Industrial and Nonfood Applications

C H A P T E R

13 Industrial and Nonfood Applications Janet Taylor1, Ke Zhang2, Donghai Wang2 1

Department of Consumer and Food Sciences and Institute for Food, Nutrition and Well-being, University of Pretoria, Pretoria, South Africa; 2Department of Biological and Agricultural Engineering, Kansas State University, Manhattan, KS, United States

1. INTRODUCTION Recently, sorghum has gained interest as a renewable resource for biofuels and bio-based products. The development of renewable energy and search for bio-based materials are motivated by various factors, including the depletion of petroleum resources, climate change, increasing emission of greenhouses gases, and the need for sustainable economic development. The United States is the world’s largest producer of bioethanol, followed by Brazil. Together these two countries produce 85% of the world’s bioethanol (Renewable Fuels Association, 2017). Europe, China, and Canada make up the majority of the remaining 15% of world production. The bioethanol industry in the United States has been largely dominated by maize grain as the primary feedstock (Kwiatkowski et al., 2006), whereas Brazil utilizes sugar cane (Renewable Fuels Association, 2017), and the European Union uses predominantly sugar beet, maize, and wheat (Deppermann et al., 2016). In 2016, the United States produced approximately 60 billion liters of bioethanol and 43 million metric tons of high-protein animal feed. Only 4% of this bioethanol was produced from sorghum, using approximately 45% of the grain sorghum produced (Renewable Fuels Association, 2017). Of the millets, pearl millet has been identified as suitable feedstock for ethanol production, with comparable yields to maize under laboratory conditions (Wu et al., 2006), but there are currently no bioethanol plants using millet as feedstock. The utilization of sorghum coproducts from sorghum processing for value-added products is crucial to maintaining the economic viability of the sorghum industry and to sustain economic development. In this chapter, we review the industrial and nonfood applications of sorghum including bioethanol from grain sorghum, sorghum biomass, and sweet sorghum; platform chemicals from sorghum; sorghum protein and starch for bioplastics, adhesives, and resins; sorghum lipids, waxes, pigments, and antioxidants for nutraceutical products and various other applications. The millets have been studied far less than sorghum for industrial and nonfood applications, but available information will be reviewed.

2. BIOETHANOL PRODUCTION 2.1 Bioethanol From Grain Sorghum and Millets 2.1.1 The Bioethanol Production Process Studies on the conversion of grain sorghum into bioethanol have been conducted for the past 20 years. Dry-grind ethanol processing is one of the most effective methods for conversion of starch to bioethanol and is the process generally used for production of bioethanol from sorghum. In this process, the sorghum starch is hydrolyzed to simple sugars using starch-degrading enzymes before yeast metabolizes these monosaccharides into ethanol. Distillers’ dried grains with solubles (DDGS) is a highly profitable coproduct of ethanol production that is left over after the distillation and is sold for inclusion in animal feed by the bioethanol companies. Fig. 13.1 shows the main five steps Sorghum and Millets, Second Edition https://doi.org/10.1016/B978-0-12-811527-5.00013-7

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Copyright © 2019 AACCI. Published by Elsevier Inc. in cooperation with AACC International. All rights reserved.

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Sorghum grain

Distillation and dehydration

Mill

Water

Centrifuge

Bioethanol

Thin stillage

Evaporators

Water

Cooking and liquefaction Wet grains

Syrup

Enzyme

Enzyme

Saccharification and fermentation

Rotary dryer

DDGS

Yeast

Carbon dioxide

Co-products

FIGURE 13.1

Flowchart of the dry-grind bioethanol production process from sorghum grain.

in the dry-grind ethanol production: milling, cooking/liquefaction, saccharification, fermentation, and distillation (Mosier and Ileleji, 2014). Milling is the first step that grinds the grain kernels down to the appropriate size (1e2 mm) using hammer mills or roller mills. In general, hammer mills are commonly used in the bioethanol industry. The purpose of milling is to break down the grain into small particles in order to allow for water penetration during cooking (Kelsall and Lyons, 2003), which is part of the liquefaction step. Prior to cooking and liquefaction, the ground meal is mixed with water to form a slurry. It is important that the water is in contact with all the particles before gelatinization. Cooking allows the starch granules to hydrate, swell greatly, and then become disrupted. This is due to water absorption at elevated temperature (90e165 C), leading to the loss of the starch crystalline structure (Kelsall and Lyons, 2003) followed by solubilization of the starch. This process enables the amylase enzymes to rapidly hydrolyze the starch molecules (liquefaction). The mash is then cooled to 75e90 C for enzymatic hydrolysis using a thermostable a-amylase. Usually, enzymic hydrolysis takes place during the cooking process with incorporation of a-amylase. Alpha-amylase only hydrolyzes the a-1,4 glucosidic linkages within the starch polymers, which results in dextrins (Delcour and Hoseney, 2010). Dextrins are short chains with a varying number of glucose molecules. Furthermore, a-amylase reduces the viscosity of the gelatinized starch (liquefaction; Zhao et al., 2008). The subsequent enzymatic hydrolysis step is known as saccharification, which is the process whereby the dextrins are hydrolyzed into individual glucose molecules. The enzyme normally used is amyloglucosidase (also known as glucoamylase). Amyloglucosidase consecutively hydrolyzes the remaining a-1,4 glucosidic linkages releasing glucose molecules. Also, this enzyme completes the hydrolysis at the a-1,6 branch points, but at a slower pace than the a-1,4 bonds (Delcour and Hoseney, 2010). Before the addition of glucoamylase, there must be a reduction in the mash temperature (to 30e32 C), and the mash must undergo a pH adjustment (4.0e5.5) using sulfuric acid or backset stillage (Kelsall and Lyons, 2003). The reason for these changes is because the prevailing temperature and pH must be favorable for the particular enzymes in each stage of the process. Another way of completing this step is to skip the saccharification tank and send the mash directly to the fermenter before adding the enzymes. This alternative method is known as simultaneous saccharification and fermentation (SSF). One of the main reasons that SSF is becoming more common is because there is less likelihood of microbial contamination, which causes toxic byproduct production and spoilage of the product (Bothast and Schlicher, 2005). Fermentation is the final step where the glucose is fermented into ethanol by the yeast Saccharomyces cerevisiae. One mole of glucose is converted into 2 Mol of ethanol and 2 Mol of carbon dioxide. It is also important to note that yeast produces carbon dioxide as

2. BIOETHANOL PRODUCTION

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a by-product. Thus, weight loss due to evolution of carbon dioxide can be measured over the period of fermentation. The weight loss curve can be used to calculate the ethanol yield. After fermentation, the fermentation broth contains ethanol, leftover solids, and water. In order to separate the ethanol from the fermentation broth, distillation by vaporization of ethanol at 78 C (Bothast and Schlicher, 2005) is performed. Once distillation is complete, the ethanol still contains more than 4% water, which forms an azeotrope (Swain, 2003). Industry regulations require further processing, with a molecular sieve system, in order to produce pure ethanol (Bothast and Schlicher, 2005). Bioethanol production via the dry-grind process is affected by many process parameters such as sorghum variety, growing environment, grain particle size, grain chemical composition, solid-to-liquid ratio, enzyme load in enzymatic hydrolysis, as well as pH and temperature during SSF. A smaller grain particle size has been found to present a larger surface area for reactions, such as water binding, solubilization, heat transfer, swelling and enzymatic action which resulted in higher sugar yield in comparison with larger particle size (Al-Rabadi et al., 2009). Barcelos et al. (2011) suggested that enzymes can be used to hydrolyze sorghum grains with maximum efficiency at a solid-toliquid ratio of 1:3 (g/mL). They also reported that the optimal enzyme loading per gram of sorghum grain is 20 mL for a-amylase and 40 mL for amyloglucosidase. 2.1.2 Factors Affecting Process Efficiency Concerning the chemical composition of sorghum grain, factors such as starch content, amylose-to-amylopectin ratio, protein content and digestibility, free amino nitrogen (FAN) content, and tannin content all affect bioethanol yield and fermentation efficiency. Wang et al. (2008) evaluated 70 sorghum grain samples with a broad range of chemical composition and physical properties for bioethanol production. In their study, starch content was a good indicator of bioethanol yield with coefficient of determination (R2) of 0.82; however, sorghum types with similar starch content could vary in bioethanol yield by as much as 7.4%. This may be attributed to the amylose-to-amylopectin ratio of sorghum starch. Sorghum types with a low amylose-to-amylopectin ratio, i.e. waxy and heterowaxy varieties, generally have a higher fermentation efficiency than nonwaxy varieties (with relatively higher amylose content) because amylose may form amyloseelipid complexes during mashing (with nonwaxy sorghums), which are resistant to enzymatic hydrolysis (Wu et al., 2007). Protein content was found as a negative factor in the dry-grind process due to the fact that some starch granules in the sorghum were embedded in a cross-linked protein matrix (Wu et al., 2008). Moreover, a strong negative linear relationship between low protein digestibility and bioethanol conversion efficiency was observed Wu et al. (2007). FAN content in sorghum grain was positively correlated with bioethanol conversion efficiency in the dry-grind process which would be expected since sufficient FAN is important nutritionally for yeast growth and proliferation in the early stage of fermentation (Zhang et al., 2017). Yan et al. (2011) reported that tannins bind with sorghum proteins to form complexes during heating and cooking, and this compromises absorption of water by the starch granules and reduces the rate of starch hydrolysis. Tannin type sorghum grain had high mash viscosity and slower conversion rate (Ai et al., 2011). Zhao et al. (2009) studied pasting properties of 68 sorghum samples, including waxy sorghum, normal sorghum, and tannin sorghum using Rapid Visco Analyser (RVA) and their effect on ethanol yield. The results showed a strong linear relationship between ethanol yield and RVA peak viscosity. Ethanol yield increased as peak viscosity decreased. Growing conditions such as irrigation, location, germination, and preharvest sprouting have effects on bioethanol conversion from sorghum grain. Liu et al. (2013) reported that sorghum grain produced under low irrigation conditions yielded 8.9% less ethanol (434.52 mL ethanol per kg sorghum) than samples produced under higher irrigation (473.32 mL ethanol per kg sorghum). Wu et al. (2008) reported that growing location substantially affected ethanol yields. Similarly, Zhan et al. (2003) reported that growing location of sorghum grain led to a 5% variation in bioethanol yield. Germination has been reported as a viable way to improve bioethanol yield from high-tannin sorghum grain. Germination could activate intrinsic enzymes to break down the protein matrix and release formerly embedded starch granules, thereby leading to increased ethanol yield, enhanced fermentation efficiency, and reduced fermentation time (Yan et al., 2009). Yan et al. (2010) also studied the effect of preharvest sprouted sorghum on bioethanol conversion. Preharvest sprouted sorghum grain only took about half the time that normal sorghum grain needed to complete the fermentation, and bioethanol yield from sprouted sorghum was higher (416e423 L/ton) than that from nonsprouted sorghum (409 L/ton). Some treatments have also been investigated to enhance bioethanol yield from sorghum grain. Ultrasound treatment has been successfully applied in liquefaction for maize starch to enhance bioethanol yield (Nikolic et al., 2010). For sorghum, Shewale and Pandit (2009) used ultrasound treatment to enhance bioethanol yield from normal healthy, germinated, and blackened sorghum grain (grain infected by insects or fungi/molds). They reported that ultrasound treatment increased liquefaction efficiency, increased dextrose (glucose) equivalents by 10%e25%,

