch, due to the structural and functional similarities between sorghum and corn starches (Eckhoff and Watson, 2009; Emmambux and Taylor, 2013). The potential for starch production makes sorghum an economically viable food crop especially in regions where it is much more cost-effective to produce sorghum compared to corn (Belton and Taylor, 2004). Industrial starch production usually employs the wet-milling process which is able to extract starch with high levels of purity along with other economically viable co-products such as fiber and protein feed (Xie and Seib, 2000). Effective wet-milling, however, requires adequate steeping which involves soaking the whole grains in aqueous solution for an extended period of time in order to soften the grain and facilitate the separation of grain components (Jackson and Shandera, 1995; Singh and Eckhoff, 1996). Recovery of starch from sorghum grain is, however, more difficult compared to corn. This is because of the fragile nature of the sorghum pericarp that disintegrates into small particles during processing and impedes the separation of starch and protein fractions during wet milling (Wang et al., 2000). Optimizing the steeping process for sorghum maximizes the efficiency of wet-milling and reduces the energy costs involved. Sorghum starch is characterized by dark off-colors mainly due to the presence of polyphenols in the grain. These polyphenols leach into the endosperm during wet-milling resulting in sorghum starch with off-colors (Subramanian et al., 1994). Alkaline hydrogen peroxide (AHP) pretreatment of sorghum has been proposed as a means to improve the color of sorghum starch prior to wet-milling. The pretreatment process reduces the pigmentation of the starch from polyphenols by oxidizing the colored compounds. To develop a wet-milling 77 procedure for AHP-pretreated sorghum, it is of practical significance to determine how the pretreatment may influence hydration of the kernels during the steeping process. Water absorption rates differ among food materials and as such, absorption data in the form of water content and uptake rate have been fitted with different mathematical models based on diffusion theories. Peleg (1988) developed an empirical and non-exponential equation which characterized moisture absorption over long periods from experimental data obtained in tests of relatively short duration. This equation has subsequently been used to model the hydration kinetics of several food materials such as amaranth grain, faba beans, sorghum, corn, millet, soybean and many others (Calzetta Resio et al., 2003; Kader, 1995; Kashiri et al., 2010; Resio et al., 2006; Sopade et al., 1992). The sorption equation proposed by Peleg is made up of two predictive parameters that estimate the movement of water through the food material over time. (Equation 7) In Equation 7, where Mt is the amount of water absorbed in the grains after time t, M0 the initial amount of water present in the grains before soaking and K1 and K2 are described as Peleg’s first and second constants. The equilibrium moisture content (Me) which is the maximum attainable moisture content as t → ∞ could also be related to the constant K2 which is called the Peleg capacity constant. (Equation 8) Following Equation 8, the momentary sorption rate could also be given as: 78 (Equation 9) The initial rate of hydration, i.e., at t = 0 could now be given as 1/K1 based on Equation 9. Sopade et al. (1992) indicated that the constant K1 was a function of the temperature whereas K2 could be considered as a constant for the food material and could be used as a characteristic sorption parameter for the food material under study. The present study was carried out to evaluate the water absorption characteristics of AHP-pretreated sorghum using the Peleg sorption model. 5.3. Materials and Methods 5.3.1. Sample Preparation Burgundy whole sorghum grain (Product number: NLSC-BRSWG-0014) provided by Nu Life Market (Scott City, Kansas) was used in this study. The grains were sealed in polyethylene bags and stored at 4oC until needed. 5.3.2. Alkaline Hydrogen Peroxide (AHP) Pretreatment Sorghum grain samples were weighed (100 g) and immersed in 200 ml of solution containing 20% (w/w) hydrogen peroxide prepared at pH 12 (adjusted with 0.1 M NaOH). The mixture was placed in a thermostatically controlled water bath at 60oC for a period of 60 min. The pretreated grains were then filtered out and washed with 500 ml of distilled water. They were then dried overnight at 40oC. Control sorghum samples were also prepared by adding 100 g 79 of sorghum grains to 200 ml of distilled water, and placing the mixture in a water bath at the same temperature and time, and then dried overnight at 40oC. 5.3.3. Physical Characteristics and Chemical Composition The pretreated sorghum samples were evaluated for moisture content, crude protein and ash content using approved laboratory methods (AOAC, 1990). The dimensions of the grain samples were determined by randomly selecting one hundred kernels and measuring the length, width and thickness using a digital micrometer screw gauge. The distance from the eye of the seed to the opposite end was considered as the length, while the width and thickness were taken as the major and minor seed diameters in the two perpendicular directions of the seed eye. The geometric mean diameter (Gmd) and degree of sphericity (ϕ) were also determined using the equations described by Kashiri et al. (2010). (Equation 10) (Equation 11) 5.3.4. Determination of Water Absorption During Soaking Water absorption of the samples was determined based on procedures developed in previous studies (Kaptso et al., 2008; Kashiri et al., 2010; Resio et al., 2006). Soaking temperatures of (30, 40, 50 and 60)oC were used in the study. The sorghum samples (~10 g) were weighed into a stainless steel mesh cage (13.46 cm x 11.68 cm x 3.56 cm) and placed in a beaker containing 200 ml of distilled water which had been previously equilibrated to the required 80 soaking temperature in a water bath. The beaker was placed in a thermostatically controlled water bath at the required temperature maintained at ±1oC. The mesh cage was removed and weighed at 10 min intervals and returned to the water bath for a total soaking time of 130 min. The water absorbed by the grains was determined from the weight gained by the grains and plotted against the time intervals. There were no corrections for the loss of solids during soaking and all soaking test were conducted in duplicates. 5.4. Results and Discussion 5.4.1. Physical and chemical properties The physical characteristics and chemical composition of the sorghum samples were determined and reported in Table 14. The total ash, crude protein and total starch contents for AHP-pretreated sorghum were determined to be 1.16%, 8.48% and 67.97%, respectively. This was compared to that of control samples determined to be 1.22%, 7.81% and 63.04%, respectively. There were no significant differences (p < 0.05) between the AHP-pretreated samples and the control sorghum samples. The results were comparable to the levels reported by the suppliers who reported total ash, crude protein and total starch values of 1.31%, 8.32% and 72.30% respectively (Nu Life Market, 2019), indicating that pretreatment did not influence the chemical composition of the sorghum grains The dimensions of the sorghum kernels; length, width and thickness, were determined to be 2.15 mm, 1.75 mm and 0.50 mm, respectively for the AHP-pretreated samples, and 2.40 mm, 2.00 mm and 0.85 mm, respectively for the control samples. There were no statistically 81 significant differences (p < 0.05) between the dimensions of the AHP-pretreated samples and control samples. The geometric mean diameter and degree of sphericity determined from the kernel dimensions were 1.22 mm and 0.56%, respectively, for AHP-pretreated sorghum, and 1.56 mm and 0.64%, respectively for the control. AHP-pretreated sorghum Control sorghum Table 14: Comparison of the physico-chemical characteristics between AHP-pretreated sorghum and untreated (control) sorghum Parameters Total ash (%) Crude protein, (N x 6.25)% Total starch (%) Length (mm) Width (mm) Thickness (mm) Geometric diameter (mm) Degree of sphericity (%) Results are reported as mean ± standard error. Values with different letters in each row are significantly different at P < 0.05 1.16 ± 0.20a 8.48 ± 0.16a 67.97 ± 4.30a 2.15 ± 0.25a 1.75 ± 0.05a 0.50 ± 0.20a 1.22 ± 0.23a 0.56 ± 0.04a 1.22 ± 0.20a 7.81 ± 0.52a 63.04 ± 9.26a 2.40 ± 0.30a 2.00 ± 0.20a 0.85 ± 0.45a 1.56 ± 0.41a 0.64 ± 0.09a 5.4.2. Water Absorption Characteristics The amount of water absorbed by AHP-pretreated sorghum and the control at four soaking temperatures during the hydration process, is shown in Figure 9. The hydration curves for all the soaked samples exhibited asymptotic behavior that was characterized by an initial rapid rate of water absorption followed gradually by a slower absorption rate in the later stages approaching equilibrium. This hydration pattern was also observed by Kashiri et al. (2010), and 82 was due to a reduction in driving force for the water transfer through the grain as the system approached equilibrium. The hydration curves also showed higher water absorption associated with increasing temperature from 30oC to 60oC for all the samples. This observation was consistent with other studies (Bello et al., 2007; Kashiri et al., 2010; Resio et al., 2006; Solutions, 1983). After 60 min of hydration, the water uptake recorded for AHP-pretreated sorghum was 42.34%, 45.34%, 55.93%, and 68.80% at the corresponding temperatures of 30oC, 40oC, 50oC, and 60oC, respectively, while that of the control sample was 38.87%, 42.55%, 53.48%, and 60.73%, at the same corresponding temperatures. Reaction with AHP delignifies cellulosic materials and removes a variable portion of the hemicellulose present in the cell walls, resulting in a disruption of the cell wall’s morphological integrity (Gould et al., 1989). This likely resulted in the increased water absorption through the pericarp of the AHP-pretreated sorghum grains. Peleg’s model, given in Equation 7, was fitted to the water absorption data and the predicted values obtained from the model were indicated by the solid lines in Figure 9. The predicted fits were close to the experimental water absorption data observed for both AHP- pretreated and the control samples. 83 (a) (b) Figure 9: Comparison of predicted and experimental water uptake of (a) control and (b) AHP-pretreated Burgundy whole grain sorghum during hydration in water at four soaking temperatures (30, 40, 50 and 60 oC). Solid lines represent predicted values based on fitted Peleg model. 