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  Isomaltooligosaccharides from leuconostoc as neutraceuticalsUSPTO Application #: 20080064657Title: Isomaltooligosaccharides from leuconostoc as neutraceuticals Abstract: Isomaltooligosaccharides (IMOs) produced by Leuconostoc mesenteroides ATCC 13146 fermentation with a sucrose:maltose ratio of 2:1 have been discovered to be effective prebiotics in mixed cultures of microbial populations, including cultures from chicken ceca. Surprisingly in mixed microbial cultures this IMO composition proved as effective as FOS. Thus, these IMOs can be used as effective prebiotics for both birds and mammals. Moreover, the IMOs were discovered to be effective non-competitive inhibitors of α-glucosidase. These IMOs also will be useful, as an α-glucosidase inhibitor, in a therapeutic application for several diseases, including obesity, diabetes mellitus, pre-diabetes, gastritis, gastric ulcer, duodenal ulcer, caries, cancer, viral disease such as hepatitis B and C, HIV, and AIDS. A diet with 5-20% IMOs was also shown to reduce the abdominal fat tissue in mammals. (end of abstract)
Agent: Patent Department Taylor, Porter, Brooks & Phillips, L.l.p - Baton Rouge, LA, US Inventors: Donal F. Day, Chang-Ho Chung USPTO Applicaton #: 20080064657 - Class: 514054000 (USPTO) Related Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), O-glycoside, Polysaccharide The Patent Description & Claims data below is from USPTO Patent Application 20080064657. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] The benefit of the May 20, 2003 filing date of provisional application Ser. No. 60/471,942 is claimed under 35 U.S.C. .sctn.119(e). [0002] This invention pertains to the use of maltosyl-isomaltooligosaccharides as a dietary supplement for birds and mammals, specifically, to promote the growth of beneficial intestinal microbes, inhibit the growth of pathogenic intestinal microbes, and for therapeutic intervention in diseases such as diabetes by inhibiting the activity of .alpha.-glucosidase to slow the rate of glucose release from carbohydrates and thereby reduce the uptake of glucose. [0003] Prebiotics are nondigestible food ingredients that selectively stimulate the growth and/or activity of beneficial microbial strains (probiotics) residing in the host intestine. See R. Barrangou et al., "Functional and comparative genomic analyses of an operon involved in fructooligosaccharide utilization by Lactobacillus acidophilus," Proc. Natl. Acad. Sci. USA, vol. 100, pp. 8957-8962 (2003). It is believed the ability of these probiotics to catabolize oligosaccharides (two to ten monosaccharide units linked with glycosidic bonds) is a key factor in bestowing beneficial health effects. Certain oligosaccharides are used as prebiotics. They are resistant to metabolism and adsorption in the small intestine and ultimately positively influence the composition of microflora in the large intestine. Oligosaccharides are also widely used in foods such as soft drinks, cookies, cereals, candies, and dairy products. Other applications for oligosaccharides such as an anti-cariogenic agent or a low sweetness humectant have been explored. See S. K. Yoo, "The production of glucooligosaccharides by Leuconostoc mesenteroides ATCC 13146 and Lipomyces starkeyi ATCC 74054, Ph.D. Dissertation, Louisiana State University (1997). [0004] Oligosaccharides used as prebiotics are currently produced either by extraction from plant sources, acid or enzymatic hydrolysis of polysaccharides or enzymatic synthesis by transglycosylation reactions. See P. Monsan et al., "Oligosaccharide feed additives," In: R. J. Wallace and A. Chesson (eds) Biotechnology in animal feeds and animal feeding, pp. 233-245, VCH Velagsgesellshaft mbH, Weinheim, Germany (1995). [0005] Types of Oligosaccharides [0006] Types of oligosaccharides include fructooligosaccharides (FOS), glucooligosaccharides (GOS), and .alpha.-galactooligosaccharides. The differences in structures are illustrated in FIG. 1. Fructooligosaccharides (FOS) have attracted serious commercial interest as prebiotics. They are composed of a D-glucopyranose unit at the non-reducing end (G) linked via an .alpha.-1,2 linkage to two or more .beta.-2,1-linked fructosyl units (F). This group includes 1-kestose (GF2), nystose (GF3), and IF-fructofuranosyl nystose (GF4). Many of the oligosaccharides marketed commercially are FOS, e.g., Raftilose and Nutraflora in the United States. [0007] .alpha.-Galactooligosaccharides, which are .alpha.-galactosyl derivatives of sucrose, are present in many legume seeds. Mono-, di-, and tri-.alpha.