Scott R. Bean

Presence of tannins in sorghum grains is conditioned by different natural alleles of Tannin1

Sorghum, an ancient old-world cereal grass, is the dietary staple of over 500 million people in more than 30 countries in the tropics and semitropics. Its C4 photosynthesis, drought resistance, wide adaptation, and high nutritional value hold the promise to alleviate hunger in Africa. Not present in other major cereals, such as rice, wheat, and maize, condensed tannins (proanthocyanidins) in the pigmented testa of some sorghum cultivars have been implicated in reducing protein digestibility but recently have been shown to promote human health because of their high antioxidant capacity and ability to fight obesity through reduced digestion. Combining quantitative trait locus mapping, meta-quantitative trait locus fine-mapping, and association mapping, we showed that the nucleotide polymorphisms in the Tan1 gene, coding a WD40 protein, control the tannin biosynthesis in sorghum. A 1-bp G deletion in the coding region, causing a frame shift and a premature stop codon, led to a nonfunctional allele, tan1-a. Likewise, a different 10-bp insertion resulted in a second nonfunctional allele, tan1-b. Transforming the sorghum Tan1 ORF into a nontannin Arabidopsis mutant restored the tannin phenotype. In addition, reduction in nucleotide diversity from wild sorghum accessions to landraces and cultivars was found at the region that codes the highly conserved WD40 repeat domains and the C-terminal region of the protein. Genetic research in crops, coupled with nutritional and medical research, could open the possibility of producing different levels and combinations of phenolic compounds to promote human health.

Effects of Sorghum (Sorghum bicolor (L.) Moench) Tannins on α‑Amylase Activity and in Vitro Digestibility of Starch in Raw and Processed Flours

The purpose of this study was to investigate the effects of tannins on starch digestion in tannin-containing sorghum extracts and wholegrain flours from 12 sorghum varieties. Extracts reduced amylase activity in a tannin concentration-dependent manner when the extract was mixed with the enzyme before substrate (amylopectin) addition, with higher molecular weight tannins showing greater reduction. Conversely, when the extract and substrate were combined before enzyme addition an enhancement in amylase activity was experienced. In uncooked, cooked, and cooked and stored wholegrain sorghum flours, rapidly digestible, slowly digestible, and resistant starches were not correlated with tannin content or molecular weight distribution. Resistant starch increased from 6.5% to 22-26% when tannins were added to starch up to 50% (starch weight). Tannin extracts both reduced and enhanced amylase activity depending on conditions, and, while these trends were clear in extracts, the effects on starch digestion in wholegrain flours was more complex.

Comparison of Waxy vs. Nonwaxy Wheats in Fuel Ethanol Fermentation

ABSTRACT Fermentation performance of eight waxy, seven nonwaxy soft, and 15 nonwaxy hard wheat cultivars was compared in a laboratory dry-grind procedure. With nitrogen supplements in the mash, the range of ethanol yields was 368–447 L/ton. Nonwaxy soft wheat had an average ethanol yield of 433 L/ton, higher than nonwaxy hard and waxy wheat. Conversion efficiencies were 91.3–96.2%. Despite having higher levels of free sugars in grain, waxy wheat had higher conversion efficiency than nonwaxy wheat. Although there was huge variation in the protein content between nonwaxy hard and soft wheat, no difference in conversion efficiency was observed. Waxy cultivars had extremely low peak viscosity during liquefaction. Novel mashing properties of waxy cultivars were related to unique pasting properties of starch granules. With nitrogen supplementation, waxy wheat had a faster fermentation rate than nonwaxy wheat. Fermentation rates for waxy cultivars without nitrogen supplementation and nonwaxy cultivars with nitro...

Evaluation of waxy grain sorghum for ethanol production.

ABSTRACT The objective of this research was to investigate the fermentation performance of waxy grain sorghum for ethanol production. Twenty-five waxy grain sorghum varieties were evaluated with a laboratory dry-grind procedure. Total starch and amylose contents were measured following colorimetric procedures. Total starch and amylose contents ranged from 65.4 to 76.3% and from 5.5 to 7.3%, respectively. Fermentation efficiencies were in the range of 86.0–92.2%, corresponding to ethanol yields of 2.61–3.03 gallons/bushel. The advantages of using waxy sorghums for ethanol production include easier gelatinization and low viscosity during liquefaction, higher starch and protein digestibility, higher free amino nitrogen (FAN) content, and shorter fermentation times. The results showed a strong linear relationship between FAN content and fermentation rate. Fermentation rate increased as FAN content increased, especially during the first 30 hr of fermentation (R2 = 0.90). Total starch content in distillers drie...