84 5.4.3. Total Solids Loss The total amount of solids lost during soaking at the four different temperatures were determined and presented in Figure 10. In general, a significant (p < 0.05) increase in total solids loss was observed for both AHP-pretreated (0.06% - 0.17%) and untreated grains (0.03% - 0.16%) as the temperature was increased from 30oC to 60oC. While a comparatively higher total solids loss was recorded for AHP-pretreated sorghum at all soaking temperatures used in the study, this increase was not significantly different (p > 0.05) at higher temperatures of 50oC and 60oC. The results indicated that AHP-pretreatment of sorghum increased the amount of soluble solids that leach into the water during soaking. The major component of such solids based on reported results could be carbohydrates (Pan and Tangratanavalee, 2003). There is also evidence that AHP-pretreatment of agricultural byproducts facilitates the dissolution of large molecular size hemicelluloses (Sun et al., 2000). In the present study, the effect of AHP-pretreatment was evident at lower temperatures of 30oC and 40oC. At the higher temperatures of 50oC and 60oC, the increasing temperature facilitated the leaching of solids during soaking (Resio et al., 2006; Wang et al., 1979). 85 Figure 10: Total solids loss from AHP-pretreated Burgundy whole grain sorghum and control (untreated sorghum) soaked in water at four temperatures. Column bars connected by the same letter are not significantly different at p < 0.05. 5.4.4. The Constant Rate of Water Absorption The parameters of the Peleg model were determined by plotting t/(Mt – M0) against the soaking time (min) (Figure 11), The rate constant (K1) and capacity constant (K2) determined at four temperatures along with the coefficient of determination (R2) are presented in Table 15. The R2 values determined for both AHP-pretreated sorghum and control samples were high and 86 varied between 0.976 to 0.996, confirming the adequacy of the Peleg equation in describing the hydration of the sorghum grain samples within the studied temperature range. The rate constants (K1) were inversely related to the temperature and indicated an increase in the rate of water absorption at higher temperatures. The sorption curves in Figure 9 show a slowing rate of water absorption, following an initially high rate of absorption at the start of soaking. Peleg’s equation allowed the determination of the momentary sorption rate dMt/dt using Equation 9 and consequently, the initial rate of hydration at the start of soaking (t = 0). The initial rate of hydration for the sorghum samples determined from application of Peleg’s model are presented in Table 15. Increasing temperature from 30oC to 60oC for all the samples corresponded with an increasing initial rate of hydration from 1.86 g/min to 5.10 g/min for AHP-pretreated sorghum, compared to the control which went from 1.56 g/min to 3.92 g/min for the control. At soaking temperatures between 30oC and 50oC, AHP-pretreatment did not have a statistically significant effect (p > 0.05) on the initial rate of hydration. A significantly higher rate of hydration was however, obtained at 60oC for AHP-pretreated sorghum grains compared to the untreated sorghum grains at p < 0.05. 87 (a) (b) Figure 11: Fitting of water absorption data during hydration of (a) the control (untreated sorghum) and (b) AHP-pretreated Burgundy whole grain sorghum, to Peleg's model. 88 Table 15: Water absorption characteristics and regression parameters following the Peleg model for AHP-pretreated Burgundy whole grain sorghum and untreated control sorghum at four soaking temperatures Soaking Temperature (oC) Capacity Constant, K2 (%-1) R2 W0 (g/min) Initial rate of hydration, Saturation Moisture Content, Me (%, dry basis) Rate Constant, K1 (min/%) 5.38E-01 5.53E-01 4.01E-01 1.96E-01 6.42E-01 6.77E-01 4.33E-01 2.55E-01 Observed AHP- pretreated sorghum Predicted from Peleg’s model 56.67 ± 5.38 de 66.48 ± 6.56 cd 74.79 ± 6.12 abc 82.37 ± 5.06 a 52.02 ± 3.41 e 59.04 ± 0.79 cd 72.11 ± 0.51 bc 76.49 ± 0.04 ab Means with different letters within the same column are significantly different at p < 0.05 using the least significant difference test. 0.990 1.86 ± 0.03 de 0.976 1.84 ± 0.32 de 0.989 2.54 ± 0.48 c 0.996 5.10 ± 0.04 a 0.986 1.56 ± 0.09 e 0.979 1.48 ± 0.03 e 0.981 2.31 ± 0.06 cd 0.996 3.92 ± 0.00 b after 24 hours 69.64 ± 3.37 bc 68.72 ± 3.00 bc 68.89 ± 1.26 bc 78.23 ± 2.38 a 66.71 ± 3.76 bc 65.21 ± 0.38 c 66.97 ± 0.58 bc 71.60 ± 0.04 b 2.30E-02 1.88E-02 1.62E-02 1.44E-02 2.52E-02 2.13E-02 1.67E-02 1.55E-02 30 40 50 60 30 40 50 60 Control 89 5.4.5. Saturation Moisture Content The capacity constants (K2) for both the AHP-pretreated sorghum and control were relatively stable over the temperature range, with an average value of 0.02%-1 for all the samples studied. Similar K2 values were determined for both AHP-pretreated sorghum and the control, indicating that the K2 constant was dependent on the nature of the sorghum grain rather than the soaking temperature or pretreatment method used. Sopade et al. (1992) also determined that the capacity constant K2 was characteristic for the food material under study and remained relatively constant over their temperature range. These observations were in agreement with the results from other studies (Abu-Ghannam and McKenna, 1997; Sopade et al., 1992; Turhan et al., 2002). The constant K2 was inversely related to the water absorption