-galactosylsucrose, known respectively as raffinose, stachyose, and verbascose, are produced by extraction from plants, particularly soybeans. These oligosaccharides are known to be, in part, responsible for the flatulence and diarrhea that follows consumption of beans, because of the absence of an .alpha.-galactosidase in the gastrointestinal tracts of humans and animals. [0008] Glucooligosaccharides (GOS) is a generic term for poly-glucose oligomers. GOS may contain a number of different linkages and are generally obtained from starch hydrolysates (maltose and maltodextrins) through the action of the .alpha.-transglucosidase (EC 2.4.1.24) from Aspergillus sp. Glucooligosaccharides can also be produced by restricting polymer size during the fermentation process. A subcategory of GOS is the .alpha.-isomaltooligosaccharides (IMO) which contain .alpha.-1,6 bonds in their main chain See H. J. Koepsell et al., "Enzymatic synthesis of dextran. acceptor specificity and chain initiation," J. Biol. Chem., vol. 200, pp. 793-801 (1952). Dextransucrase (EC 2.4.1.5), an enzyme produced mainly by species of Leuconostoc and Streptococcus, catalyzes the synthesis of high molecular weight glucans (dextrans). [0009] Oligosaccharides as Prebiotics [0010] Ingested oligosaccharides (prebiotics) are capable of reaching the colon without being digested. It has been proposed that fructooligosaccharides are preferentially utilized by Lactobacilli and Bifidobacterial species which are considered beneficial species of the human intestinal tract. See H. Kaplan et al., "Fermentation of fructooligosaccharides by lactic acid bacteria and Bifidobacteria," Appl. Environ. Microbiol., vol. 66, pp. 2682-84 (2000). Substituting fructooligosaccharides as a carbon source would preferentially increase the concentration of Lactobacillus and Bifidobacteria species with a concomitant rise in the intestinal production of lactic acid and short-chain fatty acids (SCFA). Both these products would have the net effect of lowering the pH in the large intestine. This appears to be one mode by which beneficial species can out-complete and indeed help prevent the establishment of undesirable pathogenic organisms such as Salmonella. See B. J. Juven et al., "Antagonistic effects of Lactobacilli and Pediococci to control intestinal colonization by human enteropathogens in live poultry," J. Appl. Bacteriol., vol. 70, pp. 95-103 (1991). The fructooligosaccharides may also interact with carbohydrate receptors present on the surface of either microbial or epithelial cells, affecting cell adhesion and immunomodulation. See P. J. Naughton et al., "Effects of nondigestible oligosaccharides on Salmonella enterica Serovar Typhimurium and nonpathogenic Escherichia coli in the pig small intestine in vitro," Appl. Environ. Microbiol., vol. 67, pp. 3391-95 (2001). [0011] Fructooligosaccharides, galactooligosaccharides, and soybean oligosaccharides were found not to be digested by enzymes secreted by small intestine, but to be fermented by certain microorganisms found in human and livestock intestines, especially by the Bifidobacterium sp. See. H. Tomomatsu, "Health effects of oligosaccharides," Food Technol., vol. 48, pp. 61-65 (1994). There are numerous reports regarding the stimulating effects of fructooligosaccharides on the growth of probiotic strains. See P. Monsan et al., 1995; and M. Gmeiner et al., "Influence of a symbiotic mixture consisting of Lactobacillus acidophilus 72-4 and a fructooligosaccharide preparation on the microbial ecology sustained in a simulation of the human intestinal microbial ecosystem (SHIME reactor)," Appl. Microbiol. Biot., vol. 53, pp. 219-223 (2000). Dietary FOS have been reported to be effective in reducing the numbers of the harmful bacteria, E. coli, in the intestine of piglets, but did not reduce numbers of Salmonella. See P. J. Naughton et al., "Effects of nondigestible oligosaccharides on Salmonella enterica Serovar Typhimurium and nonpathogenic Escherichia coli in the pig small intestine in vitro," Appl. Environ. Microbiol., vol. 67, pp. 3391-95 (2001). However, in the same study, commercially available glucooligosaccharides (GOS), another oligosaccharide, showed no effect on either genus of bacteria. [0012] In studies of in vitro fermentation characteristics using human fecal material, small intestinal digestibility, and effects on fecal microbial populations in dogs, GOS (containing .alpha.-1,2, .alpha.-1,4 and .alpha.