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United States Department of Agriculture

Agricultural Research Service

Research Project: DEVELOPING BIOCONVERSION PROCESSES FOR HIGH-VALUE CARBOHYDRATE PRODUCTS

Location: Renewable Product Technology Research Unit

2009 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.


3.Progress Report
Using raffinose and gentiobiose as acceptors and sucrose as a donor, novel oligosaccharides were synthesized by the lactic acid bacterial enzyme alternansucrase that have promising in vitro prebiotic activity. It was found that glucosylation of gentiobiose yields products in good yields and without the bitterness associated with gentiobiose. Chemical structures of the products were analyzed. Whereas some followed the same structural patterns as those derived from maltose, others appear to be quite different. In particular, raffinose from cottonseed meal or soy whey yielded some novel and potentially useful compounds. These results will be useful for the systematic production of valuable oligosaccharides for use in food supplements and feed additives.

The structure and production of biofilm polysaccharides produced by the lactic acid bacterium Leuconostoc mesenteroides were studied. It was found that the microbes make an insoluble polysaccharide known as mutan. Results have potential impact for research on fermentation systems for food and biofuels, utilization of carbohydrates in films and fibers, and in applications of enzymes for polysaccharide or oligosaccharide production.

Oligosaccharides of various lengths were produced from wood-product waste streams and partially purified. They were tested for prebiotic potential in collaboration with university scientists. They exhibited promise as prebiotics, which are beneficial for the selective nutrition of health-promoting gut bacteria. The oligosaccharides selectively supported bifidobacterium growth, acidified mixed cultures, and demonstrated prebiotic activity in two animal models. This work may result in low-cost nutritional supplements for animal feeds.

Likewise, oligosaccharides were produced from corn fiber, using fungal enzymes. The oligosaccharides were of the proper molecular 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. With proper modification, it may also replace imported gum arabic in certain applications.


4.Accomplishments
1. ELIMINATION OF BITTER TASTE IN CORN-DERIVED SACCHARIDES. New carbohydrate-derived functional foods are desirable from both an economic and health standpoint. The carbohydrate known as gentiobiose can be made from corn syrup, and has some potential applications in nutrition. However, gentiobiose is bitter, limiting its usefulness in foods. We have found that the bacterial enzyme known as alternansucrase can make a variety of complex carbohydrates that are useful for low-glycemic sweeteners and other nutritional supplements and ingredients by combining sugar with other carbohydrates. The reactions and products we describe show that alternansucrase can modify gentiobiose in a manner that could be useful for making food and feed ingredients. The improved prebiotic profile, lack of bitterness, and the relatively high yields (approaching 90% in unoptimized reactions) suggest promise for the use of these oligosaccharides as food or feed ingredients. This represents a potentially new product for food ingredient manufacturers, new markets for corn and sugar growers, and healthier diets for consumers.

2. NEW PREBIOTICS FROM COTTONSEED MEAL. We developed a method that uses a food-grade bacterial enzyme to modify carbohydrates from cottonseed meal or soybeans and convert them to new carbohydrates with potential applications in food and feed. Raffinose is a carbohydrate that occurs in the seeds of many agricultural crops, including soy and cotton. It is found in soy whey, a co-product of soy protein production, and can also be extracted in good yields from cottonseed meal, the residue remaining after the oil has been pressed out of the seeds. Although raffinose can enhance the growth of beneficial bacteria in the large intestine, it also causes gas production in the intestine when high-raffinose foods such as beans are eaten. This new process modifies raffinose to give novel carbohydrate that show promise as prebiotics. This process could be used in the production of valuable food and feed additives from soy whey or cottonseed meal, thus benefiting consumers, soy, sugar and cotton growers, and livestock producers.

3. POLYMERS FROM BIOFILMS. The food-grade lactic acid bacterium Leuconostoc mesenteroides strain NRRL B-1355 is becoming increasingly important as a source of enzymes, carbohydrates and polymers. This study showed that the bacteria make insoluble polymers that are very similar, if not identical, to those produced by the bacteria that form biofilms on teeth. This will help scientists understand how these bacteria form sticky insoluble masses on surfaces, and may also suggest new technological applications of the insoluble materials, such as in adhesives and fibers.

4. MODIFIED MICROBIAL GUM. New and improved methods are needed to produce value-added bioproducts from agricultural commodities. Scientists in the BBC Research Unit at USDA-ARS-NCAUR, discovered a novel enzymatic method for the production of a modified microbial gum that more closely resembles gum arabic. This work has potential impact for research to develop new uses and value-added bioproducts from agricultural commodities, and brings us closer to a potential replacement for imported gum arabic.