capacity of the grain and was used to determine the saturation moisture content (Me) based on Equation 8. The predicted Me ranged from 56.67%, d.b to 82.37%, d.b. for AHP-pretreated sorghum, whereas the control sorghum ranged from 52.02%, d.b. to 76.49%, d.b. The moisture content of the sorghum grains after 24 hours of soaking was determined and compared to the predicted Me. The observed Me for the AHP-pretreated grains ranged from 69.64%, d.b. to 78.23%, d.b. and that of the control ranged from 66.71%, d.b. to 71.60%, d.b. The predicted Me increased significantly (p < 0.05) as the soaking temperature was increased from 30oC to 60oC. The observed Me after 24 hours of soaking was however, not significantly different (p > 0.05) at soaking temperatures between 30oC and 50oC, but increased significantly (p < 0.05) at 60oC. For both the predicted and observed Me, AHP-pretreatment resulted in a significantly higher saturation moisture content for the sorghum grain samples. The effect of AHP-pretreatment on the hydration kinetics of the 90 sorghum grains were attributed to the removal of portions of hemicellulose from the grain pericarp (Brooks and Moore, 2000), which allowed more water to penetrate the sorghum grain. 5.5. Conclusion The present study evaluated the hydration of AHP-pretreated sorghum grain, which is important for efficient grain processing such as wet-milling. Soaking of the grain was characterized by an initial rapid rate of hydration which gradually slowed down as the system approached equilibrium. While AHP-pretreatment of sorghum grains prior to wet-milling has been reported to improve the color of wet-milled starch, the present study showed that it also influences the hydration of grain when soaked in water. AHP-pretreatment of whole grain sorghum increased the amount of soluble solids leached into the water and the initial rate of hydration during soaking. AHP-pretreatment did not significantly change the chemical composition and dimensions of the sorghum grains, which could have influenced its hydration. The capacity of the whole grains to hold water during soaking was characteristic of the nature of the grain and increased at higher temperatures. The saturation moisture contents determined by fitting the Peleg model parameters were close to the moisture content of the sorghum grains determined after 24 hours of soaking further indicating the reliability of the model for predicting the soaking of sorghum grains. While the present study shows higher initial rates of hydration and saturation moisture content for AHP-pretreated sorghum at 60oC, further study is required to determine how these results correlate with the recovery of wet-milling products from the grains as well as the effect on the quality of processed products after soaking. 91 6. Summary and Conclusions CHAPTER 6 The impact of alkaline hydrogen peroxide (AHP)-pretreatment of sorghum was evaluated in this dissertation as an alternative approach to improving the color of sorghum starch, which appears dull with off-colors due to the presence of polyphenols in the grains. Hydrogen peroxide under alkaline conditions produces perhydroxyl anions that are able to oxidize conjugated double bonds in colored molecules, thereby decolorizing them. In addition to removing color, AHP is also able to delignify and remove portions of hemicellulose from plant cell walls. The impact of AHP-pretreatment on sorghum was studied in terms of (1) the optimum conditions of pretreatment and its effect on the color and quality of sorghum starch, (2) the effect of AHP- pretreatment on the recovery of wet-milling products and characteristics of the wet-milled starch, and (3) hydration of AHP-pretreated grains during steeping or soaking in water. Under alkaline conditions, hydrogen peroxide produces perhydroxyl anions, which react with chromophore groups in colored molecules. This is the consensus for the mechanism by which AHP-pretreatment removes color from plant materials. The conditions necessary for effective AHP-pretreatment of sorghum were determined in Chapter 3, by varying the concentration of H2O2 and pH based on a central composite design experiment. Based on a response surface model, acceptable starch color with a degree of lightness (L-value ) at 90 and above was determined to be at optimum conditions of pH 11.84 (~12) and 20% (w/w) H2O2 concentration. Increasing the pH of the pretreatment medium corresponded with a significant increase in the degree of lightness of the sorghum starch. AHP-pretreatment of sorghum grains prior to wet-milling was capable of improving the sorghum starch color which usually appears dull and inferior. 