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disrupted the protein matrix (surrounding starch granules) and the amyloseelipid complex. Zhan et al. (2006) applied supercritical fluid extrusion for sorghum grain cooking to increase bioethanol yield by approximately 5%. Decortication (removal of the outer layers of the grain) has been studied for its effect on bioethanol conversion. Corredor et al. (2006) reported that bioethanol yields increased by 3.3%e11.1% for sorghums with 10% decortication and by 7.6%e18.1% for sorghums with 20% decortication when 20% grain solids content was used. Ethanol yields increased by 9.3%e14.2% when the substrate concentrations were higher than 20%. Alvarez et al. (2010) utilized a combination of decortication and protease treatment of sorghum grain to improve bioethanol yield by 13% and save on fermentation time by 50%. 2.1.3 The Use of Millets in Bioethanol Production Millets have been traditionally used for human food, poultry, and livestock feed and companion bird feed. The potential of millets for industrial applications has not been studied intensively. Wu et al. (2006) studied the potential of pearl millet (Pennisetum glaucum L., R. Br.) for bioethanol production. Four pearl millet genotypes were evaluated for their potential as raw materials for bioethanol production. They reported that at 35% grain solids content, the ethanol yield ranged from 15.7% to 16.8% (v/v) and fermentation efficiency ranged from 94.3% to 95.6%. The results showed that the fermentation efficiency of pearl millet, on starch basis, was similar to that of maize and grain sorghum. Rose and Santra (2013) evaluated seven proso millet (Panicum miliaceum L) cultivars and six advanced breeding lines containing waxy starch for bioethanol production. The results showed that proso millet is similar to maize in terms of ethanol yield and fermentation efficiency. They also reported that waxy proso millet lines had higher fermentation efficiency than nonwaxy lines (82.4  5.5% vs. 75.5  7.4%) with one line having the highest fermentation efficiency of 97%. Barroca et al. (2017) studied bioethanol conversion from broken grain millet through a single-step no-cook process and reported higher fermentation efficiency than a conventional multistep hightemperature process. Thus, the very limited research data indicate that millets are a potential feedstock for bioethanol production. The limitation is the crop yield and total production.

2.2 Sorghum DDGS As stated, DDGS is a coproduct from dry-grind ethanol production. The major components of sorghum DDGS are protein (30%e45%), starch (2%e6%), fiber (10%e12%), fat (7%e10%), and ash (2%e5%; Yan et al., 2011). On average, sorghum DDGS has higher crude protein, neutral detergent fiber, and acid detergent fiber content than maize DDGS (23%e32% higher protein, w10% higher fiber) and a lower fat content than maize DDGS (w10%; Al-Suwaiegh et al., 2002). The gross energy (GE) of sorghum DDGS is similar to maize DDGS, while GE digestibility is lower in sorghum DDGS (Al-Suwaiegh et al., 2002). The apparent total digestive tract digestibility for GE of maize DDGS ranges from 73.9% to 82.8%, with an average of 76.8% (Al-Suwaiegh et al., 2002). The average GE digestibility of sorghum DDGS is 74%, which is lower than the average, but within the range, for maize DDGS. The low energy digestibility of sorghum DDGS may be due to its greater concentration of fiber components compared with maize DDGS (Corredor et al., 2006). As stated, DDGS is a valuable co-product of the bioethanol process and is important to the economic viability of most commercial ethanol plants. The type of grain used is likely to have an impact on DDGS composition. As indicated, sorghum-based DDGS is similar in composition to maize DDGS, with the major exception that protein levels are higher in sorghum DDGS as a result of higher protein levels in the grain. Lower residual starch levels have been reported for waxy sorghum DDSG compared with nonwaxy sorghum DDGS (Yan et al., 2011; Wu et al., 2010a). Residual starch levels reported by both authors for nonwaxy sorghum DDGS were similar to those reported for maize (Kim et al., 2008). The other important nutritional components, protein, fat, and fiber, were similar for sorghum- and maize-based DDGS, with the exception of the protein, which was higher in sorghum DDGS (w36% vs. 24%). The nutritional quality of pearl millet DDGS is also similar to maize and sorghum DDGS, with a high protein content (up to 30%; Wu et al., 2006).

2.3 Bioethanol From Sorghum Biomass Apart from producing bioethanol from sorghum grain, sorghum plant biomass, also known as next-generation renewable energy biomass, is an alternative feedstock offering many advantages such as low production inputs and less competition with food production. Therefore there are a large number of second-generation cellulosic bioethanol plants under development and opening across the United States (Coyle, 2010). The main biomass sources include agricultural residues, woody biomass, and municipal solid waste.

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Bioethanol derived from sorghum biomass is via a biochemical process includes the following steps: size reduction, pretreatment, enzymatic hydrolysis, and fermentation (Fig. 13.2). The process begins with size reduction, where the size of sorghum biomass is reduced from typically 10e30 mm after chipping to 0.2e2 mm after milling or grinding (Sun and Cheng, 2002). The purpose of size reduction is the reduction of mass and heat transfer limitations in the biomass material during hydrolysis reactions (Schell and Harwood, 1994). In general, smaller particle size gives larger surface area, allowing enzymes in the hydrolysis step to penetrate the sorghum biomass and reach the nonstarch polysaccharides, mainly cellulose (Hamelinck et al., 2005). However, an apparent drawback is that there is a consistent increase in energy consumption as a result of particle size reduction. Pretreatment of the biomass material is a necessary process in order to reduce cellulose crystallinity, increase biomass porosity, and improve enzyme accessibility (Sun and Cheng, 2002). An effective pretreatment must enhance enzyme efficiency, minimize carbohydrate losses, and inhibit by-product formation. Inhibitory compounds commonly found in biomass hydrolysates include acetic acid, formic acid, levulinic acid, 2-furaldehyde (furfural), 5-hydroxymethyl-2-furaldehyde, vanillin, syringaldehyde, and conferyl aldehyde (Parawira and Tekere, 2011). In addition, pretreatment is recognized as a key step because of its important effects on most of the other processes, including enzymatic hydrolysis, fermentation, and final product separation (Zhang et al., 2016). Physical, physicochemical, chemical, and biological processes have been studied extensively for the pretreatment of lignocellulosic materials, and detailed descriptions of these processes are available (Mosier et al., 2005; Sun and Cheng 2002; Weil et al., 1994). Table 13.1 summarizes research related to conversion of sorghum biomass to bioethanol via pretreatment. Steam explosion is a physical pretreatment that can cause hemicellulose hydrolysis, lignin solubilization, and cellulose depolymerization to enhance bioethanol yield (Mosier et al., 2005). Manzanares et al. (2012) studied the conversion of forage sorghum biomass to bioethanol using a steam explosion pretreatment. They reported that the biomass yielded 85% glucan and 190 L/ton bioethanol at optimal steam explosion pretreatment conditions of 220 C and 7 min. Pelleting is another physical pretreatment method used to increase biomass bulk density, which improves storability and reduces transportation costs. Pelleting of biomass involves size reduction of biomass feedstock, conditioning of the milled biomass by applying heat and/or moisture, and extrusion of milled biomass through a die (Colley et al., 2006; Lam et al., 2008; Larsson et al., 2008). Theerarattananoon et al. (2012) applied pelleting and acid pretreatment to conversion of photoperiod-sensitive (PS) sorghum stalk to bioethanol. The glucan yield increased with increased die thickness and decreased with increased hammer mill screen size. Conversely, xylan yield from the pellets decreased as die thickness increased and increased as hammer mill screen size increased. Among the three combinations of pelleting conditions, sorghum biomass pelleting that utilized a die with thickness of 44.5 mm and a hammer mill screen size of 6.5 mm produced pellets with the highest sugar yield, the highest pellet durability, and the greatest bulk density of biomass (Theerarattananoon et al., 2012). Acid pretreatment is considered an effective method and has been used extensively on various biomasses in order to expose hemicellulose to effective enzymatic hydrolysis and convert solubilized hemicellulose into fermentable sugars. This method has some limitations, however, such as formation of degradation products, release of potential biomass fermentation inhibitors, and requires expensive process equipment (Leustean, 2009). Acid treatment

Sorghum biomass

Size reduction

Separation

Liquid

Xylose fermentation

Solid

Pretreatment

Glucan hydrolysis Solid lignin

Detoxification & neutraliztion

Fermentation

Distillation

FIGURE 13.2 Flowchart of utilization of sorghum biomass for bioethanol.

Bioethanol

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Processing Parameters for Production of Bioethanol From Sorghum Biomass Pretreatment Methods

Pretreatment Conditions

Results

References

Forage sorghum

Steam explosion

2e10 min reaction time, 180e230 C reaction temperature

190 L/ton bioethanol yield

Manzanares et al. (2012)

Photoperiod-sensitive sorghum stalk

Pelleting and acid

22.1 kW ring-die pellet mill with 1.5 ton capacity using 4.0 mm  31.8 mm and 6.4 mm  44.5 mm die sizes; 2% H2SO4, 10% solid loading for 30 min at 140 C

92.1% enzymatic conversion of cellulose

Theerarattananoon et al. (2012)

Sorghum stalk

Acid

0.5%e4% H2SO4, 10%e15% solid loading, 15e60 min residence time, 120e200 C pretreatment temperature

0.41 g/g sugar yield

Akanksha et al. (2014)

Sorghum (variety BRS, brown rib sorghum 655) straw

Acid

50 g/L H2SO4, 7% solid loading, 120 C reaction temperature, 15 and 30 min reaction time

100% hemicellulose and 5.5% cellulose removal

Cardoso et al. (2013)

Photoperiod-sensitive sorghum biomass

Acid

1.27% H2SO4, 6.1% solid loading, 40 min residence time, 120 Ce160 C pretreatment temperature

79.7% glucose yield

Xu et al. (2013)

Photoperiod-sensitive sorghum biomass

Acid

0.5%e1.5% H2SO4, 6.1% solid loading, 40 min residence time, 160 C pretreatment temperature

0.2 g/g bioethanol yield

Xu et al. (2011)

Sorghum straw

Acid

0%e2% H2SO4, 10% solid loading, 30e90 min residence time, 60e121 C pretreatment temperature

20% sugar yield

Vancov and McIntosh (2012)

Forage sorghum

Acid

2% H2SO4, 10% solid loading for 30 min at 140 C and for 10 min at 160 C

90% cellulose conversion rate

Theerarattananoon et al. (2010)

Photoperiod-sensitive sorghum

Acid

2% H2SO4, 10% solid loading for 30 min at 140 C and for 10 min at 160 C

97% cellulose conversion rate

Theerarattananoon et al. (2010)

