2010 Annual Report
1a.Objectives (from AD-416)
To develop biocatalytic methods for the conversion of crop derived carbohydrates to high value polysaccharides or oligosaccharides. The project will be composed of two major objectives. .
1)Develop biocatalytic methods for the conversion of starch, corn coproducts, beet sugar, or cane sugar to value-added oligosaccharides. .
2)Develop green chemistry ionic liquid-based methods for the biocatalytic production of value-added oligosaccharides. Their common feature is the use of agriculturally derived carbohydrates for the production of high-value products which utilize some of the structural features of the original carbohydrates.
1b.Approach (from AD-416)
Glycansucrases. Our research in this area will focus on the use of alternansucrase to synthesize oligosaccharides via transfer of glucosyl units from sucrose to mono- or oligo-saccharide acceptors. As alternansucrase is better at catalyzing acceptor reactions than commercial dextransucrase, yielding a better variety of mixed-linkage products, we will first focus on this enzyme. The newly synthesized oligosaccharides are expected to support the growth of specific beneficial microbes in the gastrointestinal tract. We will develop these and other products and determine their structures. The role of oligomer structure and size (degree of polymerization) in fermentability and prebiotic activity will be investigated. This is expected to yield not only new, more strain-specific prebiotics, but will also give rise to a better understanding of the mechanism of prebiotic action.
Corn coproducts. This research will utilize abundant, low-value agricultural biomass, particularly corn fiber arising as a coproduct of corn wet milling for production of starch, sweeteners, and ethanol. This is attractive as a model corn residue because it accumulates in enormous volumes in milling facilities and does not need to be collected and transported from fields as do corncobs and stover. Research will also utilize DDGS, an abundant low-value coproduct of dry grind fuel ethanol production. Both corn fiber and DDGS are rich in arabinoxylan, a complex polysaccharide with a backbone of B- (1,4) linked xylose and various side chains and other modifications. Combinations of specific enzymes will be used to cut corn fiber xylan at these linkages to produce a collection of novel oligosaccharides. Transglycosylations in ionic liquids. The approach will be to use commercial enzymes in non-aqueous ionic liquids to produce new prebiotic oligosaccharides and cyclic oligosaccharides that are unattainable in water-based systems. Various ionic liquids, enzymes, and conditions will be tested in order to optimize the non-aqueous transglycosylations. Oligosaccharides produced from this part of the project plan will be tested for prebiotic activity in vitro and in vivo.
• Patented technology was licensed by two companies for production of commercial products, including a novel low glycemic index sweetener widely available in diabetic food products. Technology transfer was honored with the 2007 ARS Tech Transfer Award and the 2008 Federal Laboratory Consortium Award for Technology Transfer.
• Oligosaccharides were produced from corn fiber using fungal enzymes. The oligosaccharides were of the proper size and distribution to suggest promise as improved prebiotics for human foods and animal feeds. This work shows that novel prebiotics can be made from corn fiber using biocatalytic processes.
• An improved enzymatic process for producing modified gums was developed. The modified gum has viscosity and solubility properties that may render it valuable for diverse applications.
• The effect of oil extraction method on the enzymatic digestion of defatted corn germ was determined. Unlike corn fiber, defatted germ was efficiently digested without a pretreatment, and regardless of oil extraction method.
• Production of the bioactive beta-glucan schizophyllan was demonstrated from the abundant wet-milling coproduct, corn fiber. Schizophyllan is clinically used as a cancer immunotherapeutic agent.
• In collaboration with scientists at Chulalongkorn University, Bangkok, Thailand, microbial germplasm from unique environments in Thailand was surveyed for production of valuable polysaccharides and enzymes.
• Oligosaccharides were produced from sugar and various other agricultural carbohydrates via acceptor reactions of glucansucrases. These enzymes from food-grade bacteria were found to make a wide variety of oligosaccharides with potential as prebiotics and low-glycemic sweeteners. One was commercialized as a low-glycemic sweetener (see 1st item), and others have been tested or are undergoing testing as prebiotics.