92 A further study was conducted in Chapter 4 to determine the effect of AHP-pretreatment and additional steeping on the recovery yields of wet-milling products and the quality of the wet- milled starch fraction obtained from four sorghum cultivars. The presence or absence of tannins in the sorghum cultivars did not influence the wet-milling characteristics of the grains. In comparison to untreated grains, AHP-pretreated grains produced higher starch fraction yields and total starch recoveries due to the ability of AHP to break down parts of the grain and release starch molecules. Tannin-containing cultivars produced comparatively darker starches than the tannin-free cultivars. In fact, they have been considered as unsuitable for wet-milling; however, AHP-pretreatment improved the color of starches from the tannin-containing cultivars and produced brighter starches compared to the tannin-free cultivars. Steeping alone in water did not improve starch color and resulted in off-colors in the starches from tannin-free cultivars. The study demonstrated that tannin-containing sorghum could be wet-milled with acceptable product quality, when pretreated with AHP. It also showed that AHP-pretreatment has little influence on the functional properties of the wet-milled starch. Steeping of the grains is an important process for wet milling because it facilitates the separation of grain components by softening the grain and allowing for the absorption of steeping reagents into the grain. As such, it is important to study the hydration characteristics of sorghum grain for consideration in wet-milling operations. The final study (Chapter 5) evaluated the effect of AHP-pretreatment on the hydration of sorghum grain during soaking in water using Peleg’s sorption model. This allowed prediction of the hydration kinetics of pretreated grains during the steeping process prior to wet-milling. The adequacy of the Peleg model was established based on high coefficient of determination values obtained from fitting the experimental data with the Peleg model. Increasing the soaking temperature also increased the 93 amount of water absorbed by the grain, which eventually slowed down as the system reached equilibrium. AHP-pretreatment increased the initial rate of hydration at 60oC. Faster hydration rates are useful for cost-effective steeping operations and the enhanced moisture capacity may have implications on the millability of the grains. There is a potential to explore wet-milling of sorghum, particularly for the extraction of starch which is an economically viable food product, and the results presented in this work show that the dull color of sorghum starch which is often a hindrance to consumer acceptance and marketability, can be mitigated with AHP-pretreatment of the grains. The minimal effect of this pretreatment approach on the functionality of the wet-milled starch is an advantage for the industry, since undesirable or irreversible changes of the wet-milled starch fractions during pretreatment was unlikely to occur. Another advantage of employing AHP-pretreatment in sorghum wet-milling is that this approach uses the same apparatus used in conventional steeping of grain and does not require additional modification of conventional wet-milling equipment. AHP-pretreatment is also less toxic and more environmentally friendly compared to chlorine- based treatments. 6.1. Future Directions Considering the increasing production of sorghum in Africa, in addition to its drought resistance and agronomic benefits, there is immense value in exploring sorghum as an alternative starch source. While the results in this work demonstrate the effectiveness of AHP-pretreatment in enhancing the sorghum wet-milling, further work is needed in the following areas: 94 1. Evaluate the practical application of AHP-pretreatment of sorghum in pilot scale wet- milling operations. 2. Investigate the influence of additional steeping reagents on the effectiveness of AHP- pretreatment on sorghum wet milling. 3. Determine how hydration of AHP-pretreated sorghum grains correlate with the texture and wet-millability of the grain, in terms of the force required and recovery yields. 4. Understand the structure-function relationship of wet-milled products from AHP- pretreated sorghum and their influence on end-use quality. 95 APPENDIX 96 7. Supplementary Materials 7.1. Characteristics of wet-milled starch after AHP pretreatment sorghum Table 16: Functional and pasting characteristics of wet-milled sorghum (Naga red) starch after alkaline hydrogen peroxide pretreatment H2O2 Conc pH Ash content (%) Starch pH WAC (g/g) OAC (g/g) Swelling power (g/g) WSI (%) Peak time (min) Pasting temperature (oC) Peak viscosity (RVU) Breakdown viscosity (RVU) Final viscosity (RVU) Setback viscosity (RVU) 20.00 12.00 0.96 ±0.05 6.96 ±0.47 1.51 ±0.21 1.68 ±0.10 10.19 ±0.09 11.20 ±8.41 9.20 ±0.40 80.75 ±2.05 139.00 ±4.17 18.00 ±5.83 181.25 ±0.83 60.25 ±10.83 11.00 10.50 0.39 ±0.28 6.81 ±0.30 1.62 ±0.26 1.77 ±0.03 9.99 ±1.44 15.69 ±2.64 8.93 ±0.20 78.20 ±0.95 151.00 ±1.50 21.33 ±1.75 186.92 ±8.67 57.25 ±5.42 2.00 12.00 0.35 ±0.10 6.67 ±0.12 1.23 ±0.08 1.38 ±0.00 11.95 ±1.02 10.15 ±6.44 8.17 ±0.23 76.13 ±0.97 158.29 ±8.63 41.67 ±6.00 179.29 ±7.71 62.67 ±5.08 0.00 10.50 0.36 ±0.04 6.50 ±0.09 1.29 ±0.08 1.48 ±0.10 11.49 ±0.11 13.45 ±0.53 8.17 ±0.23 76.55 ±1.00 157.92 ±9.25 40.29 ±4.54 180.79 ±5.71 63.17 ±1.00 23.73 10.50 0.14 ±0.09 6.27 ±0.04 1.52 ±0.01 1.48 ±0.10 10.19 ±0.37 12.85 ±1.56 9.90 ±0.23 79.53 ±1.57 138.04 ±12.13 9.96 ±1.96 167.46 ±15.88 39.37 ±5.71 20.00 9.00 0.10 ±0.03 6.30 ±0.20 1.25 ±0.13 1.78 ±0.22 11.06 ±0.12 9.09 ±0.37 9.40 ±0.07 78.35 ±0.80 142.50 ±13.83 14.88 ±3.63 173.63 ±18.21 46.00 ±8.00 2.00 9.00 0.31 ±0.15 6.64 ±0.18 1.30 ±0.11 1.27 ±0.10 11.98 ±0.34 14.91 ±2.21 8.27 ±0.20 76.55 ±1.40 172.17 ±25.42 54.63 ±17.71 185.79 ±14.04 68.25 ±6.33 11.00 12.62 0.15 ±0.02 7.00 ±0.25 1.27 ±0.12 1.77 ±0.39 11.07 ±0.96 11.20 ±3.49 8.33 ±0.27 76.90 ±0.60 161.04 ±17.63 36.13 ±10.38 187.71 ±15.21 62.79 ±7.96 11.00 10.50 0.17 ±0.02 6.72 ±0.05 1.30 ±0.11 1.58 ±0.01 11.92 ±0.35 12.02 ±0.85 8.57 ±0.03 76.93 ±0.22 150.83 ±1.42 31.46 ±0.04 176.21 ±4.79 56.83 ±3.33 11.00 8.38 0.23 ±0.15 6.56 ±0.01 1.38 ±0.18 1.58 ±0.02 11.45 ±0.63 14.08 ±0.12 8.43 ±0.03 77.13 ±0.78 146.88 ±7.13 35.96 ±4.46 172.25 ±6.83 61.33 ±4.75 Results are represented as mean ± standard error of duplicate determinations; WAC – Water absorption capacity, OAC – Oil absorption capacity. 