Brown midrib sorghum

Acid

2% H2SO4, 10% solid loading for 30 min at 140 C and for 10 min at 160 C

98% cellulose conversion rate

Theerarattananoon et al. (2010)

Grain sorghum

Acid

2% H2SO4, 10% solid loading for 30 min at 140 C and for 10 min at 160 C

81% cellulose conversion rate

Theerarattananoon et al. (2010)

Brown midrib sorghum

Acid

1.75% H2SO4, 10% solid loading for 60 min at 121 C

130 mg/g bioethanol yield

Dien et al. (2009)

Forage sorghum

Acid

25% H2SO4, 5% solid loading for 30 min at 140 C

72% hexose yield and 94% pentose yield

Corredor et al. (2009)

Sorghum straw

Acid

0.5%e2% H2SO4, 20% solid loading for 10, 30, 60 min at 121 and 140 C

0.133 g/g bioethanol yield

Mehmood et al. (2009)

Brown midrib sorghum

Acid

1%e2% H2SO4, 10e20 min residence time, 150e160 C pretreatment temperature

84% glucan yield

Kamireddy et al. (2013)

Non-brown midrib sorghum

Acid

1%e2% H2SO4, 10e20 min residence time, 150e160 C pretreatment temperature

90% glucan yield

Kamireddy et al. (2013)

Brown midrib sorghum

Alkali

0.5 g sample soaking in 30% NH4OH for 24 h at room temperature

74.4% glucan yield

Cotton et al. (2013)

Non-brown midrib sorghum

Alkali

0.5 g sample soaking in 30% NH4OH for 24 h at room temperature

61.6% glucan yield

Cotton et al. (2013)

Sorghum (variety BRS 655) straw

Alkali

50 g/L NaOH, 7% solid loading, 120 C reaction temperature, 15 and 30 min reaction time

90% lignin, 70% hemicellulose, and 25.9% cellulose removal

Cardoso et al. (2013)

Feedstock

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2. BIOETHANOL PRODUCTION

TABLE 13.1 Feedstock

Processing Parameters for Production of Bioethanol From Sorghum Biomassdcont’d Pretreatment Methods

Pretreatment Conditions 120 C

Results

References

68 g/L bioethanol yield

Sathesh-Prabu and Murugesan (2011)

Sorghum stalk

Alkali

0%e4% NaOH, at

Sorghum stalk

Alkali

28% NH4OH at 160 C for 60 min

25 g/100 g bioethanol yield

Salvi et al. (2010)

Sorghum stalk

Alkali

0%e2% NaOH, 5% solids loading, 30e90 min residence time, 121 C pretreatment temperature

95% saccharification efficiency

McIntosh and Vancov (2010)

for 60 min

commonly uses sulfuric acid, hydrochloric acid, nitric acid, or phosphoric acid, but dilute sulfuric acid has been studied extensively because it is inexpensive and has proven effective, resulting in up to 80% cellulose conversion efficiency (Zheng et al., 2007). Akanksha et al. (2014) reported that sorghum stalk yielded 0.41 g/g reducing sugars after dilute acid pretreatment when conditions were optimized by response surface methodology. In a study by Cardoso et al. (2013) using sorghum stalks, a 50 g/L H2SO4 pretreatment extracted 100% hemicellulose and 5.5% cellulose at a 120 C reaction temperature over 30 min. Xu et al. (2011, 2013) used PS sorghum (a sorghum type with a high soluble sugar content, high mass yield, and high drought tolerance under rainfed cultivation) biomass as the feedstock and produced 79.7% glucose (Xu et al., 2011) and 0.2 g bioethanol per gram sorghum biomass (Xu et al., 2013) using 1.27% H2SO4 pretreatment with 6.1% solids loading, 40-min residence time, and 120e160 C pretreatment temperature. Kamireddy et al. (2013) compared Brown midrib (BMR)-type sorghum biomass and non-BMR sorghum biomass as feedstock for bioethanol using a dilute acid pretreatment. Under similar pretreatment conditions, the overall glucan saccharification yield after enzymatic hydrolysis for non-BMR sorghum biomass was 90 and 84 wt% for BMR sorghum. In another study, BMR sorghum was considered a promising feedstock for bioethanol production because it has less lignin, and BMR was able to yield 130 mg/g bioethanol using a 1.75% H2SO4 pretreatment with a 10% solids loading for 60 min at 121 C (Dien et al., 2009). Theerarattananoon et al. (2010) compared four types of sorghum biomass: forage sorghum, PS sorghum, BMR sorghum, and grain sorghum in terms of cellulose conversion rate after 2% H2SO4 pretreatment with a 10% solids loading for 30 min at 140 C and for 10 min at 160 C. BMR gave the highest cellulose conversion rate of 98%, following by PS sorghum with 97%, forage sorghum with 90%, and grain sorghum with 81%. Alkaline pretreatment involves the use of sodium hydroxide, liquid ammonia, aqueous ammonia, lime, or other alkalis for pretreatment of lignocelluloses. Alkali pretreatment swells biomass, reduces polymerization, delignifies lignocelluloses, and increases biomass surface area. In general, the alkaline pretreatment process has been used under conditions of relatively low temperatures and over long duration, usually hours or days (Zhang et al., 2015). Cardoso et al. (2013) reported that a 50 g/L NaOH pretreatment with a 7% solids loading, 120 C reaction temperature, 15- and 30-min reaction time of sorghum stalks removed 90% lignin, 70% hemicellulose, and 25.9% cellulose for bioethanol conversion. Sathesh-Prabu and Murugesan (2011) reported that sorghum stover yielded 68 g/L bioethanol using a 4% NaOH pretreatment at 6.5 kg pressure for 60 min. Salvi et al. (2010) reported that a 25 g/100 g bioethanol yield was achieved from sorghum stalks by a 28% NH4OH pretreatment at 160 C for 60 min. A comparison of BMR and non-BMR sorghum biomass with regard to bioethanol conversion using alkali pretreatment (soaking in 30% NH4OH for 24 h) was conducted by Cotton et al. (2013). BMR sorghum biomass yielded 74.4% glucan, whereas non-BMR sorghum biomass yielded 61.6% glucan. Enzymatic hydrolysis utilizes mild conditions (pH 4.5 and approximately 50 C), as opposed to conventional hydrolysis techniques that use alkali or acid. Duff and Murray (1996) reported that fungal cellulases have the best potential for commercial-scale use. Cellulases are produced by bacterial species such as Clostridium, Cellulomonas, and Bacillus (Bisaria, 1998). In a complex system of three specific enzymes, cellulases act synergistically to hydrolyze cellulose. The three enzyme components are b-glucosidase (EC 3.2.1.21), 1,4-b-d-glucan cellobiohydrolase (EC 3.2.1.91), and 1,4-b-d-glucan glucanohydrolase (EC 3.2.1.74) (Ladisch et al., 1983; Wright et al., 1988), or cellobiase, endoglucanase, and exoglucanase, respectively. In order to produce glucose, the endoglucanase randomly cleaves cellulose chains to release cellobiose units. These cellobiose units are then attacked at the nonreducing end by exoglucanase to release fermentable glucose units. Because cellobiose accumulation results in cellulase inhibition, fungal cellulases may be supplemented with b-glucosidase to reduce cellobiose inhibition (Ryu and Mandels, 1980). Fermentation is a process that metabolizes the fermentable sugars released by enzymatic hydrolysis to ethanol by microorganisms. Glucose and xylose are the dominant sugars in the hydrolysis mixture. Saccharomyces cerevisiae and

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Zymomonas mobilis are capable of efficiently fermenting glucose into ethanol but are unable to ferment xylose. Other yeasts, such as Pachysolen tannophilus, Pichia stipitis, Entamoeba coli, and Candida shehate, can ferment xylose into ethanol (McMillan, 1993; Wang et al., 1980). Du Preez (1994) and Hahn-Ha¨gerdal et al. (1994) noted the difficulties associated with commercial use of xylose-fermenting yeasts, which include low ethanol tolerance, difficulty in optimization of fermentation parameters, and slow rate of fermentation. An alternative approach is to convert xylose into an isomer called xylulose using xylose isomerase (Chiang et al. 1981a,b; Gong et al., 1981); xylulose can then be fermented by traditional yeasts. However, Saha (2003) found that this approach is not cost-effective and that the development of genetically engineered microorganisms capable of fermenting hexoses and pentoses into ethanol should be a priority. S. cerevisiae is of particular interest, and these two review articles detail efforts to improve pentose sugar fermentation using this microorganism (Chu and Lee, 2007; Hahn-Ha¨gerdal et al., 2007). In addition to separate hydrolysis and fermentation process unit operations, other approaches include direct microbial conversion (DMC) and SSF. DMC utilizes microorganisms that simultaneously produce cellulase to hydrolyze cellulose and ferment the resulting sugars into ethanol. Clostridium thermocellum and Clostridium thermosaccharoliticum have been used in DMC studies (Wyman, 1994), but significant by-product formation and low ethanol tolerance are limitations of this approach. In SSF, enzymatic hydrolysis and fermentation take place in the same vessel. The rationale for this approach is that because cellulase activity is inhibited by glucose, rapid fermentation into ethanol increases the rate and efficiency of the overall process. The use of pearl millet biomass as a biofuel feedstock has intensified in recent years due to its high drought tolerance and use as a rotation crop (Be´lair et al., 2006). Sweet pearl millet produced high dry matter (DM) yields ranging from 10 to 19 Mg/ha under the unfavorable environmental condition in Eastern Canada (Leblanc et al., 2012). Dos Passos Bernardes et al. (2015) reported that sweet pearl millet produced 20.4 ton/ha biomass with 127.9 g/kg DM nonstructural carbohydrate concentration (NSC) and 120.9 g/kg DM water-soluble carbohydrate concentration (WSC). Moreover, delaying harvest substantially increased biomass yield, NSC, and WSC. Thivierge et al. (2015) evaluated the bioethanol production potential of such sweet pearl millet biomass. They reported that the theoretical bioethanol yield can be up to 2040 L/ha.

