Sorghum Protein Extraction by Sonication and Its Relationship to Ethanol Fermentation

Cereal Chem. 85(6):837–842 The objectives of this research were to develop a rapid method for extracting proteins from mashed and nonmashed sorghum meal using sonication (ultrasound), and to determine the relationships between the levels of extractable proteins and ethanol fermentation properties. Nine grain sorghum hybrids with a broad range of ethanol fermentation efficiencies were used. Proteins were extracted in an alkaline borate buffer using sonication and characterized and quantified by size-exclusion HPLC. A 30-sec sonication treatment extracted a lower level of proteins from nonmashed sorghum meal than extracting the proteins for 24 hr with buffer only (no sonication). However, more protein was extracted by sonication from the mashed samples than from the buffer-only 24-hr extraction. In addition, sonication extracted more polymeric proteins from both the mashed and nonmashed samples compared with the buffer-only extraction method. Confocal laser-scanning microscopy images showed that the web-like protein microstructures were disrupted during sonication. The results showed that there were strong relationships between extractable proteins and fermentation parameters. Ethanol yield increased and conversion efficiency improved significantly as the amount of extractable proteins from sonication of mashed samples increased. The absolute amount of polymeric proteins extracted through sonication were also highly related to ethanol fermentation. Thus, the SE-HPLC area of proteins extracted from mashed sorghum using sonication could be used as an indicator for predicting fermentation quality of sorghum. Sorghum (Sorghum bicolor L. Moench) is a drought-resistant and low-input cereal grain grown throughout the world, and interest in using it for bioindustrial applications is now growing in the United States (Farrell et al 2006). Although currently only ≈2.5% of fuel ethanol is produced from grain sorghum, annual consumption of sorghum by the ethanol industry is steadily increasing from 11.25% in 2004 to 15% in 2005 and 26% in 2006 (Renewable Fuels Association 2005, 2006, 2007). Researchers and ethanol producers have shown that grain sorghum is a viable feedstock (technically acceptable, fits the infrastructure, and can be economically viable) for ethanol, and could make a larger contribution to the nation’s fuel ethanol requirements. Starch and protein are the two major components in sorghum grain. Recent research has shown that starch content is a good indicator of ethanol yield in the dry-grind process but starch content itself could not explain conversion efficiency well (Wu et al 2007). Sorghum varies in protein content from 6 to 18%, with 70–90% of the total protein belonging to the storage proteins (kafirins) (Lookhart et al 2000). According to previous research with 68 sorghum hybrids, a strong negative correlation was observed between ethanol yield and protein content (R = 0.60, P < 0.01) (unpublished data), which is similar to data reported for soft wheat cultivars (Swanston et al 2007). However, multiple linear regression, including both starch and protein content as predictors, verified that protein content did not significantly contribute to ethanol yield (P = 0.395). The effect of protein content on conversion efficiency was statistically significant (P = 0.015) but represented only 8.6% of variation in efficiency (unpublished data). Recently, we investigated the role of protein cross-linking to determine its impact on ethanol production. Protein digestibility, solubility, and microstructures were characterized for insight into protein cross-linking occurring during the mashing process (Zhao et al 2008). Protein digestibility decreased significantly during mashing to levels lower than found in cooked sorghum foods. Likewise, protein solubility in an alkaline borate buffer containing SDS decreased substantially after mashing. Confocal laser-scanning microscopy (CFLSM) images showed that web-like protein cross-links formed during mashing could trap oligosaccharides, polysaccharides, or starch and reduce the availability to enzymes during ethanol production (Wu et al 2007; Zhao et al 2008). Therefore, protein cross-linking does have a significant effect on production of ethanol from sorghum (Zhao et al 2008). Protein digestibility, used as a marker of protein cross-linking of nonmashed sorghum meal, and protein solubility parameters showed positive correlation with conversion efficiency of sorghum. It is certain that most of the proteins soluble in the borate buffer would not be digested by yeast directly (Berry and Brown 1987). The role of protein solubility to predict ethanol fermentation could be related to protein structures which can determine the access of enzymes to native and gelatinized starch, polysaccharides, or oligosaccharides (Rooney and Pflugfelder 1986; Zhao et al 2008). The amount of total area under SE-HPLC had a better correlation with fermentation parameters than protein solubility and could be used as an indicator to predict ethanol-production quality of sorghum (Zhao et al 2008). It is promising that the two indicators of degree of cross-linking occurring during mashing, protein digestibility and solubility, were highly correlated to fermentation parameters. To date, there is no rapid method that can be used to predict conversion efficiency of sorghum except for direct laboratory fermentation procedures. SEHPLC has been widely used and provides automatic analysis, high accuracy, and utilizes only a small amount of sample. Therefore, SE-HPLC could be considered a good system for rapid characterization and quantization of proteins extracted from sorghum for predicting ethanol fermentation parameters. Sonication was used as a rapid method for extraction of unreduced wheat flour proteins by breaking down the glutenin fraction with the largest molecular size (Morel et al 2000; Singh and MacRitchie 2001; Singh et al 1990), presumably by the mechanical shear degradation (MacRitchie 1975; Singh et al 1990). Ultrasound has also been used to enhance the extraction of sorghum proteins (EI Nour et al 1998; Bean et al 2006). A method for isolating sorghum starch was also developed using sonication combined with buffers (Park et al 2006), that proved the effectiveness of sonication to separate starch from protein matrix rapidly. 1 Department of Biological and Agricultural Engineering, Kansas State University, Manhattan, KS 66506. 2 Grain Marketing and Production Research Center, Agricultural Research Service, United States Department of Agriculture (USDA), Manhattan, KS 66502. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may also be suitable. 3 Corresponding author. Phone: +1 785-776-2725. Fax: +1 785-537-5534. E-mail: [email protected] doi:10.1094 / CCHEM-85-6-0837 This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. AACC International, Inc., 2008.