-1,6 linkages) and FOS produced short chain fatty acids in human fecal material more rapidly than other substrates, such as gum arabic, guar gum and guar hydrolysate. GOS also appeared to be indigestible in the small intestine, while supplying a carbon source for bacterial fermentations in the large intestine of cannulated dogs. See E. A. Flickinger et al., "Glucose-based oligosaccharides exhibit different in vitro fermentation patterns and affect in vivo apparent nutrient digestibility and microbial populations in dogs," J. Nutr., vol. 130, pp. 1267-1273 (2000). When the viable count of Bifidobacterium infantis and B. longum, and changes in pH due to various carbohydrate-supplemented soymilks were monitored, B. longum showed a significantly (P<0.05) higher count on a crude isomaltooligosaccharide (75%) supplemented soymilk than in the control (soymilk without the added supplement) at the end of fermentation. See C-C. Chou et al., "Growth of Bifidobacteria in soymilk and their survival in the fermented soymilk drink during storage," Int. J. Food Microbiol., vol. 56, pp. 113-121 (2000). Another study showed that GOS was only 20% digested by germfree rats. See P. Valette et al., "Bioavailability of new synthesized glucooligosaccharides in the intestinal tract of gnotobiotic rats," J. Sci. Food Agric., vol. 62, pp. 121-127 (1993). Dietary isomaltooligosaccharides (13.5g/day for 14 days) were reported to increase fecal Bifidobacteria levels (P<0.05) in healthy adult males. See T. Kohmoto et al., "Effect of isomalto-oligosaccharides on human fecal flora Bifidobacteria," Microflora, vol. 7, pp. 61 69 (1988). Another study investigated the ability of several human gut bacteria to break the .alpha.-1,2 and .alpha.-1,6 glycosidic linkages in .alpha.-glucooligosaccharides, in vitro, in substrate utilization tests. See Z. Djouzi et al., "Degradation and fermentation of .alpha.-gluco-oligosaccharides by bacterial strains from human colon: in vitro and in vivo studies in gnotobiotic rats," J. Appl. Bact., vol. 79, pp. 117-127 (1995). Branched oligomers were resistant to both gastrointestinal enzymes and utilization by pathogenic microorganisms. They also reported that .alpha.-1,2 glucosidic bonds were more resistant than .alpha.-1,6 linkages in kinetic studies of glucooligosaccharide hydrolysis in pH-regulated fermentations. This study indicated the differences in utilization, and thus effectiveness, of GOS based on types and degree of branching. [0013] Production of Glucooligosaccharides [0014] Glucansucrases have been extensively studied because of their role in the production of dextran and its role in the cariogenic process. Glucansucrases (EC 2.4.5.1), usually extracellular but in some cases cell-associated, are primarily produced by various species of soil bacteria. Those produced by Leuconostoc sp. are called dextransucrase. Those produced by Streptococcus sp. and other lactic bacteria, Lactococci, are called glucosyltransferases. Streptococcal glucansucrases synthesize primarily .alpha.-1,3 rich polysaccharides. Leuconostoc glucansucrases produce .alpha.-1,6 rich polysaccharides. [0015] Glucansucrases catalyze the synthesis of high molecular weight D-glucose polymers from sucrose. In the presence of efficient acceptors, e.g., maltose, they may catalyze the synthesis of low molecular weight oligosaccharides. See F. Paul, "Acceptor reaction of a highly purified dextransucrase with maltose and oligosaccharides: Application to the synthesis of controlled-molecular-weight dextrans," Carbohydr. Res., vol. 149, pp. 433-441 (1986). [0016] Dextransucrases catalyze the synthesis of high molecular weight glucans (dextrans) according to the reaction: [0017] Dextran is a D-glucose polymer composed mainly of .alpha.-1,6 linked backbones in a linear chain and .alpha.-1,2, .alpha.-1,3, and/or .alpha.-1,4 branch linkages. See U.S. Pat. No. 5,229,277. The chemical structure of the dextran is specific to the glucansucrase of the producing strain of microbes (Table 1). See J. F. Robyt, "Dextran," In: Encyclopedia of Polymer Science and Engineering," (H. F. Mark et al., eds.), Vol. 4, pp. 752-767, John Wiley & Sons, New York (1986). The dextransucrase from L. mesenteroides NRRL B-1299 can produce .alpha.-glucooligosaccharides (GOS) containing one or more D-glucopyranosyl branch units linked via .alpha.-1,2 glycosidic bonds if maltose supplied as an acceptor. See F. Paul et al., "Method for the production of .alpha.-1,2 oligodextrans using Leuconostoc mesenteroides B-1299," U.S. Pat. No. 5,141,858. However, dextransucrase from L. mesenteroides B-742 (ATCC 13146)produces two dextrans; one with .alpha.-1,6 and .alpha.-1,3 linkages, and another with .alpha.-1,6 and .alpha.