97 7.2. Details of Experimental Methods 7.2.1. Determination of Starch Color Wet milled starch fractions were evaluated for color directly using a chroma meter (Konica Minolta CR-410, Osaka, Japan). The instrument was calibrated with a white standard (L= 92.32, a= -0.36, b= 4.00). The L value from the Hunter Lab color space was used to determine the brightness of the starch fractions on a scale of 0 to 100, where 100 was indicative of white and 0 was for black. 7.2.2. Determination of Moisture Content Moisture content determinations were conducted using the Air Oven Method (AACC, 2000 Method 44-19.01). Finely ground samples were accurately weighed into aluminum dishes and dried in a forced air oven at 135oC for 2 h. The dishes with the dried samples were then cooled in a desiccator and weighed. The moisture content was determined from the loss in weight of the fresh sample. 7.2.3. Starch pH Determination All wet-milled starch fractions were evaluated for pH using the method described by Benesi et al. (2004). The starch fractions (5 g) were accurately weighed into beakers and mixed with 20 ml of distilled water. The mixtures were stirred for 5 min and allowed to settle to the bottom of the beaker. The temperature of the aqueous layer was monitored to ensure that it was 98 at room temperature. The top layer was then evaluated for pH using a calibrated pH meter (Oakion ion 6+ pH meter) 7.2.4. Determination of Crude Protein by Boric Acid Titration Crude protein content was determined using the Kjeldahl Method (AOAC, 1990). Finely ground samples were accurately weighed (1 g) and digested with 25 ml concentrated sulfuric acid by heating on a digestion block along with selenium catalyst. The cooling sample was distilled with boric acid-methyl red-methylene blue indicator using a distillation unit set up with concentrated NaOH. The distillate was titrated to neutrality with 0.1 N sulfuric acid. The percentage protein was calculated by multiplying the amount of nitrogen by a factor of 6.25 (Buffo et al., 1998). 7.2.5. Determination of Crude Protein by Phenate method The amount of crude protein in wet-milled starch fractions were determined using a variation of the method 976.06 described in AOAC (1990). Finely ground samples were accurately weighed (100 mg) and digested along with 4 ml of hydrogen peroxide and 6 ml of sulfuric acid using the Anton Paar Microwave Digester (Multiwave 3000 with rotor 16HF100). Digestion was completed in 1 hour and after allowing to cool for 20 min, the samples was transferred into a 50 ml volumetric flask, slowly bringing the volume to the mark with distilled water. In approximately 30 ml of water in a beaker, 1 ml of the digested sample was pippeted and adjusted to pH 6.5 – 7.5 using 1 M Na2CO3. The neutralized sample was transferred into a 50 ml volumetric flask and volume brought up to the mark with distilled water. Serial dilutions of 99 ammonium chloride standard (5 μmg NH3-N) were prepared and to 1 ml of each standard and sample, 2 ml of reagent 1(10 g Phenol, 50 mg sodium pentacyanonitrosyl ferrate dehydrate per liter) and reagents 2 (15 g sodium hydroxide, 10 mL sodium hypochlorite per liter) were added and mixed thoroughly. The mixtures were incubated for 40 min at 50oC in a thermostatically controlled water bath. The absorbance of the incubated samples and standards were read with a spectrophotometer at a waveleght of 640 nm. A standard curve was created by plotting absorbance vs the concentration of nitrogen in the standard solution (Figure 12). The amount of nitrogen in the samples were calculated and multiplied by a factor of 6.25 to determine the amount of crude protein per 100 g of dry starch fraction. Figure 12: Standard curve for concentration of NH3-N using ammonium chloride standard against absorbance at 640 nm. 7.2.6. Determination of Crude Fat Fat content was determined using AOAC approved methods (AOAC, 1990). The crude fat was extracted from 2 g of the ground sample with petroleum ether using a Soxhlet apparatus. 100 Excess solvent was removed by evaporation after which the extract, and the previously dried and weighed beakers, were dried in an oven at 100 oC for 30 min. The dried extract was cooled in a desiccator and weighed. The crude fat content was reported as the percentage of petroleum ether extract. 7.2.7. Determination of Ash Content The amount of inorganic or mineral residue in the starch fractions was determined as the ash content (AACC, 2000 Method 08-01). Clean empty crucibles were placed in a muffle furnace at 600 oC for an hour, cooled to room temperature in a desiccator, and then weighed. Approximately 3 g (±0.01 g) of each sample was weighed into the crucibles and placed in a muffle furnace at 500 oC for 5 h. The appearances of light gray ash indicated complete oxidation of all organic matter in the sample. The incinerated samples were cooled to room temperature in a desiccator and weighed. 