5. IMPROVED MICROBIAL GERMPLASM. Improved microbial germplasm is needed to enhance bioconversions of agricultural materials to high-value bioproducts. In collaboration with university scientists in Thailand, scientists in the BBC Research Unit at USDA-ARS-NCAUR surveyed microbial germplasm from unique environments in Thailand and for the first time used molecular tools to identify classes of organisms producing valuable polysaccharides and enzymes. This work has potential impact for research to develop new uses and value-added bioproducts from agricultural commodities by providing useful information and tools for scientists researching new bioproducts and bioprocesses.

6. LARGE-SCALE PRODUCTION AND STRUCTURAL CHARACTERIZATION OF GALACTOGLUCOMANNAN OLIGOSACCHARIDES (GGMOs). We developed methods for the large-scale production and purification of galactoglucomannan oligosaccharides (GGMOs) from hemicellulose molasses. Sufficient quantities (several grams) of GGMO oligosaccharides of various lengths (dp 4 – 13) were produced by size-exclusion column separation, and have been tested for prebiotic potential in collaboration with university scientists. Prebiotics are beneficial for the selective nutrition of health-promoting gut bacteria. 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 helps finds new markets for co-products from the forest products industry.

7. PREBIOTIC BIOLOGICAL TESTING: GENOME AND METABOLOME OF HUMAN GUT BIFIDOBACTERIA. Probiotic bacteria in the lower gut are beneficial to health. In collaboration with university scientists, the complete genome of the human infant gut bacterium (Bifidobacterium infantis) has been completed and reported (see Accomplishments). Several islands of genes needed for the metabolism of human milk oligosaccharides (HMOs) were identified in this study. Also, using metabolomic-based assays, the metabolism of HMOs has been studied directly. The new understanding of bacterial metabolism indicates the importance of prebiotic HMOs to the diet of babies and suggests ways to new "tailored" oligosaccharides for human health. This provides a useful tool for scientists researching dietary needs and for the development of new, beneficial food ingredients.

8. NEW METHODS FOR CHEMICAL ANALYSIS OF OLIGOSACCHARIDES. High-value dietary carbohydrates are an important component of our diet. To understand carbohydrate function and metabolism we need detailed knowledge of their chemistry. Three new carbohydrate methods have been developed in the previous year. The first, called diffusion magnetic resonance allows sugars to be visualized according to their molecular size, including carbohydrate complexes and sugar-based detergent foams. Second, methods to "tag" carbohydrates with colored or fluorescent groups have been developed based on ARS "locked-ring sugar" technology. These methods may have uses in the field for the detection of carbohydrate-degrading microorganisms or pathogens. Third, improvements have been made to enumerate the hydroxyl group on sugars, resulting in two publications. This work has impact for US researchers and companies developing new uses and value-added sugars from agricultural commodities and byproducts.


6.Technology Transfer

Number of the New/Active MTAs (providing only)2
Number of Invention Disclosures Submitted1
Number of New Patent Applications Filed1
Number of Other Technology Transfer1

Review Publications
Price, N.P., Ray, K.J., Vermillion, K., Kuo, T. 2008. MALDI-TOF Mass Spectrometry of Naturally-Occurring Mixtures of Mono- and Di-rhamnolipids. Carbohydrate Research. 344:204-209.

Cote, G.L. 2009. Acceptor products of alternansucrase with gentiobiose. Carbohydrate Research. 344:187-190.

Leathers, T.D., Nunnally, M.S., Cote, G.L. 2009. Modification of alternan by dextranase. Biotechnology Letters. 31:289-293.

Cote, G.L., Leathers, T.D. 2009. Insoluble glucans from planktonic and biofilm cultures of mutants of Leuconostoc mesenteroides NRRL B-1355. Applied Microbiology and Biotechnology. 82:149-154.

Adeuya, A., Price, N.P. 2009. Electron Impact Ion Fragmentation Pathways of Peracetylated C-glycoside Ketones Derived from Cyclic 1,3-diketones. Rapid Communications in Mass Spectrometry. 23:1-10.

Sela, D.A., Chapman, J., Adeuya, A., Kim, J.H., Chen, F., Whitehead, T.R., Lapidus, A., Rokhsar, D.S., Lebrilla, C.B., German, J.B., Price, N.P., Richardson, P.M., Mills, D.A. 2008. The Genome Sequence of Bifidobacterium longum subsp. infantis Reveals Adaptations for Milk Utilization Within the Infant Microbiome. Proceedings of the National Academy of Sciences. 105(48):18964-18969.

Vermillion, K., Price, N.P. 2009. Stable Isotope-enhanced Two- and Three-Dimensional Diffusion Ordered 13C-NMR Spectroscopy (SIE-DOSY 13C-NMR). Journal of Magnetic Resonance. 198:209-214.

Last Modified: 9/23/2014
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