• In collaboration with scientists in the Plant Polymer Research Unit of the National Center for Agricultural Utilization Research, microbial gums were developed which can prevent corrosion on steel. These gums are produced from sugar, and are biodegradable.
ANTICORROSIVE POLYSACCHARIDES. Renewable and ecologically friendly agents are highly sought-after for the protection of metals from corrosion. In a collaboration between Plant Polymer Research Unit (PPL) scientists and the Renewable Product Technology Research Unit (RPT) scientists at the National Center for Agricultural Utilization Research (NCAUR), in Peoria, Illinois, microbial gums were developed which can prevent corrosion on steel. These gums are produced from sugar, and are biodegradable. This work represents a significant step toward replacing petrochemicals with bio-based materials.
PREBIOTIC GALACTOGLUCOMANNAN OLIGOSACCHARIDES (GGMOs). In previous years, Renewable Product Technology Research Unit (RPT) scientists at the National Center for Agricultural Utilization Research (NCAUR) in Peoria, Illinois reported the large-scale production and purification of galactoglucomannan oligosaccharides (GGMOs) from a molasses by-product of fiber board manufacture. Various lengths of GGMOs (dp 4 – 13) were produced and have been tested for prebiotic activity by our collaborators at the University of Illinois. The GGMOs selectively support bifidobacterium growth, acidify mixed cultures, and also have prebiotic activity in vitro in two animal models. This provides a new source of beneficial dietary ingredients for livestock and poultry producers, and for the pet food market.
NOVEL MICROBIAL GERMPLASM. Novel microbial germplasm is needed to enhance bioconversions of agricultural materials to high-value bioproducts. In collaboration with a visiting scientist from Thailand, under TFCA 58-3620-0-082F, Renewable Product Technology Research Unit (RPT) scientists at the National Center for Agricultural Utilization Research (NCAUR), Peoria, Illinois, surveyed microbial germplasm from unique environments in Thailand and used molecular tools to identify classes of organisms producing valuable biopolymers. This work has potential impact for research to develop new uses and value-added bioproducts from agricultural commodities.
PREBIOTIC OLIGOSACCHARIDES FROM RAFFINOSE. Raffinose is a carbohydrate that can be extracted from cottonseed meal or soy whey, both low-value agricultural coproducts. Anything that can enhance the value of these coproducts will enhance the value of soybeans or cotton. Raffinose can be used in food and feed to increase the growth of beneficial bacteria in the lower GI tract, but has the undesirable traits of low solubility and it can cause unwanted gas and bloating. Renewable Product Technology Research Unit (RPT) scientists at the National Center for Agricultural Utilization Research (NCAUR) in Peoria, Illinois, developed a way to use food-grade bacteria and enzymes to increase the solubility and potentially decrease gas production from raffinose. These new products are currently being evaluated by our collaborators in the UK and Spain. If successful, they represent a potential new market for cottonseed meal or soy whey, and a new prebiotic for animal feed and human food.
NEW METHODS FOR CHEMICAL SUGAR ANALYSIS. Sugars are an important component of our diet. To understand carbohydrate structure and metabolism, requires new sugar chemistry methods. New carbohydrate methods have been developed by the Renewable Product Technology Research Unit (RPT) scientists at the National Center for Agricultural Utilization Research (NCAUR), in Peoria, Illinois based on ARS patented "locked-ring sugar" technology. These methods are used to label carbohydrates with colored or fluorescent tags. Also new ways of immobilizing sugars hydrogels and onto inert beads have been developed. This technology has potential for the detection of carbohydrate-degrading microorganisms, pathogens, or enzymes. This work has impact for U.S. researchers and companies developing new uses and value-added sugars from agricultural commodities and byproducts.