7.2.8. Determination of Total Starch Content Finely ground samples were assayed enzymatically for total starch content using the amyloglucosidase/α-amylase assay kit (Megazyme, Ireland) (AACC, 2000 method 76-11). Aqueous ethanol (0.2 ml, 80% v/v) was added to approximately 100 mg of the starch fraction in propylene test tubes to aid wetting. Diluted thermostable α-amylase solution (3 ml, 40 U) was added immediately added and incubated in a boiling water bath for 12 min while stirring at 4 min intervals. The tubes were removed and placed in a water bath maintained at 50oC. Amyloglucosidase (0.1 ml, 330 U) was added to the tubes, stirred and incubated at 50oC for 30 101 min. Aliquots (0.1 ml) of the tube contents were diluted to 10 ml and 0.1 ml of the diluted solution transferred into glass test tubes. Glucose oxidase-peroxidase-4-aminoantipyrine reagent (3 ml) was added to each tube, stirred and incubated at 50oC for 20 min. Absorbance at 510 nm was measured against a reagent blank (0.1 ml water) and D-glucose solution (1 mg/ml) was used as control. Total starch was measured as the glucose derived from hydrolyzed starch and expressed as a percentage of total sample weight. For the starch fractions, starch yield obtained from the wet-milling process was determined based on the mass of the wet-milled grain whereas the starch recovery was based on the starch mass in the grain samples. 7.2.9. Determination of Amylose Content of Starch Fractions The amylose content of the starch fraction samples obtained from wet-milling was determined colorimetrically based on amylose-iodine binding as decribed by Beta et al. (2001a). The samples (100 mg) were suspended in a flask containing 100 ml of distilled water at room temperature and stirred vigorously. An aliquot (5 ml) of the resulting solution was taken into a glass test tube and a mixed with 1 ml of 1 M acetic acid and 2 ml iodine solution. The contents were mixed and allowed to stand for 20 minutes. The absorbance was read at 620 nm. Amylose standard mixtures were prepared from corn starch reference samples (Megazyme, Ireland) to represent 0 to 60% amylose. 7.2.10. Test for Presence of Tannins The qualitative “bleach” test described by Waniska et al. (1992) was used to determine the presence of tannins in the sorghum samples by dissolving the pericarp of the grain, thereby 102 revealing the presence or absence of a pigmented testa layer. A 1 L beaker was filled with 200 ml of distilled water, then heated and maintained at 60oC on a heated magnetic stir plate. For each of the sorghum samples, 15 g of grains were accurately weighed into a separate 250 ml beaker containing 7.5 g KOH and 70 ml NaOCl. A stir bar was placed into the beaker containing the mixture which was in turn placed in the larger 1 L beaker containing the pre-heated distilled water. The magnetic stirrer was adjusted to maximum agitation and after 7 min, the sorghum samples were carefully removed and rinsed with a steady stream of distilled water. The kernels that had turned light yellow to white were considered non-tannin-containing cultivars (Type I) and those that had turned black were tannin-containing cultivars (Type II/III). 7.2.11. Thousand Kernel Weight The thousand kernel weights (TKW) for each of the sorghum grain samples were determined by randomly selecting 100 intact kernels and weighing them on an analytical balance to an accuracy of 0.001 g. TKW was calculated by multiplying the weight of the grains by 10. 7.2.12. Determination of Water Absorption Capacity Water absorption capacity (WAC) of the starch fractions was determined using the method outlined by (Awolu, 2017). Each dried starch fraction was carefully weighed (0.5 g) into a test tube and dispersed in 10 ml of distilled water. The test tube was vortexed for 30 seconds and allowed to settle at room temperature for 30 min. They were subsequently centrifuged at 2800 x g for 25 min. The supernatant was filtered with Whatman No 1 filter paper and the volume of water obtained was accurately measured. The water absorption capacity at room 103 temperature was then determined as a ratio of the amount of water absorbed and the weight of the starch fraction. 7.2.13. Determination of Oil Absorption Capacity The oil absorption capacity of each starch fraction was also determined as the volume of oil (ml) absorbed per gram of sample as described by (Awolu, 2017). Each sample (1 g) was accurately weighed into test tube and thoroughly mixed with 10 ml of refined sunflower oil with a density of 0.9178 g/ml using a vortex mixer. The mixtures were allowed to stand for 30 min after which they were centrifuged at 2800 x g for 30 min. The supernatant was carefully decanted into a 10 ml graduated cylinder and the volume was recorded. 7.2.14. Determination of Swelling Power and Water Solubility Index The swelling power and solubility of each wet-milled starch fraction were determined according to procedures described by (Boudries et al., 2009). Starch fraction suspensions (2%, w/v) were prepared and preheated in a water bath at 90oC ,for 30 min. They were then allowed to cool for 10 min at room temperature and centrifuged at 2000 x g for 20 min. The supernatants were carefully decanted and the sediments accurately weighed. Aliquots of the supernatant were dried at 100oC in a convective oven for approximately 1 hour until a constant weight was reached. The water solubility index (WSI) was reported as the ratio of weight of the dry mater supernatant to weight of the dry starch sample whereas the swelling power (SP) was reported as the ratio of weight of swelling starch granule sediment to weight of the dry starch. 