2.4 Bioethanol From Sweet Sorghum Sweet sorghum, also known as sweet-stemmed sorghum, has a sap rich in sucrose like sugar cane. It was originally developed as a natural sweetener in the form of a syrup, which is produced from sweet sorghum juice using an evaporation process. The fermentable sugars in the juice (sap) (53%e85% sucrose, 9%e33% glucose, and 6%e21% fructose) can be directly fermented into bioethanol (Serna-Saldı´var et al., 2012). Apart from the juice from the sweet sorghum stem, the grain in the head contains 60%e74% starch which can be hydrolyzed and fermented into bioethanol. The bagasse, the dry fibrous lignocellulosic stem material, and the residues from the head can also be used as biomass for bioethanol production. Fig. 13.3 shows a flowchart for utilization of sweet sorghum for bioethanol via three pathways: (1) direct fermentation of extracted sweet sorghum juice (glucose, sucrose, and fructose) into ethanol; (2) enzymatic hydrolysis of pretreated lignocellulosic sweet sorghum biomass (leaves, bagasse, and seed head residuals) into glucose and xylose and fermentation of these sugars into ethanol; (3) enzymatic hydrolysis and fermentation of grain from sweet sorghum panicle into ethanol. Since pathways (2) and (3) are similar to bioethanol conversion from sorghum biomass and sorghum grain, which have been comprehensively discussed earlier, this section will focus on the process of conversion of sweet sorghum juice into bioethanol. Traditionally, sweet sorghum juice is extracted by crushing the harvested stalks after the panicle is removed using roller mills (with two or three rollers in tandem) or using a hydraulic press to squeeze the juice from stalks that have been chipped (Rao et al., 2013). A press efficiency (juice extraction) of 45%e55% has been reported (Caffrey et al., 2014). Sweet sorghum juice can be directly fermented to ethanol using S. cerevisiae. Wu et al. (2010b) studied sweet sorghum biomass yield and the fermentation efficiency. Sweet sorghum variety M81, cultivated in different locations in Kansas, USA, gave biomass yields of 18,000 to 32,000 kg/ha, with sugar and grain accounting for about 40% of the total dry mass yield. The authors reported that the fermentation efficiency ranged from 93% to 94% in laboratory flask shaking fermentation tests. Wang et al. (2011) applied a BoxeBehnken response surface methodology central composite design to optimize ethanol fermentation from sweet sorghum juice using brewing instant dry yeast with fermentation parameters of temperature (25e35 C), pH (4e6), and yeast inoculation rate of 1%e5%. The optimum fermentation conditions were 27.7 C, pH 5.4, 5% inoculation, giving a 9.5% ethanol yield. Using response surface methodology, Phutela and Kaur (2014) reported an ethanol yield of 8.83% (v/v) from sweet sorghum juice with a fermentation efficiency of 87.3% using S. cerevisiae strain NRRL Y-2034 under optimized

401

2. BIOETHANOL PRODUCTION

Sweet sorghum

Stalk(75%)

Leaves(10%)

Milling

Panicles(15%)

Residues(30%)

Grain(70%)

Mill

Solid

Bagasse(30–40%) Liquid

Liquefaction Pretreatment

Juice(60–70%)

Hydrolysis

Hydrolysis

Sugar from biomass Sugar from juice

Sugar from grain

Fermentation

Distillation

Bioethanol

FIGURE 13.3

Flowchart of utilization of sweet sorghum for bioethanol production.

conditions of temperature (30 C), agitation rate (50 rpm), and yeast inoculum concentration (7.5% v/v). In addition to S. cerevisiae, Mucor hiemalis strains have been shown to be good candidates for ethanol fermentation from sweet sorghum (Goshadrou et al., 2011). Sweet sorghum juice can also be incorporated into the current dry-grind bioethanol process for improved ethanol yield and water efficiency (Appiah-Nkansah et al., 2015). These authors reported that the ethanol yield from sweet sorghum juice with the optimum grain sorghum loading was 28% higher than that from the conventional ethanol process.

402

13. INDUSTRIAL AND NONFOOD APPLICATIONS

Solid-state fermentation, a fermentation process with solid substrates under damp conditions, has also been studied for conversion of sweet sorghum juice to ethanol, as the process requires lower energy input and produces less wastewater. Several studies have been conducted on solid-state fermentation for ethanol production from sweet sorghum using S. cerevisiae (Du et al., 2014; Shen and Liu, 2008), S. cerevisiae strain AF37X (Yu and Tan, 2008), strain TSH1 (Li et al., 2013), and a thermotolerant yeast strain (Yu and Tan, 2008). Li et al. (2013) demonstrated the use of advanced solid-state fermentation using S. cerevisiae strain TSH1 on sweet sorghum stems. In their study, the stems were pulverized into particle sizes of 1e2 mm diameter and 3e50 mm length, heated to 28 C and combined with the S. cerevisiae TSH1 culture liquid in a continuous rotary drum fermenter and incubated for 2 weeks. The dosage rate was 1e2 mg dry cell weight/g dry sweet sorghum. Sweet sorghum, culture liquid and steam were constantly supplied to the fermenter. Sixteen tons of sweet sorghum stems yielded 1 ton of ethanol (99.5%, v/v). At a stem feed rate of 3.72 ton/h, the fermentation yielded 1.54 ton/h crude ethanol. The cost of fuel ethanol production was estimated at US$615.4 per ton (49 cent/L) on the premise that the cost of the sweet sorghum stems was US$30 per ton, which is cost competitive compared with wheat-based fuel ethanol (US$869.9 per ton), maize-based fuel ethanol (US$841.7 per ton), and cassava-based fuel ethanol (US$778.1 per ton). Du et al. (2014) isolated S. cerevisiae strain TSH1 from samples of soil in which sweet sorghum stalks were stored. It was cultured with crushed feedstocks (96 tons), and the ethanol fermentation process took place in a 550 m3 industrial rotary-drum fermenter at 30 C for 21 h. The fermentation process was completed after 15 h, reaching a theoretical yield of about 88% at a 10 kg/ton/h production rate. The cost of ethanol per ton was competitive compared with other ethanol production feedstocks such as maize and cassava. Their findings indicated the strong potential of industrial-scale, solid-state fermentation of S. cerevisiae strain TSH1 on sweet sorghum feedstocks. In addition to solid-state fermentation, very high gravity (VHG) fermentation is considered as an emerging fermentation method for bioethanol production because of its high ethanol yield and lower water use (Wang et al., 2007). Laopaiboon et al. (2009) studied the bioethanol fermentation efficiency from sweet sorghum juice supplemented with sucrose or sugarcane molasses as carbon sources and yeast extract and peptone or ammonium sulfate as nitrogen sources. Ethanol yield from VHG fermentation of sweet sorghum with addition of sucrose was 15% higher than that with sugarcane molasses as an additive in batch mode at 30 C, under static conditions. The data obtained were applied to develop kinetic models to demonstrate bioethanol fermentation from sweet sorghum juice using the VHG technique in the batch operation, continuous operation, and fed-batch operation modes (Thangprompan et al., 2013). An ethanol yield of 90 g/L was obtained using the continuous batch mode and 96 g/L from fed-batch production.

3. VALUE-ADDED PRODUCTS FROM SORGHUM AND MILLET PROTEINS 3.1 Sorghum and Millet Bioplastics Bioplastics have a very long history. Glyptodon was an early herbivorous mammal that lived in the South Americas between the Pleistocene and Modern eras (2 millione10,000 years ago; Wikipedia, 2017). Glyptodon resembled a large slow-moving armadillo and was the size and weight of a Volkswagen Beetle car, with a protective shell made up of more than a thousand 2.5 cm-thick plates. These plates were made from the biopolymer keratin. There is some evidence that the earliest South American settlers sheltered from adverse weather under Glyptodon shells, which represents an early example of man’s use of a biomaterial. Unfortunately, there is also some evidence that early man hunted Glyptodon to extinction and so lost their early source of a renewable biomaterial. Moving forward in time, zein, the prolamin protein of maize, was identified as a potential raw material for polymer applications by Gorham in 1821 (Lawton, 2002). This was even before the discovery of kafirin, the prolamin protein of sorghum, which occurred in 1916 (Johns and Brewster, 1916). According to Lawton (2002), from the time zein was discovered in the early 1900s, a number of patents were granted for the extraction and use of zein. Commercial production of zein began in the 1930s, and its production peaked in the late 1950s, with applications ranging from coatings, fibers, adhesives, printing inks, and binders. During this period, much of the patent literature for zein and its applications were broadened to other prolamins, including kafirin but not the millet prolamins. However, it does not appear that kafirin was commercially produced and used at this time. With the rise of modern petroleum-based synthetic plastics, zein production has fallen, and present-day commercial applications are extremely limited, with none at all for kafirin or the millet prolamins. This is in spite of the recent resurgence in interest in plant prolamins as renewable, biodegradable resources for bioplastic materials. Concerns that

3. VALUE-ADDED PRODUCTS FROM SORGHUM AND MILLET PROTEINS

403

fossil fuel feed-stocks are finite and limited, and that disposal of synthetic nonbiodegradable biopolymer plastics result in environmental problems has driven interest in these proteins (Taylor et al., 2013). Consequently, there is a huge body of published research literature, mainly on zein bioplastics, with a lesser amount on kafirin and other plant prolamin bioplastics. However, currently, this body of research remains largely in the laboratory. High-cost and inferior functional properties of prolamin bioplastics are the main reasons given for the lack of commercial products (Lagrain et al., 2010; Lawton, 2002). The availability of very large amounts of protein-rich coproducts from bioethanol production using maize or sorghum has potential to improve the economics of zein and kafirin extraction (Anderson and Lamsal, 2011; Wang et al., 2009) and potentially improve the chances of commercial prolamin bioplastic products becoming economically viable. But as stated, for now, there is very little zein commercially produced, and regardless of its potentially superior properties (Schober et al., 2011), almost no kafirin is extracted outside the laboratory. The superiority of kafirin over zein as a starting material for bioplastic production is due to its higher degree of hydrophobicity and its slow digestibility which is decreased further in the presence of wet heat (Belton et al., 2006; Duodu et al., 2003). Details of how kafirin and millet prolamins can be extracted, their chemistry and functionality are described in Chapter 6 “Starch and Protein Chemistry and Functional Properties.” A number of different forms of bioplastic can be produced from kafirin, including films and coatings, microparticles, fibers, foams, emulsions, mats, and scaffolds. The form taken is dependent on the preparation conditions. Taylor et al. (2013) reviewed the various self-assembly mechanisms that have been proposed for the various bioplastic forms. Millet prolamins have been studied far less than kafirin, but millet bioplastic films have been produced and their properties determined (Gillgren et al., 2011; Gόmez-Martίnez et al., 2012). Table 13.2 summarizes potential nonfood applications of various forms of sorghum and millet bioplastics and the technologies used for their preparation. The text that follows will detail how these different bioplastics are made and what attempts have been made to improve their functionality.

