2008 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 dried distillers grains (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.
In collaboration with Slovak Academy of Science, we discovered that the leaves and roots of germinating corn contain inhibitors to enzymes used to digest corn fiber. This work has potential impact for research to developing new uses and value-added bioproducts from corn.
The biofilm polysaccharides produced by the lactic acid bacterium Leuconostoc mesenteroides were studied. It was found that the polysaccharides produced in biofilms differ from those produced in planktonic cultures. Results have potential impact for research to control biofilm contamination in agricultural processing industries and for utilization in applications of enzymes for polysaccharide production.
Using fructose, raffinose, and gentiobiose as acceptors and sucrose as a donor, oligosaccharides were synthesized by alternansucrase. They proved to be promising prebiotics when tested in vitro. Chemical structures of the products were analyzed. Whereas some followed the same structural patterns as those derived from maltose, others appear to be different. Some were prepared in large amounts and submitted to Agricultural Research Service (ARS) partner labs for testing in animal feed. These results will be useful for production of valuable oligosaccharides for use in food supplements and feed additives.
Under a Trust Fund agreement with a private-sector partner, galactoglucomannan oligosaccharides (GGMOs) with potential prebiotic activity have been purified from a wood molasses produced as an industrial waste material. The GGMOs have been purified and structurally characterized, and component oligosaccharides separated from coproduct arabinoxylans. The GGMOs were fractionated and tested for prebiotic activity and improved bifidobacterial growth at the University of Illinois.
In collaboration with the University California, Davis, we have sequenced the genome of Bifidobacterium infantis, a human infant gut bacterium. We have demonstrated that B. infantis uses the Bifido shunt to metabolize complex sugars to acetate. This finding on the metabolism of oligosaccharides by the infant strain of Bifidobacterium may provide insights for the design of potential new prebiotics.
New mass spectrometric techniques were developed for the analysis of most complex carbohydrate products. Methods were developed to evaluate the number of hydroxyl groups on polyols. "Locked ring" sugar C-glycosides have been developed to immobilize prebiotic oligosaccharide products for binding/separation of carbohydrate-binding proteins.
In collaboration with the Southern Regional Research Laboratory, polysaccharides were isolated from hard-to-boil massecuites obtained from Louisiana sugar mills. Results show that there are multiple polysaccharides, some arising from sugar cane itself and some from icrobial contaminants. This study may help sugar farmers and refiners avoid these costly problems in the future.
Our work in the area of nutritive oligosaccharides was recognized in fiscal year 08 with an ARS Technology Transfer Award for Superior Effort and with a Federal Laboratory Consortium Technology Transfer Award. This progress directly addresses NP 306, Component 2.
BIOFILM FORMATION BY POLYSACCHARIDE MUTANTS. Fundamental information is needed on how biofilms form. We found that polysaccharide mutations greatly affected the appearance of the biofilms. Results have potential impact for research to control biofilm contamination in agricultural processing industries and for utilization in applications of enzymes for polysaccharide production. This accomplishment directly addresses NP 306, Component 2, Problem Area 2b.
ALTERNANSUCRASE OLIGOSACCHARIDES. Practical new methods are needed to produce sugar-based products from low-value agricultural commodities such as corn starch and sugar. Using fructose, raffinose, and gentiobiose as acceptors and sucrose as a donor, novel oligosaccharides were synthesized by the lactic acid bacterial enzyme alternansucrase. They proved to be promising prebiotics when tested in vitro. Chemical structures of the products were analyzed. These results will be useful for the systematic production of valuable oligosaccharides for use in food supplements and feed additives. This accomplishment directly addresses NP 306, Component 2, Problem Area 2b.
PURIFICATION AND PREBIOTIC TESTING OF TEMULOSE "BROWN SUGAR" GALACTOGLUCOMANNAN OLIGOSACCHARIDES. Practical new methods are needed to produce value-added products from low-value agricultural residues. Under a Trust Fund Agreement with Temple-Inland Inc., Diboll, TX, galactoglucomannan oligosaccharides (GGMOs) with potential prebiotic activity have been purified from Temulose, a wood molasses produced as an industrial waste material on a multiple ton scale daily. This work has potential impact for production of valuable food supplements and feed additives. This accomplishment directly addresses NP 306, Component 2, Problem Area 2b.
