Effects of Amylose, Corn Protein, and Corn Fiber Contents on Production of Ethanol from Starch-Rich Media1 X. Wu,2 R. Zhao,2 D. Wang,2,3 S. R. Bean,4 P. A. Seib, 5 M. R. Tuinstra,6 M. Campbell,6 and A. O’Brien7 ABSTRACT Cereal Chem. 83(5):569–575 The effects of amylose, protein, and fiber contents on ethanol yields either. Conversion efficiencies increased as the amylose content de- were evaluated using artificially formulated media made from commer- creased, especially when the amylose content was >35%. The reduced cial corn starches with different contents of amylose, corn protein, and quadratic model fits the conversion efficiency data better than the full corn fiber, as well as media made from different cereal sources including quadratic model does. Fermentation tests on mashes made from corn, corn, sorghum, and wheat with different amylose contents. Second-order sorghum, and wheat samples with different amylose contents confirmed response-surface regression models were used to study the effects and the adverse effect of amylose content on fermentation efficiency. High- interactions of amylose, protein, and fiber contents on ethanol yield and temperature cooking with agitation significantly increased the conversion conversion efficiency. The results showed that the amylose content of efficiencies on mashes made from high-amylose (35–70%) ground corn starches had a significant (P < 0.001) effect on ethanol conversion effi- and starches. A cooking temperature of ≥160°C was needed on high- ciency. No significant effect of protein content on ethanol production was amylose corn and starches to obtain a conversion efficiency equal to that observed. Fiber did not show a significant effect on ethanol fermentation of normal corn and starch. A great amount of research recently has been conducted to much larger and highly branched polysaccharide with up to 3×106 increase ethanol yield and conversion efficiency from starch-rich glucose units and a MW of ≈5×108 and linked by ≈95% α-1,4, sources. For example, plant breeders have made a great effort to and 5% α-1,6 bonds. In general, normal cereal starches contain develop new and improved corn hybrids with higher starch 20–30% amylose and 70–80% amylopectin. Starches with <5% content to increase ethanol yields (Bothast and Schlicher 2005). and >35% amylose are defined as waxy and high-amylose starch, Wang et al (1997, 1998) studied the saccharification and fermen- respectively (Tester et al 2004b). Cereal cultivars with various tation characteristics of rye and triticale for ethanol production. amylose contents have been developed in corn, rice, wheat, barley, The saccharification and fermentation efficiencies of oats, barley, and sorghum (Jacobs and Delcour 1998; Tester et al 2004a,b; wheat, and pearl millet have also been investigated (Thomas and Goesaert et al 2005). Ingledew 1990, 1995; Thomas et al 1995; Sosulski et al 1997; Wu Many researchers have studied the structure and physical prop- et al 2006). These authors reported conversion efficiencies of erties of high-amylose starches. High-amylose starches had higher starch to ethanol in the above-mentioned cereal grains were ≈90%. gelatinization temperatures (Shi et al 1998) and formed stronger The effects of other factors such as fermentation temperatures, gels (Case et al 1998). Starch gels with different amylose contents free amino nitrogen, nitrogen sources, bacterial contamination, had different continuous matrix structure (Leloup et al 1991). and preprocessing of feedstock on ethanol fermentation have also Higher cooking temperatures and branched starch molecules could been investigated (Thomas and Ingledew 1990; O’Connor-Cox et retard the reassociation of starch molecules, phase separation, and al 1991; Jones and Ingledew 1994a,b; Sosulski et al 1997; Naren- network development processes during cooling (Case et al 1998; dranath et al 2000). But the relationships among the chemical Klucinec and Thompson 1999). The resistance of high-amylose composition of grains and ethanol production have not suffi- starches to α-amylase was also investigated (Sievert and Pomeranz ciently been studied. 1989, 1990; Richardson et al 2000; Brumovsky and Thompson The major components of cereal grains are starch, protein, fiber, 2001; Evans and Thompson 2004). They reported that the resid- and lipids. The bioavailability of starch may differ among grain ual resistant starches found after amylolytic hydrolysis of gela- cultivars and may affect the conversion rate and final yield of etha- tinized starches consisted mainly of retrograded amylose. Reid et nol (Moorthy 2002). Starch is a polymer of glucose, composed of al (1998) reported that the amylose-to-amylopectin ratio of starches various genetically determined ratios of amylose and amylopectin. significantly affected its fermentation to fatty acid by Clostridium Amylose is basically a linear polymer with ≈200 to 6,000 glucose butyricum, especially after pancreatin digestion and retrograda- units (MW 105–106) linked mainly by α-1,4 bonds (≈99%) and tion. But there is no information about the effects of amylose few α-1,6 bonds (<1%). Amylopectin, on the other hand, is a content in starches and grains on the production of ethanol and other bioproducts. The objective of this study was to determine the effects of amylose contents of starches, protein, and fiber con- 1 Contribution No 06-173-J from the Kansas Agricultural Experiment Station, tents, as well as their interactions, on yeast fermentation of starchy Manhattan, KS 66506. 