3.2 Sorghum Protein (Kafirin) and Millet Protein Films and Coatings Research into the production and functional properties of free-standing kafirin films has largely paralleled and been compared with that of work on zein films made from commercially available zein (commercial zein). All kafirin films have been made from laboratory-extracted kafirin. In early work, kafirin films were made from kafirin extracted from the protein-rich fractions remaining after wet milling of sorghum, using 95% (w/w) aqueous ethanol at a temperature of 65 C, with no reducing agent (Buffo et al., 1997). Film tensile properties and water vapor barrier properties were found to be similar to those of films made from commercial zein. When sorghum dry milling fractions, including bran, were used for kafirin extraction, films were found to be stronger and less extensible than commercial zein films (Da Silva and Taylor, 2005). Water vapor permeability (WVP) of these films was higher than that of commercial zein films. This was attributed to greater film thickness and the presence of microcracks. These films were also variable in color, depending on the milling fractions used for kafirin extraction (Fig. 13.4). Films made from kafirin extracted from bran of red pericarp sorghum were darker in color than those from flour or white pericarp sorghum. This was attributed to coextracted phenolics which were present in the bran. With kafirin extracted from decorticated red nontannin sorghum, Gillgren and Stading, (2008) found that film tensile properties were similar to those found by Da Silva and Taylor (2005) but WVP was found to be significantly lower than that of commercial zein films depending on the level of plasticization used. Inclusion of natural antimicrobial agents and polyphenols into kafirin films was found to reduce oxygen permeability but did not affect WVP (Giteru et al., 2015). Inclusion of citral decreased tensile strength but increased film elongation, whereas quercetin addition did not change film tensile properties. Films containing citral showed strong antimicrobial activity against a range of microorganisms. Pearl millet prolamin (pennisetin) films were found to be similar in strength and extensibility, and WVP to kafirin films and either equal in strength or stronger but less extensible with lower WVP than commercial zein films, when the same amount of plasticizer was used (Gillgren et al., 2011). It has become clear, that at least in part, the functional properties of prolamin films are dependent on the composition of the prolamins in terms of their prolamin subclasses (Schober et al., 2011). This composition is determined by the methodology used for the prolamin extraction (Chapter 6). Kafirin used in the Da Silva and Taylor (2005) and Gillgren and Stading (2008) studies were prepared with a reducing agent as part of the extractant. This consequently enabled the reduction of disulfide linkages and resulted in the extraction of more kafirin subclasses, which in turn enabled films to be formed with different functional properties to those made by Buffo et al. (1997). Another factor responsible for the different functional properties of these kafirin films was the use of different plasticizers. Films without plasticizers are brittle. Plasticization improves film extensibility by reducing proteineprotein interactions,

404

TABLE 13.2

Summary of the Forms of Sorghum and Millet Bioplastics, the Technologies Used, and Potential Applications Suggested Potential Application

Material

Technology

Properties

References

Cast films

Kafirin

Cast from aqueous ethanol

Similar to commercial zein films. No reducing agent included in extractant.

Biopolymer for edible or nonedible applications

Buffo et al. (1997)

Stronger and less extensible with lower water vapor permeability (WVP) than commercial zein films. Bran films highly colored due to coextracted phenolics. Reducing agent included in extractant.

Fruit and nut coatings

Da Silva and Taylor (2005) and Gillgren and Stading (2008)

Kafirin extracted with different solvents and dried under different conditions. Stronger and less extensible with lower WVP than commercial zein films. Heat dried kafirin caused protein aggregation and films with inferior functional properties

Alternative to zeinbased materials

Gao et al. (2005)

Tannin cross-linked films: stronger and less extensible, less digestible and slower biodegradation than untreated kafirin films

None given

Emmambux et al. (2004)

Cast from glacial acetic acid

Microwave heat treatment of kafirin films was more effective at increasing tensile strength than wet heating kafirin prior to film formation. Both resulted in intermolecular disulfide crosslinking of kafirin monomers and increased b-sheet structure

None given

Byaruhanga et al. (2005, 2006, 2007)

Cast from aqueous ethanol with inclusion of antimicrobial agents

Inclusion of citral decreased tensile strength but increased film elongation. Quercetin addition did not change film tensile properties. Both lowered oxygen permeability of films but did not affect WVP. Citral showed a wide range of antibacterial activity

Bioactive packaging to improve food safety and quality

Giteru et al. (2015) and Taylor et al. (2007)

Pennisetin (pearl millet prolamin)

Cast from aqueous ethanol

100 mm thick; similar to zein and kafirin films.

None given.

Gillgren et al. (2011)

Sorghum starch/flour

Cast

Inclusion of antimicrobial agent, nisin was found to show inhibitory activity against Lactobacillus delbrueckii strain ATCC11842

Active packaging

Schause et al. (2002)

Sorghum starch

Cast from aqueous solution with glycerol as plasticizer

Oxidation and acid-oxidation modifications improved film tensile strength, reduced elongation but resulted in higher WVP

Biodegradable films

Biduski et al. (2017)

13. INDUSTRIAL AND NONFOOD APPLICATIONS

Form of Bioplastic

Coatings

Kafirin

Dipping

Decreased fruit respiration rate and reduced ethylene production resulting in retarded senescence and increased shelf-life.

Extension of shelf-life of climacteric fruits including pears and avocado

Buchner et al. (2011) and Taylor et al. (2016)

Coating retained its integrity at acidic pH and disintegrated at basic pH allowing drug release

Enteric coating on capsules for controlled drug release

Lal et al. (2016)

Kafirin

Compression

Tablets had acceptable hardness and friability. Drug release was greater at acidic pH (pH 1.3) than at pH 6.8

Tablet excipient

Elkhalifa et al. (2009)

Fibers and mats

Kafirin/kafirin polycaprolactone

Electrospinning

Cylindrical fibers, 300e500 nm with hydrophilic surface

Controlled release, wound healing, tissue engineering

Xiao et al. (2016b)

Microparticles

Kafirin

Coacervation from a solution of kafirin in glacial acetic acid

Spherical porous structures 10e20 mm diameter Not suitable as biomaterial - can cause chronic inflammatory response when implanted in animal model

Encapsulation of antioxidants; Binding bone morphogenetic protein-2 (BMP-2) for bioactive scaffolds

Anyango et al. (2012) and Taylor et al. (2009a,c, 2015)

Kafirin

Coacervation from a solution of kafirin in aqueous ethanol

Encapsulated sorghum condensed tannins (SCT) was effective at inhibiting digestive amylases and retained the inhibitory activity during a simulated digestion. Oral starch tolerance test using a rat model, showed encapsulated SCT prevented a glucose spike and did not cause elevation in insulin levels

Nutraceutical as antihyperglycemic agent for treatment of Type 2 diabetes

Links et al. (2015, 2016)

Kafirin

Coacervation from a solution of kafirin in aqueous ethanol, sodium chloride used for phase separation

In vitro release profiles indicated that the kafirin microparticles may have potential for delayed release of prednisolone

Oral nutraceutical and drug delivery system

Lau et al. (2015a,b)

Microparticle films

Kafirin

Cast from colloidal suspension of kafirin microparticles in diluted acetic acid

Microparticle films 20 mm thick; water stable; slowly biodegradable; nonallergenic, No abnormal inflammatory response when implanted in rodent model

Bioactive biomaterial scaffolds.

Anyango et al. (2011) and Taylor et al. (2009a, 2015)

Nanoparticles

Kafirin, kafirin/ carboxymethylchitosan

Coacervation from a solution of kafirin in aqueous ethanol

Spherical nanoparticles. Chitosan/kafirin nanoparticles were larger, had better encapsulation and loading efficiency, better photo stability, improved dissolution profile during simulated digestion and better cellular uptake of curcumin than kafirin nanoparticles

Delivery system for bioactive compounds

Xiao et al. (2015a)

3. VALUE-ADDED PRODUCTS FROM SORGHUM AND MILLET PROTEINS

Tablets

Continued

405

406

TABLE 13.2

Summary of the Forms of Sorghum and Millet Bioplastics, the Technologies Used, and Potential Applicationsdcont’d Suggested Potential Application

Material

Technology

Properties

Nanoparticle stabilized Pickering emulsions

Kafirin

Coacervation from a solution of kafirin in glacial acetic acid, then emulsified with an oil

Protect curcumin against ultraviolet light, retard lipid oxidation but not resistant to high pH and temperature nor pepsin digestion. Improvements with stabilization in hydrogel but reduced curcumin bioavailability

Oral administration of Pickering emulsions as bioactive encapsulation agents

Xiao et al. (2015b, 2016a, 2017a,b)

Thermoplastic materials

Kafirin

Thermoplastic molding

Compression molded slabs had similar tensile properties to cast kafirin films using the same plasticizers but at lower plasticizer level

None given

Di Maio et al. (2010)

Pennisetin (pearl millet prolamin)

Thermoplastic molding

Could form viscoelastic melts, evidence of plasticizer phase separation

None given

Go´mez-Martı´nez et al. (2012)

Sorghum flour

Extrusion and compression molding

Flexible slabs from chemical-modified flours had better mechanical properties than unmodified flours but inferior to oil-based plastics.

Thermoplastic materials

Trujillo-de Santiago et al. (2015)

Sorghum DDGS

Grafting with methacrylates followed by compression molding

Films had good strength and wet stability

Thermoplastic materials

Reddy et al. (2014)

Millet flour composite with zein

Extrusion

Foams densities of 350e500 kg/m3

Foam-based material suitable for packaging

Filli et al. (2011)

Sorghum starch composite with Yucca schidigera extract

Extrusion and blown tubular films

Yucca extract added as a natural surfactant to improve film mechanical properties and reduce WVP

None given

Rodrı´guez-Castellanos et al. (2013)

Blown tubular films

References

13. INDUSTRIAL AND NONFOOD APPLICATIONS

Form of Bioplastic

3. VALUE-ADDED PRODUCTS FROM SORGHUM AND MILLET PROTEINS

(A)

(B)

Whole grain

Commercial zein

Whole grain

90% extraction

75% extraction

90% extraction

75% extraction

25% bran

10% bran

25% bran

Commercial zein

407

10% bran

FIGURE 13.4 Films prepared from defatted commercial zein and kafirin extracted from a (A) white, tan-plant nontannin sorghum (B) red (gray in print version), non-tannin sorghum milling fractions (whole grain flour, 90% and 75% extraction rate flour, and 25% and 10% brans). From Da Silva, L.S. and Taylor, J.R.N., 2005. Physical, Mechanical, and Barrier Properties of Kafirin Films from Red and White Sorghum Milling Fractions. Cereal Chemistry, American Association of Cereal Chemists Inc. Fig . 1, p. 10. http:// aaccipublications.aaccnet.org/doi/abs/10.1094/CC-82-0009.