-1,4 linkages. (Table 1) Usually a high molecular weight dextran (10.sup.6-10.sup.7 Da) is produced. This is the case, for example, of the enzyme from L. mesenteroides NRRL-512F, which is used to produce dextran polymers of industrial interest including chromatography supports, photographic emulsions, iron carriers, and blood plasma substitutes (Robyt, 1986). TABLE-US-00001 TABLE 1 Linkages in different dextrans as obtained by methylation analysis Linkages % Dextran.sup.a Solubility .alpha.-1.fwdarw.6 .alpha.-1.fwdarw.3 .alpha.-1.fwdarw.3 Br.sup.b .alpha.-1.fwdarw.2 Br.sup.b .alpha.-1.fwdarw.4 Br.sup.b L. m. B-512F Soluble 95 5 L. m. B-742 Soluble 50 50 L. m. B-742 Less soluble 87 13 L. m. B-1299 Soluble 65 35 L. m. B-1299 Less soluble 66 1 27 L. m. B-1355 Soluble 54 35 11 L. m. B-1355 Less soluble 95 5 S. m. 6715 Soluble 64 36 S. m. 6715 Insoluble 4 94 2 .sup.aL. m., Leuconostoc mesenteroides; S. m., Streptococcus mutans. .sup.bBr, Branch linkage. Adapted from Robyt, 1986. [0018] The synthesis of oligosaccharides using dextransucrase can be induced at the expense of dextran synthesis. In the presence of sucrose, the introduction into the reaction medium of molecules, like maltose, isomaltose, and O-.alpha.-methylglucoside, shifts the pathway of glucan synthesis towards the production of oligosaccharides. See Paul, 1986; and M. Remaud et al., "Characterization of .alpha.-1,3 branched oligosaccharides synthesized by acceptor reaction with the extracellular glucosyltransferases from L. mesenteriodes NRRL B-742," J. Carbohyd. Chem., vol. 11, pp. 359-378 (1992); Koepsell et al., 1952; and J. Robyt et al., "Relative, quantitative effects of acceptors in the reaction of Leuconostoc mesenteroides B-512F dextransucrase," Carbohydr. Res., vol. 121, pp. 279-286 (1983). The molecular weight and polydiversity of this oligosaccharide product are dependent upon the sucrose to acceptor ratio, the strain of bacteria, and on the characteristics of the intermediate oligosaccharides in the reaction. The ratio of sucrose to maltose affects the composition and yield of the oligosaccharides produced by the acceptor reaction. When the maltose to sucrose ratio was 2, a partially purified dextransucrase from L. mesenteroides NRRL B-512F produced 85% of ththeheoretical yield of polysaccharide as oligosaccharides, with an average degree of polymerization (DP) of 4. See U.S. Pat. No. 5,141,858; and Paul, 1986. [0019] Leuconostoc mesteroides B-742 ATCC 13146 [0020] Leuconostoc mesenteroides ATCC 13146 was isolated from spoiled canned-tomatoes. (Robyt, 1986) The dextran produced by this (B-742) Leuconostoc strain is highly branched, containing as much as 50% .alpha.-1,3 linkages. Leuconostoc mesenteroides ATCC 13146 actually produces two exocellular .alpha.-D-glucans, a fraction L, which is precipitated at an ethanol concentration of 39%, and a fraction S, which is precipitated at a concentration of 45% ethanol (Robyt, 1986). Fraction L consists of an .alpha.-1,6 backbone with .alpha.-1,4 branch-points, and fraction S consists of an .alpha.-1,6 backbone with .alpha.-1,3 branch-points. The L fraction from Leuconostoc mesenteroides ATCC 13146 contains 87% .alpha.-1,6 linkages and 13% .alpha.-1,4 linkages. The percentage of .alpha.-1,3 branch-points in the fraction S glucan is variable, dependant on the conditions under which it is synthesized from sucrose. The .alpha.-1,3 linkages of the S fraction of L. mesenteroides ATCC 13146 are all branched linkages. This dextran demonstrates extreme resistance to endodextranase. This property seems related to its structure that has the highest possible degree of branching and exhibits a comb-like structure with main chains of consecutive .alpha.-1,6 linked glucose residues to which single .alpha.-1,3 linked glucosyl residues are attached. Any change in reaction conditions that affects the rate of acceptor reaction relative to chain elongation also affects the degree of branching in ATCC 13146 fraction S dextran. [0021] The acceptor reaction of L. mesenteroides ATCC 13146 was investigated and found that branch formation in this strain, when maltose was the acceptor, was dependant upon reaction conditions. L. mesenteroides ATCC 13146 in the presence of maltose produced 90% of the theoretical yield of polymer as isomaltooligosaccharides, under optimum conditions for sucrose fermentation. See Yoo, 1997; S. K. Yoo et al., "Co-production of dextran and mannitol by Leuconostoc mesenteroides, J. Microbiol. Biotechnol., vol. 11, pp. 880-883 (2001); and S. K. Yoo et al., "Highly branched glucooligosaccharide and mannitol production by mixed culture fermentation of Leuconostoc mesenteroides and Lipomyces starkeyi, J. Microbiol. Biotechnol., vol. 