KR20190117447A - Method for producing agarotriose and prebiotics use thereof - Google Patents

Abstract

The present invention relates to a method for manufacturing seaweed-derived agarotriose derived and uses thereof as prebiotics. More specifically, the present invention identifies properties of agarotriose as prebiotics selectively metabolized by probiotic microorganisms, thereby using the same as an anticancer or anti-inflammatory material in food and medicine fields, and also can obtain a high yield of agarotriose through efficient purification that minimizes loss after enzymatic hydrolysis of red algae-derived polysaccharide without pretreatment.

Description

Method for producing agarotriose derived from seaweed and its use as a prebiotic {Method for producing agarotriose and prebiotics use thereof}

The present invention relates to a method for producing agarotriose derived from seaweed and to its use as a prebiotic.

Prebiotics refer to substances that are selectively fermented by beneficial bacteria in the intestine to improve the intestinal flora and are beneficial to human health. Recently, as studies on the correlation between human disease and intestinal flora are reported, the intestinal flora is recognized as the second human genome, and research in this field is rapidly developing. In particular, research results showing that obesity, diabetes, and immune function are improved according to an increase in the distribution of beneficial flora in the intestine is drawing more attention.

The prebiotic effect of the hydrolyzate of agarose, a major constituent polysaccharide in red algae, has been predicted through animal experiments. It was confirmed that the distribution of bifidobacteria, a beneficial bacteria in the intestine, was increased as a result of oral administration of agarooligosaccharide mixture to the rats inducing obesity through a high fat diet. In addition, it promoted the synthesis of low-molecular fatty acids in the intestine and induced expression of genes related to immune and anti-inflammatory functions. In addition, in the case of a neo-agaro-oligosaccharide mixture produced through two types of endotype beta-agarose enzyme reactions of agarose, it was confirmed that Bifidobacteria and Lactobacillus grow under the carbon source condition of the neo-agaro-oligosaccharide mixture. However, in this experiment, the growth test of Bifidobacteria and Lactobacillus in the absence of the neo-agaro-oligosaccharide mixture, which is the control group, was not conducted, so it is possible to accurately determine whether the growth was caused by the metabolism of neo-agaro-oligosaccharides. There is no problem. In addition, it was confirmed that the distribution of Bifidobacteria and Lactobacillus increased when a neoagarooligosaccharide mixture was administered to a rat model.

As previously described, studies on the prebiotic functionality of oligosaccharides derived from red algae have been conducted using a mixture rather than a purified standard material. Nothing was known. In addition, it is not known how oligosaccharides derived from agarose are metabolized by effective probiotic microorganisms in the gut.

On the other hand, the main polysaccharide constituting red algae is agarose, and agarose is 3,6-anhydro-L-galactose (hereinafter referred to as '3,6-AHG') and D-galactose (hereinafter referred to as'D-Gal'). Is a polymer in which alpha-1,3-bonds and beta-1,4-links are alternately connected. In previous studies, a process for the production of AHG with anti-caries, anti-inflammatory, whitening and moisturizing functions was established. Agarooligosaccharides are obtained through pretreatment of the substrate using acetic acid, which is a weak acid in agarose or agar substrate, or a low concentration of neutral buffer Tris-HCl buffer (pH 7.4). Neoagarobiose was produced through -type β-agarase Ⅱ) enzyme reaction. At this time, there is a disadvantage that agarotriose is produced together as a by-product.In order to decompose it into monosaccharides AHG and galactose, an additional enzyme called agarolytic β-galactosidase (ABG) is required. Was necessary. However, agarotriose in the form of Gal-AHG-Gal, an oligosaccharide, which was considered as a by-product for AHG production in previous studies, is known through various literatures that it has a prebiotic effect and enhances the beneficial bacteria of the body's intestinal function. However, the detailed process technology for obtaining purified pure agarotriose is not known in Korea.

Bindels et al. (2015) Nat Rev Gastroenterol Hepatol 12(5):303-310 Hu et al. (2006) Anaerobe 12(5-6):260-266 Higashimura et al. (2016) Am J Physiol Gastrointest Liver Physiol 310(6):G367-375 Miaomiao Li et. al. PLOS ONE, vol.9(3), e91106(2014.03)

An object of the present invention is to provide a use as a medical or food material by determining the metabolism of agarotriose by effective probiotic microorganisms in the intestine.

Another object of the present invention is to provide a method for producing agarotriose through enzymatic hydrolysis and purification.

In order to achieve the above object, the present invention is one or more substrates selected from the group consisting of agar, agarose, neo-agarohexaose and agarotriose; Bacteroides plebeius strain; And it provides a pharmaceutical composition comprising a strain of Bifidobacterium (Bifidobacterium).

The present invention also provides a method of treating cancer or an inflammatory disease by administering an effective amount of the pharmaceutical composition to a subject.

The present invention also includes at least one substrate selected from the group consisting of agar, agarose, neoagarohexaose and agarotriose; Bacteroides plebeius strain; And it provides a food composition comprising the Bifidobacterium (Bifidobacterium) strain.