lowering the Tg (glass transition temperature) of the protein, and increasing free volume (Taylor et al., 2013). Glycerol is the most widely used plasticizer for prolamin films. For kafirin films, glycerol is usually used in combination with one or more other plasticizers. These additional plasticizers include polyethylene glycol 400 (Buffo et al., 1997), polyethylene glycol 300 (Lal et al., 2016), lactic acid and polyethylene glycol 400 (Byaruhanga et al., 2006; Da Silva and Taylor, 2005; Emmambux et al., 2004; Gillgren et al., 2011; Giteru et al., 2015), citric acid (Gillgren et al., 2011), and glucono-delta-lactone (GDL) (Buchner et al., 2011; Taylor et al., 2016). While film extensibility is increased with plasticization, film strength and water barrier properties are decreased (McHugh and Krochta, 1994). These properties are further affected when glycerol and other hygroscopic plasticizers are used, since water has a major plasticization effect on prolamins (Gontard et al., 1993) by further lowering their Tg (Lawton, 2002) and making the films even more extensible. In addition, glycerol does not bind well to kafirin and so over time leaches out of the films, resulting in less extensible films with greater water permeability (Anyango et al., 2011). However, it has been noted that when very low levels of glycerol are used to plasticize films, the glycerol adsorbs onto or into the protein structure becoming what is known as an internal plasticizer (Gao et al., 2006; Gillgren et al., 2009). Regardless of the extraction method or the type of plasticization used, kafirin bioplastic films still have poor functional properties compared with synthetic plastics (Gillgren and Stading, 2008). Prolamin polypeptides are thought to be held together by weak forces, with few covalent disulfide linkages (Taylor et al., 2013). Methods used to improve kafirin film functionality have been largely limited to the use of heat to induce additional disulfide linkages (Byaruhanga et al. 2005, 2006, 2007) and cross-linking with natural compounds such as tannins (Emmambux et al., 2004; Taylor et al., 2007). This was done to ensure any potential application could be classified as food compatible and to remain in keeping with the “natural” or “green” image of prolamin bioplastics. When kafirin was wet microwave heat treated prior to film formation, film tensile strength was increased, but extensibility and WVP of the films both decreased due to intermolecular disulfide cross-linking of kafirin monomers (Byaruhanga et al., 2005). Similar film functional properties were found when films were made from extracted kafirin dried at elevated temperatures (Gao et al., 2005). Film functional property changes were associated with increased levels of b-sheet structure in the kafirin (Byaruhanga et al., 2006; Gao et al., 2005) and in the kafirin films (Byaruhanga et al., 2006). Microwave heat treatment applied to preformed films was found to be more effective at increasing film tensile strength than heat treatment of the kafirin prior to film formation (Byaruhanga et al., 2007). There was no effect on film WVP, but biodegradation of the films was slowed. When kafirin films were cross-linked with sorghum tannins, they had higher stress, lower strain, lower oxygen permeability (Emmambux et al., 2004), and were less digestible, with slower biodegradation (Taylor et al., 2007) than untreated films. Kafirin structural changes induced by tannin cross-linking were different from those induced

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by heat (Byaruhanga et al., 2006). Tanninekafirin complex formation resulted in kafirin films with decreased b-sheet conformation. The mechanism of cross-linking with tannins was also found to be different to that of heat treatment. Sorghum condensed tannins (SCT) are thought to bind by hydrogen bonding to proline-dense regions of prolamins (Emmambux et al., 2004). In kafirin, it is the proline-rich g-kafirin that preferentially binds with tannins (Taylor et al., 2007) and consequently is responsible for the observed functional changes in kafirin films. The research described so far concerned free-standing kafirin and millet prolamin films produced by casting. This involves dissolving the protein in a suitable solvent, usually aqueous ethanol at elevated temperature (Buffo et al., 1997) or glacial acetic acid at ambient temperature (Taylor et al., 2005), pouring the protein solution into a petri dish or similar and allowing the solvent to evaporate. This methodology is used as a laboratory process and gives valuable information on film functional properties using small amounts of protein. However, it is difficult to see how film casting could be upscaled for use as food packaging or any of the industrial applications of prolamin bioplastics set out in Table 13.2. A potentially more attractive way of making prolamin bioplastic materials is to apply manufacturing technologies used for conventional synthetic thermoplastic polymers (Di Maio et al., 2010). Heat and shear applied to synthetic polymers enable melt-processing. However, the highly a-helical structure of prolamin proteins leading to strong inter- and intra-molecular interactions is very different from the simple and regular structure of synthetic polymers and leads to thermal degradation before a melt can be formed (Di Maio et al., 2010). The addition of suitable plasticizers can allow techniques such as extrusion, various types of molding, and film blowing to be used. The feasibility of processing kafirin and pennisetin into bioplastic materials using melt mixing technologies has been investigated (Di Maio et al., 2010; Go´mez-Martı´nez et al., 2012). It was found that both kafirin and pennisetin in the presence of plasticizers can form viscoelastic melts. Compression molded slabs of kafirin had similar tensile properties to those of cast films with the same plasticizers but at lower plasticizer content (Di Maio et al., 2010). Even at the small-scale investigated (50 g per mix), the lack of sufficient kafirin or pennisetin has limited further work in this area. Perhaps, the area of research into kafirin bioplastics films closest to a commercial product is that of coatings of climacteric fruits to increase shelf life and extend eat-ripe quality (Buchner et al., 2011; Taylor et al., 2016) and as enteric coatings for drug release (Lal et al., 2016). When kafirin coatings, including GDL and propylene glycol (PG) as plasticizers, were used to coat pears and avocados, descriptive sensory evaluation indicated the eat-ripe quality of pears was extended between 1 and 2 weeks (Buchner et al., 2011) and avocados by 1 week (Taylor et al., 2016) compared with untreated controls. This quality improvement was due to reduced respiration rate and consequent reduction in ethylene production of the coated fruit. The addition of GDL was thought to improve kafirin solvation and so improve the coating effectiveness over and above that of kafirin coatings containing just PG (Taylor et al., 2016). There were some negative aspects of the effect of kafirin coatings on fruit quality. Coated pears appeared to shrivel over the whole pear surface faster than uncoated pears (Buchner et al., 2011). Free-standing kafirin films are noted to have poor water barrier properties, and this became evident when kafirin coatings failed to reduce moisture loss in pears. The shriveling of the pears was thought to be due to the elasticity of the kafirin coating squeezing the pears on moisture loss, in a similar way to a deflating balloon held in a net. Kafirin coatings also impacted negatively on the visual appearance of litchi, darkening the fruit peel, and also forming an unsightly white deposit on the peel (Taylor et al., 2006). In the pharmaceutical area, kafirin film formulations prepared from aqueous ethanol containing sodium hydroxide and polyethylene glycol as plasticizer, have been used to coat gelatin and hydroxypropylmethyl cellulose capsules containing paracetamol (Lal et al., 2016). Simulated drug release studies showed that the coatings retained their integrity, with no drug release at pH 1.2 after 2 h and disintegrated at basic pH which allowed gradual drug release when exposed to pH 6.8 phosphate buffer.

3.3 Kafirin Microparticles and Nanoparticles Another form of kafirin bioplastic material which has potential application in the pharmaceutical and biomedical areas is kafirin microparticles and films that can be made from them. Kafirin microparticles have been made by a simple phase separation/coacervation process, by dissolving the kafirin in a suitable solvent, either aqueous ethanol or glacial acetic acid, and then adding water to precipitate out the kafirin as microparticles (Taylor et al., 2009a). The morphology of the microparticles differed depending on the solvent used. Those made with the use of glacial acetic acid were spherical or irregular in shape, ranging in size between 1 and 10 mm, with a rough porous surface and numerous internal holes or vacuoles. The holes appeared to be the “footprint” of air bubbles entrapped during microparticle formation and resulted in structures with a large surface area. Varying the final acetic acid concentration caused changes to the microparticle structures from porous spheres to an open matrix (Fig. 13.5). These changes

3. VALUE-ADDED PRODUCTS FROM SORGHUM AND MILLET PROTEINS

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FIGURE 13.5

Light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) images of kafirin microparticles showing the effects of different acetic acid concentrations on microparticle structure, LM: (AeC); SEM: (DeF); TEM: (GeI). (A, D and G): 5.4%; (B, E and H): 21.6%; (C, F and I): 40% acid concentrations, respectively.

in form were accompanied by protein secondary structural changes from a-helical to b-sheet, indicative of protein aggregation. In contrast, microparticles made using aqueous ethanol as the initial kafirin solvent were small, smooth spheres, 1e3 mm diameter, with very few or no internal holes. A large surface area for bioactive binding is advantageous for nutraceutical or drug delivery. Exploiting the large surface area of acetic acideprepared kafirin microparticles, encapsulation of the bioactive polyphenols, catechin, and SCT was investigated as a potential delivery vehicle for controlled release of dietary antioxidants (Taylor et al., 2009b). The antioxidant release profiles of the encapsulated substances were studied under simulated gastric conditions. Over a period of 4 h, catechin and sorghum condensed tannin-encapsulated kafirin microparticles showed virtually no protein digestion but released approximately 70% and 50%, respectively, of total antioxidant activity. Furthermore, since bound SCT have been found to retain at least 50% of their antioxidant activity (Riedl and Hagerman, 2001), it was speculated that a further 25% of the antioxidant activity of kafirin-encapsulated SCT would be available to act as a free radical sink in the gastrointestinal tract (Taylor et al., 2009b). Building on these findings, kafirin microparticles were investigated as an oral delivery system for SCT as a potential agent to attenuate hyperglycemia (Links et al., 2015). Plant polyphenols are capable of inhibiting digestive amylase enzymes, a-amylase, and a-glucosidase (Kim et al., 2011; Kim and Park, 2012) and thus can reduce glucose absorption. Prevention of glucose absorption decreases postprandial hyperglycemia and has been identified as an appropriate therapeutic approach to treating Type 2 diabetes (Shobana et al., 2009). Kafirin microparticles encapsulating SCT, which were produced by an alcoholic coacervation method, were found to be more highly effective (approximately 20,000 times) at inhibiting a-glucosidase than acarbose, an antidiabetic drug (Links et al., 2015). However, acarbose was found to be a better a-amylase inhibitor. Moreover, the kafirin-encapsulated SCT retained its inhibitory activity