11, pp. 700-703 (2001). The fermentation was essentially complete in 24 hours, with oligosaccharide production being linked to growth. The production rate was about 0.9 g/L hr. The maltose to sucrose ratio was able not only to alter the yield of oligosaccharide but also to change the relative proportion of different size oligosaccharides produced by the fermentation. The highest yields of isomaltooligosaccharides were obtained when the ratio of sucrose to maltose in the fermentation was two. This is the same ratio reported for optimum oligosaccharide production in vitro by the dextransucrase of L. mesenteroides B-512F (See Paul et al., 1986). Several Leuconostoc strains were tested to check for oligosaccharide size profiles produced in response to maltose, because individual Leuconostoc species synthesize different dextransucrases in response to various acceptors. The isomaltooligosaccharides produced by L. mesenteroides ATCC 13146 were mostly DP (degree of polymerization) 3-5 by chemical analysis. Isomaltooligosaccharides prepared by alcohol-precipitated, cell-free culture broths had greater amounts of higher branched isomaltooligosaccharides up to DP 7, than commercial preparations and had no glucose and less maltose (Yoo, 1997). These isomaltooligosaccharides were found to affect isolated, single microbial cultures by suppressing growth of Salmonella enteritidis, Salmonella typhimurium, Staphylococcus aureus, Staphylococcus epidermidis, and Clostridium perfringenes, and supporting growth of two Bifidobacterium species. (Yoo, 1997). [0022] D-mannitol is a sugar-alcohol derived from mannose or fructose by dehydrogenation. In sucrose fermentations, mannitol is produced as an end product, as fructose can be used as an electron acceptor, but the levels of mannitol produced vary with the strain. See Yoo, 1997; and C. Y. Kim et al., "Production of mannitol using Leuconostoc mesenteroides NRRL B-1149," Biotechnol. Bioprocess Eng., vol. 7, pp. 234-236 (2002). Mannitol was found as one of the major end products in this Leuconostoc fermentation. It is necessary to separate the mannitol from the oligosaccharides if they are to be used as prebiotics, because mannitol can act as an additional carbon source. Its presence would hinder the ability to ascribe the essential and unique role of oligosaccharides on intestinal microflora. (Yoo, 1997) [0023] Oligosaccharides as Antibiotic Alternatives in Animals [0024] Antibiotic resistance among known pathogens such as Salmonella and Escherichia coli is expanding due to the wide use of antibiotics in areas ranging from medicine to animal feed. Although only specific antibiotics are used in feed preparations and are exclusive to non-human use, their chemical similarity to antibiotics prescribed for humans has raised concern that resistance will spread more rapidly, since resistant mechanisms generally affect an entire class of antibiotics (ex: penicillinases to inhibit the Penicillins). This, coupled with public pressure to remove antibiotics from animal feeds, has created a need for safe alternatives that can effectively control the growth of bacterial pathogens in the human food supply. Selected fructooligosaccharides and glucooligosaccharides have shown potential as alternatives to antibiotics. See P. Monsan et al., (1995); J. V. Loo et al., "Functional food properties of non-digestible oligosaccharides: a consensus report from the ENDO project (DGXII AIRII-CT94-1095)," Brit. J. Nutr., vol. 81, pp: 121-132 (1999); and P. Valette et al., "Bioavailability of new synthesized glucooligosaccharides in the intestinal tract of gnotobiotic rats," J. Sci. Food Agric., vol. 62, pp. 121-127 (1993). However, not all oligosaccharides have been found effective. Although FOS is generally considered to be effective in regulating and reducing pathogenic microbial populations, conflicting reports exist about the effectiveness of GOS. See Naughton et al., 2001; and Yoo, 1997. These conflicting reports may be due to variability in the composition of the GOS (the degree of branching, the size, the amount of mannitol, or the acceptor used in fermentation production), or whether the GOS was tested on single microbial cultures, mixed microbial cultures, or in vivo. There may also be differences depending on the animal tested. Continue reading... Full patent description for Isomaltooligosaccharides from leuconostoc as neutraceuticals Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Isomaltooligosaccharides from leuconostoc as neutraceuticals patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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