104 7.2.15. Determination of Starch Pasting Properties The pasting properties of the each wet-milled sorghum starch fraction was determined using a Rapid Visco Analyzer (Perten RVA 4500) with procedures described by Crosbie and Ross (2007). Each starch fraction was accurately measured and suspended in distilled water (14% moisture basis). A 23-min heating and cooling cycle was programmed to hold the suspensions (3 g sample/ 24 g water) at 50 °C for 1 min, then heating to 95 °C in 7.5 min, holding at 95 °C for 5 min before cooling back to 50 °C in 7.5 min and holding at 50 °C for 1 min. The starch viscosity parameters measured were pasting temperature, peak viscosity, peak time, holding strength, breakdown viscosity, final viscosity and setback viscosity (Figure 13). Figure 13: Representative viscosity parameters measured on a Rapid Visco Analyzer (RVA) pasting curve with heating and cooling rate of ~6oC/min 105 7.3. Analysis of variance for the effect of grain cultivar and treatment on wet-milling and starch functional properties N DF Sum of Squares F Ratio Prob > F Remarks 0.0215 0.0006 0.0325 444.4168 1040.446 891.3577 Significant Significant Significant Table 17: ANOVA results for effect of grain cultivar and treatment on recovery yields of wet- milling fractions Source Coarse Fraction Grain cultivar Treatment Grain cultivar*Treatment Fine Fraction Grain cultivar Treatment Grain cultivar*Treatment Starch Fraction Grain cultivar Treatment Grain cultivar*Treatment Total Solids Recovered Grain cultivar Treatment Grain cultivar*Treatment 502.33906 493.31024 375.03714 64.2981 1652.4759 537.4201 633.2055 46.34644 557.02059 Significant 0.0397 0.8552 Not significant 0.4585 Not significant 0.0581 Not Significant 0.061 Not significant 0.6511 Not significant 0.9109 Not significant 0.0176 0.8598 Not Significant Significant 3.5113 0.257 1.0296 3.0653 3.0102 0.7628 4.2665 9.9885 2.8524 0.1762 4.5292 0.491 3 3 9 3 3 9 3 3 9 3 3 9 3 3 9 3 3 9 3 3 9 3 3 9 106 N DF Sum of Squares F Ratio Prob > F 0.2818 5.3396 0.5404 213.9257 4053.7272 1230.8498 0.8378 Not significant 0.0097 0.8245 Not significant Significant Table 18: ANOVA results for effect of grain cultivar and treatment on starch recovery, protein residue, amylose, and starch color Source Starch Recovery Grain cultivar Treatment Grain cultivar*Treatment Amylose Grain cultivar Treatment Grain cultivar*Treatment Protein Content Grain cultivar Treatment Grain cultivar*Treatment Starch color Grain cultivar 0.9482 Not significant 0.6498 Not significant 0.4647 Not significant 0.1293 Not significant 0.3314 Not significant 0.2694 Not significant 11.14816 52.74976 288.89086 2.3860731 1.3416452 4.5613202 0.1181 0.5588 1.0201 2.1869 1.2297 1.3935 0.6208 Not significant 34.93793 0.6058 Remarks 3 3 9 3 3 9 3 3 9 3 3 3 9 3 3 9 3 3 9 3 Treatment Grain cultivar*Treatment 3 9 3 9 652.71058 11.3175 0.0003 441.1764 2.5499 0.0492 Significant Significant 107 N DF Sum of Squares F Ratio Prob > F Remarks 0.8277 5.3595 1.4436 0.09725038 0.62969544 0.50883221 Table 19: ANOVA results for effect of grain cultivar and treatments on starch functional properties Source Water Absorption Capacity Grain cultivar Treatment Grain cultivar*Treatment Oil Absorption Capacity Grain cultivar Treatment Grain cultivar*Treatment Swelling Power Grain cultivar Treatment Grain cultivar*Treatment Water Solubility Index Grain cultivar Treatment Grain cultivar*Treatment 0.23347206 0.30540228 0.54665226 211.87257 321.52536 301.2131 2.433173 9.532877 14.563461 0.641 2.5112 1.2788 1.6543 2.5104 0.7839 0.789 1.0321 0.6158 3 3 9 3 3 9 3 3 9 3 3 9 3 3 9 3 3 9 3 3 9 3 3 9 0.5997 Not significant 0.0955 Not significant 0.3197 Not significant 0.2167 Not significant 0.0956 Not significant 0.6346 Not significant 0.4978 Not significant 0.0095 0.2499 Not significant Significant 0.5175 Not significant 0.4049 Not significant 0.7671 Not significant 108 Remarks 0.0789 Not significant 0.0008 0.0321 Significant Significant 0.1045 Not significant 0.0202 0.0274 Significant Significant F Ratio Prob > F 3 3 9 3 3 9 3 3 9 3 3 9 3 3 9 3 3 9 N DF Sum of Squares 2.7214 9.3798 2.8599 2.4142 4.3468 2.9803 42.14648 145.26336 132.87258 1.14375 2.0593056 4.2356944 Table 20: ANOVA results for effect of grain cultivar and treatments on the starch pasting properties Source Pasting Temperature Grain cultivar Treatment Grain cultivar*Treatment Peak Time Grain cultivar Treatment Grain cultivar*Treatment Peak Viscosity Grain cultivar Treatment Grain cultivar*Treatment Beakdown Viscosity Grain cultivar Treatment Grain cultivar*Treatment Final Viscosity Grain cultivar Treatment Grain cultivar*Treatment Setback Viscosity Grain cultivar Treatment Grain cultivar*Treatment 1303.8288 7663.9225 31.8603 <.0001 2247.8336 14055.155 48.8903 <.0001 3708.086 234.6611 1569.6616 1794.1992 184.81076 253.76562 506.1875 2.1201 2.9112 1.9356 1.3344 8.9262 3.401 3 3 9 3 3 9 3 3 9 3 3 9 758.01 2.6367 3.1149 0.0229 5.4203 0.0091 3 3 9 3 3 9 Significant Significant Significant 0.1378 Not significant 0.0666 0.1194 Not significant Significant 0.0852 Not significant 4.2995 0.0055 Significant Significant 0.2982 Not significant 0.001 0.0159 Significant Significant 109 REFERENCES 110 REFERENCES AACC, 2000. 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