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during simulated digestion, whereas unencapsulated SCT lost most of its enzyme inhibitory activity. This work was extended to include an in vivo oral starch tolerance test using healthy rats (Links et al., 2016). Kafirin-encapsulated SCT was found to prevent a glucose spike and was effective at reducing blood glucose levels by 11.8% when compared with water controls. This reduction was the same as that of acarbose. Neither kafirin-encapsulated SCT nor acarbose caused elevation in insulin levels. Furthermore, the kafirin encapsulation process was able to mask the bitterness and astringency usually associated with SCT. This very preliminary work indicated that encapsulating SCT in kafirin microparticles has potential as an affordable nutraceutical treatment for hyperglycemia management. Kafirin microparticles have also been identified as potential carriers for other drugs and bioactive compounds. Prednisolone, an anti-inflammatory drug, was successfully loaded into kafirin microparticles using kafirin extracted from different sources (Lau et al. 2015a,b). Regardless of the source of kafirin, in vitro release profiles indicated that the kafirin microparticles may have potentially delayed release of this drug. Kafirin nanoparticles and kafirin/ carboxymethyl-chitosan nanoparticles were used to encapsulate curcumin (Xiao et al., 2015a), a lipid-soluble yellow pigment with potential health benefits including anti-inflammatory, anticarcinogenic, and antioxidant activities derived from its polyphenolic content (Xiao et al., 2015b). Both types of encapsulated nanoparticles were spherical in morphology. The chitosan-containing particles were slightly larger and had better encapsulation and loading efficiency than the kafirin nanoparticles. In addition, the kafirin/carboxymethyl-chitosan nanoparticles showed better photostability of the curcumin, had improved dissolution profile during simulated digestion, and gave better cellular uptake of curcumin. Another method studied for encapsulating curcumin for controlled release was using kafirin nanoparticlese stabilized Pickering emulsions (Xiao et al., 2015b). Pickering emulsifiers function by providing a physical barrier at the droplet interphase. They are characterized by having good stability against coalescence and Ostwald ripening, as well as the ability to stabilize emulsions with large droplet size. Kafirin nanoparticles were made in a similar way to that of the acetic acideformed microparticles (Taylor et al., 2009b). Briefly, a solution of kafirin in glacial acetic acid was added drop-wise to bulk water, resulting in the precipitation of a nanoparticle suspension (Xiao et al., 2015b). After dialysis to remove excess acetic acid, the nanoparticle suspension was homogenized with a solution of curcumin in vegetable oil to form the Pickering emulsion. Initial work showed these emulsions could protect curcumin against ultraviolet light and were able to retard lipid oxidation (Xiao et al., 2015b). However, they were not resistant to high pH and temperature and were unable to withstand pepsin digestion, resulting in early lipid phase release (Xiao et al. 2015a, 2016b). To improve the functional properties of Pickering emulsions for oral administration, a double Pickering emulsion using kafirin nanoparticles as an outer layer stabilizer was prepared (Xiao et al., 2017a). The stability of the double Pickering emulsion was similar to that of the single Pickering emulsion stabilized using kafirin nanoparticles under simulated intestinal conditions (Xiao et al., 2017b). Kafirin microparticle-stabilized Pickering emulsions were then immobilized within a calcium cross-linked alginate hydrogel in a further attempt to improve the functional properties of these emulsions as encapsulating agents for controlled release (Xiao et al., 2017a). Results showed emulsion droplet coalescence observed previously under alkaline or heating conditions was largely prevented when the Pickering emulsion was immobilized within the hydrogel. Also under simulated digestion, coalescence was less with pepsin digestion, and lipid phase release took place in simulated intestinal fluid. However, curcumin bioavailability was reduced due to free-curcumin retention on the hydrogel. Investigation into other potential hydrogel formulations was proposed to improve this methodology as a means of oral administration of Pickering emulsions as bioactive encapsulation agents. With a view to using kafirin microparticles as a three-dimensional scaffold for hard tissue repair, vacuolated, kafirin microparticles formed by coacervation from acetic acid were cross-linked with either glutaraldehyde or heat, in order to make larger structures (Anyango et al., 2012). After formation, these larger microparticle structures were loaded with bone morphogenetic protein-2 (BMP-2) agents, which are able to induce the formation of bone and cartilage. Both wet heat and glutaraldehyde treatments resulted in larger kafirin microparticles than those without treatment. Different mechanisms of cross-linking were identified for heat and glutaraldehyde. Heat treatment induced polymerization by disulfide bonding and resulted in structures with large vacuoles that appeared to be formed from coalesced, round nanostructures. The mechanism of cross-linking with glutaraldehyde treatment was not by disulfide cross-linking, and the structures formed were comprised of spindle-shaped nanostructures. Both cross-linking treatments increased the amount of BMP-2 that could be bound to the modified kafirin microparticles, probably due to their increased size. In a study to determine the safety and biocompatibility of kafirin microparticles for use as an implantable biomaterial, kafirin microparticles that had not been cross-linked were found to induce chronic inflammation, abnormal

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red blood cells, and gross fibrin formation 1 week post a subcutaneous injection of the kafirin microparticles (Taylor et al., 2015). This adverse reaction was thought to be due to the release of hydrolysis products, such as glutamate, during the rapid degradation of the kafirin microparticles, which was a consequence of their large surface area. It appears that the form of kafirin biomaterial to be implanted needs to be considered when assaying the safety of such materials (Taylor et al., 2015). Very thin (15 mm), free-standing bioplastic films can be made from kafirin microparticles by a casting method (Taylor et al., 2009c). When implanted subcutaneously in rats, no abnormal inflammatory response was found, and the films were only partially degraded after 28 days (Taylor et al., 2015). The functional properties of these films were superior to films cast directly from kafirin at the same protein content. Kafirin microparticle films, when compared with films cast directly from kafirin, were found to be relatively strong, weakly extensible with lower digestibility (Taylor et al., 2009c) and better water stability (Anyango et al., 2011; Taylor et al., 2009c). When these films were cross-linked with heat or transglutaminase, the films were thicker, and their tensile strength was reduced (Anyango et al., 2011). Glutaraldehyde treatments increased the film tensile strength considerably and also led to improved water stability. Thus, it appears that while kafirin microparticles are not as safe as an implantable biomaterial, the preliminary works suggest that films made from kafirin microparticles are safe and so may find applications as an implantable biomaterial.

3.4 Kafirin Fibers and Mats Another method of making a biomaterial suitable for controlled release, wound healing, or tissue engineering is the production of electro-spun fiber mats, as described by Xiao et al. (2016a). Different proportions of kafirin and polycaprolactone (PCL) were used. The kafirin/PCL mixture was dissolved in a 4:1 solution of acetic acid/dichloromethane and then electro-spun into fibers which were collected as fiber mats. For drug release studies, carnosic acid, a water-soluble natural diterpene found in sage and rosemary, with anti-inflammatory, antioxidant, and antitumor properties, was codissolved with the kafirin/PCL in the spinning solution and encapsulated during the spinning process. The mats consisted of cylindrical fibers of diameters in the range of 300e500 nm with a hydrophilic surface. Mats with a higher kafirin content swelled more in phosphate buffer solution than those containing a larger PLC component. The most suitable mechanical properties for the intended applications were obtained when a 1:2 ratio of kafirin to PLC was used. Drug release was by diffusion, with a rapid burst release up to 30 min for all three of the kafirin/PCL ratios studied. After 5 h, mats made from 1:1 kafirin:PLC had released the greatest amount of carnosic acid (58.1%), whereas mats from a 1:3 ratio released the least (43.5%).

3.5 Kafirin Tablets The potential of kafirin for use as a tablet excipient has been investigated (Elkhalifa et al., 2009). Tablets consisting of kafirin, calcium hydrogen orthophosphate, and magnesium stearate with caffeine as a model drug were formed by compression. The tablets were of acceptable hardness and friability. Drug release over several hours showed greater caffeine release at acidic pH (pH 1.3) than in either pH 6.8 phosphate buffer or distilled water. This was thought to be due to deamidation of the kafirin at acid pH. It was concluded that kafirin could have potential application for use as a tablet for drug delivery.

3.6 Sorghum and Millet Films From Flour, Starch, and Sorghum DDGS With a view to avoiding the prolamin extraction step, some research has focused on using sorghum or pearl millet flour, sorghum or Japanese Barnyard millet starch and sorghum distillers dried grains to produce bioplastic materials (Biduski et al., 2017; Filli et al., 2011; Reddy et al., 2014; Rodrı´guez-Castellanos et al., 2013; Schause et al., 2002; Cao et al., 2017; Trujillo-de Santiago et al., 2015). Cast sorghum starch and flour films containing the antimicrobial agent nisin were found to show inhibitory activity against Lactobacillus delbrueckii strain ATCC11842 (Schause et al., 2002). Sorghum flour was said to form better matrices than starch due to the presence of kafirins and glutelins acting as cross-linking agents. This, in combination with low cost and ready availability, makes sorghum flour a better raw material for starch-based films. However, to improve the functional properties of cast sorghum starch films, the effect of acid and oxidation was studied (Biduski et al., 2017). Oxidation and acid modification followed by oxidation resulted in films with higher tensile strength, lower elongation, and higher WVP than untreated starch films. These changes were due to inclusion of carbonyl and carboxyl groups in the starch. Yucca schidigera, also known as the Mojave yucca, is an agave native to desert

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regions of the United States (Rodrı´guez-Castellanos et al., 2013). It contains saponins, mainly sarsasapogenin and smilagenin, and its extract is permitted for food use in the United States. When an extract from Yucca schidigera was composited with sorghum starch then extruded and blown, tubular films were formed (Rodrı´guez-Castellanos et al., 2013). The yucca extract was added as a natural surfactant to improve film mechanical properties and reduce WVP. A formulation of 78.5% sorghum starch, 20% glycerol, 1.5% yucca extract, and 20% water was found to produce continuous extruded films, with few pores and with the highest tensile strength and elongation when compared with control extruded films. When this formulation was used to make blown tubular films, the films were flexible and were not brittle when dry and had lower values of WVP than the control. Blown tubular films were less extensible with greater tensile strength and higher Young modulus than extruded films. Japanese Barnyard millet starch has been used to make edible films (Cao et al., 2017). When borage seed oil was incorporated into the films, the films exhibited antioxidant activity proportional to the amount of borage seed oil added. Films containing borage oils had improved functional properties when compared with control Japanese Barnyard millet starch films. They were darker in color, had improved water barrier properties, and were less water soluble, more extensible but weaker than control films. Unfortunately, it is not possible to say whether these films had superior functional properties to other starch films as no comparison was made. Composites of zein and pearl millet flour were extruded into foams with a view to producing a foam-based material suitable for packaging, which would provide both insulation and protection of goods (Filli et al., 2011). Processing conditions were identified, and foams with densities of 350e500 kg/m3 were produced. Sorghum flour and flours that had been chemically modified with maleic anhydride were identified as suitable raw materials for producing thermoplastic materials (Trujillo-de Santiago et al., 2015). Extrusion processing followed by compression molding was used. Flexible slabs made from modified flours had better mechanical properties and more uniform microstructure than those made from unmodified flours but were still inferior to those of oil-based plastics. Another type of chemical modification that has been applied was to graft synthetic monomers on to biopolymers to make them thermoplastic (Reddy et al., 2014). This technique was used in combination with compression molding to produce thermoplastic films from sorghum distillers’ dried grain (DDG) by grafting with various methacrylates. At a grafting ratio of 40%, butyl methacrylateegrafted films had good wet and dry strength and stability. Thus, grafting appears to have the ability to overcome the brittleness and poor water stability of biopolymer films.

3.7 Sorghum Protein Adhesives and Resins Li et al. (2011) developed sorghum protein adhesives with high adhesion properties and high water resistance. The authors compared the adhesive performance of three types of sorghum proteins: acetic acideextracted sorghum protein from DDGS (PI), aqueous ethanol-extracted sorghum protein from DDGS (PII), and acetic acide extracted sorghum protein from sorghum flour (PF). PI had the best adhesion performance in terms of dry-, wet-, and soaked adhesion strength, followed by PF and PII. At a protein concentration of 12%, the wet adhesion strength of PI was 3.15 MPa, compared with 2.17 and 2.59 MPa for PII and PF, respectively. The curing condition was 150 C for 10 min. According to the differential scanning calorimetry thermograms, PF protein contained a higher amount of carbohydrates than PI and PII, and those nonprotein contaminants might be the reason for the low adhesion strength of PF. In addition, the authors suggested that more hydrophobic amino acids may be aligned at the protein-wood interface than in PII, which would contribute to the better water resistance of PI. The effects of sorghum protein concentration and curing conditions on the adhesive properties of sorghum protein were also investigated in this study. The optimum sorghum protein concentration and cure temperature for best adhesion strength was 12% and 150 C, respectively. Compared with a native soy protein isolate, acetic acide extracted PI from sorghum DDGS had significantly higher wet adhesion strength of 3.15 MPa (1.63 MPa for soy protein). Amino acid analysis showed that PI had up to 57% of hydrophobic amino acids, which was likely a key factor in the greater water resistance than soy protein (36% hydrophobic amino acids). Hojilla-Evangelista and Bean (2011) used sorghum flour as alternative protein extender in plywood adhesives and obtained an adhesion strength of 1.37 MPa. In addition to sorghum protein being investigated as an adhesive, sorghum lignin has been investigated to improve the adhesion strength of soy protein isolates (Xiao et al., 2013). Sorghum lignin and extruded sorghum lignin both improved the shear strength and water resistance of adhesive composites made with soy protein isolates. Also, natural rubber composites have been produced using milled sorghum stalks as a re-enforcing filler (Yakubu et al., 2010). A cationic pretreatment of the sorghum stalk material improved the properties of the rubber composites in terms of increased density and resulted in the highest strength and hardness at low inclusion levels.