GENOMICS AND SUGAR METABOLISM OF PREBIOTIC HUMAN GUT BIFIDOBACTERIA. Fundamental information is needed to understand how probiotic Bifidobacterium are beneficial to improved resistance to intestinal disorders. In collaboration with the University of California, Davis, we have sequenced the complete genome of Bifidobacterium infantis and demonstrated that this genome encodes all genes required to metabolize human milk oligosaccharides. This work has potential impact for research to design new prebiotics. This accomplishment directly addresses NP 306, Component 2, Problem Area 2b.
METHODS FOR SEPARATION AND ANALYSIS OF OLIGOSACCHARIDES. Predicting how carbohydrates are degraded requires detailed knowledge of their chemical composition. New mass spectrometric techniques were developed for the analysis of most complex carbohydrate products. Methods were developed to evaluate the number of hydroxyl groups on polyols. "Locked ring" sugar C-glycosides have been developed to immobilize of prebiotic oligosaccharide products onto hydrazine-functionalized beads, for binding/separation of carbohydrate-binding proteins. These results will be useful for scientists who analyze complex carbohydrates for food and non-food applications. This accomplishment directly addresses NP 306, Component 2, Problem Area 2b.
ENZYME INHIBITORS IN GERMINATING CORN. Practical new methods are needed to produce sugar-based products from low-value agricultural residues such as corn fiber. In collaboration with scientists at the Slovak Academy of Science, we discovered for the first time that the leaves and roots of germinating corn contain inhibitors to enzymes used to digest corn fiber. This work has potential impact for research to developing new uses and value-added bioproducts from corn. This accomplishment directly addresses NP 306, Component 2, Problem Area 2b.
5.Significant Activities that Support Special Target Populations
|Number of Non-Peer Reviewed Presentations and Proceedings||5|
|Number of Newspaper Articles and Other Presentations for Non-Science Audiences||2|
|Number of Other Technology Transfer||1|
Holt, S.M., Teresi, J., Cote, G.L. 2008. Influence of alternansucrase-derived oligosaccharides and other carbohydrates on alpha-gelactosidase and alpha-glucosidase activity in Bifidobacterium adolescentis. Letters in Applied Microbiology. 46(1):73-79.
Cote, G.L., Sheng, S., Dunlap, C.A. 2008. Alternansucrase acceptor products. Biocatalysis and Biotransformation. 26(1-2):161-168.
Leathers, T.D., Cote, G.L. 2008. Biofilm formation by exopolysaccharide mutants of Leuconostoc mesenteroides strain NRRL B-1355. Applied Microbiology and Biotechnology. 78(6):1025-1031.
Adeuya, A., Price, N.P. 2007. Rapid characterization of polyalcohols by silylation and MALDI-TOF mass spectrometry. Rapid Communications in Mass Spectrometry. 21:3977-3981.
Whitehead, T.R., Price, N.P., Drake, H.L., Cotta, M.A. 2008. Catabolic pathway for the production of skatole and indoleacetic acid by the acetogen Clostridium drakei, Clostridium scatologenes, and swine manure. Applied and Environmental Microbiology. 74(6):1950-1953.
Price, N.P., Tsvetanova, B. 2007. Biosynthesis of the Tunicamycins: a review. Journal of Antibiotics. 60(8):485-491.
Biely, P., Leathers, T.D., Cziszarova, M., Vrsanska, M., Cotta, M.A. 2008. Endo-beta-1,4-xylanase inhibitors in leaves and roots of germinated maize. Journal of Cereal Science. 48(1):27-32.
Price, N.P. 2008. Permethylation linkage analysis techniques for residual carbohydrates. Applied Biochemistry and Biotechnology. 148(1-3):271-276.