MODIFIED MICROBIAL GUM. New and improved methods are needed to produce value-added bioproducts from agricultural commodities. Renewable Product Technology Research Unit (RPT) scientists at the National Center for Agricultural Utilization Research (NCAUR), Peoria, Illinois, optimized process conditions for the enzymatic modification of a microbial gum such that it more closely resembles gum arabic. This work has potential impact for research to develop new uses and value-added bioproducts from agricultural commodities.
Manitchotpisit, P., Leathers, T.D., Peterson, S.W., Kurtzman, C.P., Li, X., Eveleigh, D.E., Lotrakul, P., Prasongsuk, S., Dunlap, C.A., Vermillion, K., Punnapayak, H. 2009. Multilocus Phylogenetic Analyses, Pullulan Production and Xylanase Activity of Tropical Isolates of Aureobasidium pullulans. Mycological Research. 113:1107-1120.
Cote, G.L., Dunlap, C.A., Vermillion, K. 2009. Glucosylation of raffinose via alternansucrase acceptor reactions. Carbohydrate Research. 344:1951-1959.
Striegel, A.M., Isenberg, S.L., Cote, G.L. 2009. An SEC/MALS study of alternan degradation during size-exclusion chromatographic analysis. Analytical and Bioanalytical Chemistry. 394:1887-1893.
Leathers, T.D., Nunnally, M.S., Cote, G.L. 2010. Optimization of process conditions for enzymatic modification of alternan using dextranase from Chaetomium erraticum. Carbohydrate Polymers. 81(3):732-736.
Liu, S., Bischoff, K.M., Hughes, S.R., Leathers, T.D., Price, N.P., Qureshi, N., Rich, J.O. 2009. Conversion of biomass hydrolysates and other substrates to ethanol and other chemicals by Lactobacillus buchneri. Letters of Applied Microbiology. 48(3):337-342.
Rooney, A.P., Price, N.P., Erhardt, C., Swezey, J.L., Bannan, J.D. 2009. Phylogeny and Molecular Taxonomy of the Bacillus subtilis species Complex and the Description of Bacillus subtilis subsp. inaquosorum subsp. nov. International Journal of Systematic and Evolutionary Microbiology. 59(Pt 10):2429-2436.
Rooney, A.P., Price, N.P., Ray, K.J., Kuo, T. 2009. Isolation and Characterization of Rhamnolipid-Producing Bacterial Strains from a Biodiesel Facility. FEMS Microbiological Letters. 295(1):82-87.
Kitova, A.E., Leathers, T.D., Reshetilov, A.N. 2009. Screening Substrate Properties of Microorganisms for Biosensor Detection of Oligosaccharides. Water:Chemistry and Ecology. 5:24-31 (In Russian).
Kitova, A., Reshetilov, A., Ponamoreva, O., Leathers, T.D. 2010. Microbial biosensors for selective detection of disaccharides. The Internet Journal of Microbiology. 8(2).
Labeda, D.P., Price, N.P., Tan, G.A., Goodfellow, M., Klenk, H. 2010. Emendation of the Genus Actinokineospora Hasegawa 1988 and Transfer of Amycolatopsis fastidiosa Henssen et al. 1987 as Actinokineospora fastidiosa comb. nov. International Journal of Systematic and Evolutionary Microbiology. 60(6):1444-1449.
Labeda, D.P., Price, N.P., Donahue, J.M., Williams, N.M., Sells, S.F. 2009. Streptomyces atriruber sp. nov. and Streptomyces silaceus sp. nov.: New Species of Equine Origin. International Journal of Systematic and Evolutionary Microbiology. 59(11):2899-2903.
Price, N.P., Bowman, M.J., Legall, S., Berhow, M.A., Kendra, D.F., Lerouge, P. 2010. Functionalized C-glycoside ketohydrazones: Carbohydrate derivatization that retains the ring integrity of the terminal reducing sugar. Analytical Chemistry. 82(7):2893-2899.