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4. FATS/OILS AND WAXES, PIGMENTS, AND ANTIOXIDANTS AS NUTRACEUTICAL PRODUCTS 4.1 Lipids and Waxes Here the lipids concerned are either oils or waxes that coat the external surface of the kernel. The kernel oils have been investigated for use as edible oils (Mehmood et al., 2008). Both sorghum kernel oils and waxes contain bioactive agents (Althwab et al., 2015). Phytosterols are present in sorghum kernel oils, and policosanols are present in the waxes. The potential health benefits of these substances are described in Chapter 8. Waxes and lipids have also been extracted from sorghum DDGS (Johnston and Moreau, 2017; Wang et al., 2005). These oils are known as postfermentation oils, as they are extracted from DDGS, the residue remaining after the bioethanol fermentation process. They can be used for biodiesel production or added to animal feed diets to provide important nutrients (Johnston and Moreau, 2017). The sorghum kernel pericarp wax has similar properties to that of carnauba palm wax, which is used in food applications (Taylor et al., 2006). Both waxes have similar melting points but different acid values and saponification numbers (Hwang et al., 2002). The melting point range for sorghum waxes is 77e85 C and 78e86 C for carnauba. Sorghum wax has a lower saponification number (16e49) than carnauba (77e95) and a higher acid value (10e16) versus 2e10. Sorghum waxes have been combined with medium chain triglyceride oil and used to coat gelatin-based candies (Weller et al., 1998b). The coated candies were less soluble in water and were slower to melt than uncoated candies. However, their sensory properties were inferior to uncoated candies, being less shiny, more opaque, chalkier, with more intense off-flavors and aftertaste. Using a more refined sorghum wax was suggested as a way to improve these sensory properties. Overall, it was concluded that sorghum wax had potential as an edible coating for confectionary. Bilayer films were made using a similar combination of triglyceride oil and sorghum wax to coat plasticized zein films (Weller et al., 1998a). These bilayer films had improved water vapor barrier properties and similar tensile properties to films made from plasticized zein alone. Inclusion of sorghum waxes into the casting solution of soy protein isolate films again resulted in films with improved water vapor barrier properties (Kim et al. 2002, 2003). But in this case, the films had reduced extensibility and water solubility. When the plasticizers glycerol and sorbitol were included with sorghum wax in the soy protein films, response surface methodology revealed that all three components affected the WVP, tensile properties, and water solubility. Sorghum wax was responsible for a decrease in film WVP and elongation at break.

4.2 Pigments A bright red colorant has been traditionally extracted from the leaf sheaths of what is known as dye sorghum in Nigeria, Benin, and other African countries (Akogou et al., 2018; Kayode´ et al., 2012; Oluwaniyi et al., 2009). These extracts have been used as natural food colorants, colorants for leather, wickerwork, and ornamental calabashes and also in traditional medicine (Kayode´ et al., 2012). The dyes consist predominantly of 3-deoxyanthocyanins, mainly apigeninidin and luteolinidin. Ethanol extraction at elevated temperature has been shown to double the yield of 3-deoxyanthocyanins when compared with the traditional aqueous extraction process. The dyes have also been shown to exhibit a much higher antioxidant activity than either cereal bran or fruits and vegetables. Whole grain, dehulled (decorticated) sorghum, and sorghum bran have been compared with rice as substrates for the solid-state fermentation of Monascus purpureus, an edible fungus that produces pigments as secondary metabolites (Srianta and Harijono, 2015; Srianta et al., 2016). These red, orange, and yellow pigments have been used as natural food colorants, as food supplements, and in traditional medicine (Srianta et al., 2016). Of the sorghum substrates used, dehulled sorghum gave the highest growth yield and pigment production, bettered only by rice. The major pigments produced with dehulled sorghum were identified as rubropunctamine (red) (66%), rubropunctatin (orange) (18%), and xanthomonascin (yellow) (4%). Rubropunctamine has been shown to have antiinflammatory and anticancer activities, whereas rubropunctatin possesses antimicrobial properties. It was suggested that when present, the outer bran layer of the sorghum kernel inhibited fungal mycelia penetration into the grain. Consequently, it was concluded that dehulled sorghum could be used as an alternative substrate for M. purpureus.

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4.3 Antioxidants There is a large body of literature that describes the measurement of antioxidant activity of various extracts from sorghum and the different millets. There is also literature on the potential health benefits of these antioxidant extracts and of antioxidant-rich sorghum and millet foods which are described in Chapter 8 Phytochemical-related health-promoting attributes of sorghum and millets. It has been suggested that sorghum extracts from sorghum grain or bran with high antioxidant activity may have applications as natural food colorants (Awika et al., 2005; Kil et al., 2009) and as natural antioxidants for prevention of lipid rancidity (Sikwese and Duodu, 2007). A crude phenolic extract made from sorghum bran was shown to be effective at reducing sunflower oil primary and secondary oxidation products compared with control samples. The extract was less effective at inhibiting primary oxidation products than the commonly used synthetic antioxidant TBHQ (tert-butylhydroquinone) in the presence or absence of ferric ions. However, compared with TBHQ in the presence of ferric ions, the crude phenolic extract was better able to inhibit the production of secondary oxidation products. This was probably due to the extracts’ ability to chelate ferric ions, thus preventing the ferric ions from decomposing hydroperoxides to secondary oxidation products.

5. MISCELLANEOUS USES Historically, in traditional societies where sorghum is grown as a subsistence crop, the dried stems, stalks, and roots are used as fuels, building materials, including thatched roofs and fences, brooms (National Research Council, 1996), and as dyes (described above). There are now examples of small family-owned businesses that have taken on the challenge of manufacturing and selling products made from sorghum plant materials. In Eastern Europe, Thailand, and China, small companies are manufacturing brooms made from sorghum stalks (Ðurciansky, 2017; EcoBrooms, 2017; Objectsofuse, 2017). Internet sales have enabled businesses access niche markets, selling sorghum and millet seed heads for flower arrangements, scarves dyed with sorghum dyes, and resin jewelry containing sorghum seeds (Etsy, 2017). Recently, the use of sorghum stalks for production of eco-friendly building materials has gained popularity. Sorghum stalks are woven and then heat pressed with a urea- and formaldehyde-free adhesive to produce a board, which has been used as wall covering, flooring, cabinetry, and furniture (Kireiusa, 2017). The boards are said to be resistant to chipping, with low water absorption so that they do not warp or deform (Chlorofill, 2017). Lastly, an Indian company has developed edible spoons made from sorghum flour blended with rice and wheat and baked at high temperatures (Bakeys, 2017). The company plans to extend its range of natural, biodegradable products to include other cutlery items and salad bowls.

6. CONCLUSIONS For industrial and nonfood applications, sorghum has potential to become a new-generation bioenergy crop due to its wide adaptability to varied agro-climatic conditions. While research into bioethanol production from millets lags behind that of sorghum, millets do appear to have potential as a bioenergy feedstock. More research is needed to confirm this. In addition to the bioethanol produced, bioethanol plants using sorghum grain, biomass, and sweet sorghum generate large amounts of by-products including DDGS and lignin. To improve the economic viability of the bioethanol process, ways must be sought to utilize these by-products. DDGS is rich in kafirin protein (30%e40%), which, as stated is more hydrophobic and less digestible than other cereal prolamins. These unique kafirin properties enable the production of biomaterials with superior functional properties when compared with those made from, for example, zein or gluten. Lignin, the most abundant aromatic polymer, can be used for heat generation and can be depolymerized into phenolic compounds for chemicals and biofuels. For sorghum to be utilized as a bioethanol feedstock in preference to maize, sorghum production yields, new and high-yielding varieties such as the multiseeded mutant sorghums that produce more than twice the amount of seed weight per panicle as the parent (BTx623) are needed (Zhang et al., 2017). Since millets have yield and production limitations, they could potentially be used as supplementary feedstock for maize and sorghum bioethanol production. Sorghum genotypes with high conversion efficiency are also required. For example, waxy (high amylopectin) sorghum has the advantage of rapid starch conversion rate when compared with regular sorghum. The use of sweet (sweet stemmed) sorghum for bioethanol production, with its high concentration of soluble sugars in its stalk plus

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grain in the panicle, also has considerable potential. Again, better yielding varieties with good agronomic properties are needed. Fully utilizing all fermentable sugars including starch in the grain and nonstarch polysaccharides in the stalk is critical to the viability of the biofuel industry. To achieve this, more efficient conversion processes must be developed. For biobased products, the documentation of research into sorghum and millet protein and starch bioplastics is, unfortunately, something of a shopping list where the preparation of various materials and their properties is described at a laboratory scale only. Notwithstanding this, the most promising application for kafirin bioplastics has been identified in the biomedical area as biomaterials (materials that can be introduced into living tissue) (Taylor et al., 2013). These high-value products are less sensitive to high production costs, and the superior functional properties of kafirin biomaterials, including low hydrophilicity, potential bio- and cyto-compatibility, and slow biodegradability are advantageous (Reddy and Yang, 2011). However, confirmation that these materials are safe (nonimmunogenic) when used, for example, as implants in living tissue, is needed (Taylor et al., 2013). Furthermore, in order for any of these applications to be exploited, an inexpensive and consistent source of kafirin of consistent quality is needed. The major research focus in this area then should be the development of advanced kafirin extraction techniques that are cost-effective. Research into novel and more effective modification methods for improvement of kafirin bioplastic functional properties and performance in use are required. Kafirin has also shown considerable potential for application in wood veneer adhesives with high water adhesion strength. Improvement of adhesion performance for large-scale production and commercialization is, however, still needed. Lastly, with the aim of further improving the economic viability of bioethanol production, high-value chemicals extracted from sorghum, so-called platform chemicals, especially from sorghum DDGS, may have great potential as novel functional or health-promoting ingredients for food, feed, medical, and industrial uses that can be easily commercialized.

Acknowledgments Dr Scott Bean of the United States Department of Agriculture for his helpful comments on the manuscript.

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