2 Department of Biological and Agricultural Engineering, Kansas State University, materials to ethanol. Manhattan, KS 66506. 3 Corresponding author. Phone: 785-532-2919, Fax: 785-532-5825. Email: dwang@ MATERIALS AND METHODS ksu.edu 4 USDA-ARS Grain Marketing & Production Research Center, Manhattan, KS 66502. Names are necessary to report factually on available data; however, the USDA Starch and Cereal Samples neither guarantees nor warrants the standard of the product, and the use of the The starch samples used in this study were Amioca (essentially name by the USDA implies no approval of the product to the exclusion of others pure amylopectin), Melojel (≈28% amylose), Hylon-V (≈50% that may also be suitable. amylose), and Hylon-VII (≈70% amylose), which were of corn 5 Department of Grain Science and Industry, Kansas State University, Manhattan, KS 66506. origin. They were kindly provided by the National Starch and 6 Department of Agronomy, Kansas State University, Manhattan, KS 66506. Chemical Co. (Bridgewater, NJ). High-amylose (corn-70, corn- 7 Science Division, Truman State University, Kirksville, MO 63501. 55, and corn-35), normal, and waxy corn samples were obtained from Mark Campbell’s 2004 summer breeding nursery at the DOI: 10.1094/ CC-83-0569 This article is in the public domain and not copyrightable. It may be freely re- Truman State University Agricultural Research Farm at Kirks- printed with customary crediting of the source. AACC International, Inc., 2006. ville, MO. Corn-70 represents an S5 inbred line derived from the Vol. 83, No. 5, 2006 569 cross GUAT209:S13 × (OH43ae × H99ae) possessing ≈70% starch model development and model analysis. Starches and cereal sam- amylose and developed cooperatively between Truman State Uni- ples with different amylose contents were used to verify the results versity and the Germplasm Enhancement of Maize program. from the response-surface design tests. All experiments were Corn-55 and corn-35 were developed from an open-pollinated conducted in triplicate. Results were presented as averages of synthetic cultivar Hsyn-99 that was backcross-converted to replicates. possess the recessive starch-altering alleles amylose extender (ae) and dull (du) sugary-2 (su2), respectively. Both corn-55 and corn- Preparation of Fermentation Media from Starches 35 were developed by David Glover at Purdue University. Normal and Grain Samples and waxy sorghum samples were obtained from the Department The liquefaction and saccharification processes were the same of Agronomy, Kansas State University (Manhattan, KS). Normal as those described by Wu et al (2006). The components of the and waxy wheat samples were from the USDA-ARS (Lincoln, formulated media, containing 20.0 g of starch or 30.0 g of cereal NE). The grain samples were ground to a fine meal (≈99% passed samples, were mixed with 100 mL of distilled water in 250-mL a 1.19-mm sieve) using a Magic Mill III Plus grain mill (Magic Erlenmeyer flasks. The mixed slurries were digested in a water Mill Products & Appliances, Monsey, NY). The chemical compo- bath shaker at 95°C for 45 min after the addition of 10 μL of sition of the corn, wheat, and sorghum samples are listed in Table I. Liquozyme SC DS (240 KNU/g, 1.25 g/mL; Novozyme, Frank- linton, NC), an enzyme preparation containing a thermostable α- Central-Composite Design amylase as major component. During the early stage of digestion, The central-composite design approach was used to study the flasks were shaken manually to prevent the formation of a gel. effects and interactions of corn amylose, protein, and fiber con- The digested mashes were taken out of the water bath after 45 tents on ethanol yield and conversion efficiency. The central-com- min and cooled to 80°C, and a second dose of 10 μL of Liquo- posite design is a type of response-surface methodology (RSM) zyme was added to each flask. The liquefaction step was con- that is focused on characteristics of the fit response function tinued in the water bath shaker at 120 rpm for an additional 30 where optimum estimated response values occur. Five concentra- min at 80°C. Then saccharification was conducted in a 60°C tions of amylose (5.56–64.4%), corn gluten (8.5–15.5%) (Sigma, water bath shaker at 120 rpm for 30 min after 100 μL of Spirizyme St. Louis, MO), and corn fiber (8.5–15.5%) were used in the formu- (750 AGU/g, 1.15 g/mL; Novozyme, Franklinton, NC), an lated fermentation media.