Assessing Fermentation Quality of Grain Sorghum for Fuel Ethanol Production Using Rapid Visco Analyzer

ABSTRACT The Rapid Visco-Analyzer (RVA) was used to characterize the pasting properties of 68 sorghum grains with a standard 23-min temperature profile. The results showed a strong linear relationship between ethanol yield and final viscosity as well as setback. Ethanol yield increased as final viscosity decreased. A modified RVA procedure (10 min) with an application of α-amylase was developed to simulate the liquefaction step in dry-grind ethanol production. There was a remarkable difference in mashing properties among the sorghum samples with the normal dosage of α-amylase. The sorghum samples which were difficult to liquefy in the mashing step had much higher peak viscosities than the samples that were easily liquefied. The results also showed that the relationship between conversion efficiency and mashing property was significant. Tannins cause high mash viscosities. There was a strong linear relationship between tannin content and final viscosity as well as peak viscosity. The modified RVA procedure...

Separation of Kafirins on Surface Porous Reversed-Phase High-Performance Liquid Chromatography Columns

Surface porous high-performance liquid chromatography (HPLC) columns were investigated for the separation of kafirins, storage proteins of grain sorghum. Kafirins were successfully separated using C3, C8, and C18 surface porous stationary phases in less than 17 min. Separations using a monolithic C18 stationary phase were also developed and were slightly faster than those achieved on the surface porous C18 stationary phase. However, the resolution was higher on the latter column. Using an ammonium hydroxide/acetonitrile mobile phase, separations were performed on a novel, alkaline stable surface porous C18 stationary phase. The resolution at alkaline pH was not as high, however, as with the traditional acidic acetonitrile mobile phases. In comparison to fully porous stationary phases, the surface porous phases provided higher resolution with much lower separation times (17 versus 40 min). Total peak areas were correlated to total protein content of sorghum (r(2) = 0.96; n = 10), and a method to measure in vitro pepsin digestibility using reversed-phase (RP)-HPLC peak areas showed good correlation to the traditional nitrogen combustion method (r(2) = 0.82; n = 20). Thus, the surface porous stationary phases could be used not only for more rapid separations but also to provide simultaneous information on total protein content and digestibility.

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 2 = 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).

Ethanol-Production Performance of Ozone-Treated Tannin Grain Sorghum Flour1

ABSTRACT Ozone has been reported as being able to degrade macromolecules such as cellulose, starch, lignins, and tannins in the textile, pulping, and water-treatment industries. Thus, we hypothesized that ozone treatment may also inactivate tannin activity and increase fermentation efficiency of tannin sorghum lines. The objective of this research was to study the physicochemical properties of ozone-treated whole tannin grain sorghum flour and its fermentation performance in ethanol production. Results showed that the ethanol yields from ozone-treated tannin grain sorghums were significantly higher than yields from the untreated flour. The fermentation efficiency of ozone-treated tannin grain sorghum was approximately 90%, which was 8–14% higher than that of untreated samples at the 36th hr of fermentation. At the end of 72 hr of fermentation, the efficiencies of ozone-treated sorghum flour were 2–5% higher than those of untreated samples. Measured tannin levels of ozone-treated samples decreased signific...

Improved characterization of sorghum tannins using size-exclusion chromatography.

L. Moench) is a native grass spe-cies of the arid and semi-arid regions of Africa (Kimber 2000). Sorghum is a genetically diverse crop and contains some geno-types that have a pigmented testa and therefore contain tannins (Rooney et al 1980; Rooney and Miller 1982). Such lines have the dominant B1_B2_ genes and are the only sorghum types with tannins (Blakeley et al 1979), despite common misperceptions that all sorghums have tannins or that the presence of tannins is linked to seed color (Boren and Waniska 1992). Due to the high antioxidant activity, sorghum tannins have recently been exam-ined for potential as a nutraceutical, especially cancer prevention (Awika and Rooney 2004). Generally, tannins which are polymers, are characterized on the basis of their monomeric composition, and their degree of polym-erization, both of which can impact their functionality (Asquith et al 1983). Many analytical methods have been used to study their composition such as reversed phase (RP) HPLC (Putman and Butler 1989; Prior and Gu 2005), normal-phase (NP) HPLC (Gu et al 2002; Awika et al 2003), size-exclusion chromatography (SEC) (Williams et al 1983; Karchesy et al 1988; Kennedy and Taylor 2003), capillary electrophoresis (CE) (Bae et al 1994; Ci-fuentes et al 2001; Bonoli et al 2004), and mass spectrometry (Cheynier and Fulcrand 2003; Kruger et al 2003). It is generally agreed that sorghum tannins are composed of monomeric flavan-3-ols. Thus, recent research has focused on the degree of polymerization of these subunits, with one limitation being how well the largest polymers (i.e., a high degree of polym-erization [DP]) can be resolved. Recently, NP-HPLC has resolved tannins to a DP of >10 and has significantly improved the separa-tion and characterization of tannins from a number of sources, including sorghum (Hammerstone et al 1999; Gu et al 2002). While NP-HPLC was capable of excellent resolution, run times were lengthy (40 min) and NP-HPLC must be conducted in the total absence of water, which complicates switching instruments between types of HPLC such as RP-HPLC. Thus, the objectives of this study were to investigate the use of improved SEC procedures for analyzing sorghum tannins that required no special sample preparation (such as derivatization, tannin purification, etc.) and that would provide information on the M