The present invention also provides a reaction product obtained after reacting any one of agar, agarose, or neo-agarohexaose with the beta-agarase of SEQ ID NO: 1 or 5, and the neo- Reacting with agarobiose hydrolase; And it provides a method for producing agarotriose comprising the step of purifying agarotriose from the reaction product to a size exclusion column.

The present invention is an effect that can be used as an anti-cancer or anti-inflammatory material in the field of medicine and food by identifying the characteristics of agarotriose as a prebiotic that is selectively metabolized by probiotic microorganisms such as bacteroid and bifidobacterium. There is.

In addition, the present invention has the effect of obtaining a high yield of agarotriose through efficient purification that minimizes loss after enzymatic hydrolysis of polysaccharides derived from red algae without pretreatment.

Figure 1 shows a schematic diagram for producing oligosaccharides, neo-agarobiose, and AHG of various degrees of polymerization through an enzymatic reaction from agarose.
Figure 2 is S. degradans (S. degradans) 2-40 ^T- derived endotype beta-agarase, Aga16B, exotype beta-agarase, Aga50D, and alpha-neo agarobiose hydrolase, SdNABH, purified recombinant protein (A), agar through enzymatic reaction Oligosaccharides of various degrees of polymerization from Roth, Neoagarobiose and 3,6-AHG production results (B), Bacteroides plebeius , endotype beta-agaraase enzyme derived from DSM 17135, BpGH16A (BACPLE_01670) and Neoagaro The results (C) of sugar purification through size-exclusion chromatography of the enzyme reaction product of the bios hydrolase BpGH117 (BACPLE_01671) with agarose are shown.
Figure 3 is an intestinal microorganism Bacteroides plebeius (Bacteroides plebeius) Endotype beta-agarase enzyme derived from DSM 17135 BpGH16A (BACPLE_01670), BpGH50 (BACPLE_01683), and neo-agarobiose hydrolase BpGH117 (BACPLE_01671) respectively. The recombinant enzyme was expressed in E. coli and then purified (A) and the enzyme reaction test result (B).
FIG. 4 is an agar through an enzymatic reaction of BpGH16A (BACPLE_01670), an endotype beta-agarase enzyme derived from Bacteroides plebeius DSM 17135 from agarose substrate, and BpGH117 (BACPLE_01671), a neoagarobiose hydrolase. The results of the production of rotriose and AHG are shown.
Figure 5 illustrates the effect of a carbon source of per each tablet of using a Bioscreen C, and culturing the probiotic microorganism is Bifidobacterium ronggeom Infante tooth (Bifidobacterium longum subsp. Infantis) ATCC 15697 strain ( A: AHG, B: NeoDP2, C: AgaDP3, D: NeoDP4, E: AgaDP5, F: NeoDP6, G: Glucose, H: Galactose, I: 2FL).
6 is Bifidobacterium ronggeom Infante tooth (. Bifidobacterium longum subsp infantis) shows the analysis results of the agar lot Rios fermentation of ATCC 15697 strain profile (A: the result of the AgaDP3, NeoDP2 and galactose as a substrate, B: Cells Density, as a result of using acetate and lactate as substrates).
Figure 7 illustrates a Bifidobacterium ronggeom Infante tooth (Bifidobacterium longum subsp. Infantis) using a crude enzyme solution of the ATCC 15697 strain agar lot Rios (A), and neo-agar BIOS (B) as a result of experimental resolution.
8 is Bifidobacterium ronggeom Infante Tees (Bifidobacterium longum subsp infantis.) ATCC on 15,697 isolates four types of beta-cloned galactosidase City Days coding genes (Blon_2016, Blon_2123, Blon_2334 and Blon_2416) and production of recombinant proteins in E. coli And the result of enzymatic reaction after purification (A: gel picture, B: TLC result, C: β-galactosidase specific activity of each enzyme on agarotriose substrate).
9 is Bifidobacterium ronggeom Infante tooth (Bifidobacterium longum subsp. Infantis) other strains of Bifidobacterium ronggeom Infante tooth (Bifidobacterium longum subsp. Infantis) belonging to ATCC 17930, Bifidobacterium ronggeom Infante tooth (Bifidobacterium longum subsp . infantis) shows a baby lot Rios to efficacy results of the ATCC 15702 strain (a: Bifidobacterium ronggeom Infante tooth (Bifidobacterium longum subsp infantis.) ATCC 17930, B: Bifidobacterium ronggeom Infante tooth (Bifidobacterium longum subsp.infantis ) ATCC 15702).
Figure 10 is Bifidobacterium bifidum (B. bifidum ) DSM 20082 and Bifidobacterium Kashiwanohense ( B. kashiwanohense ) shows the experimental results of agarotriose fermentation ability of DSM 21854 strains (A: Bifidobacterium B. bifidum ) DSM 20082, B: Bifidobacterium B. kashiwanohense DSM 21854).
Fig. 11 shows the results of stability test of agarotriose against artificial gastric juice (A: TLC results are shown in a graph, B: HPLC results).
12 illustrates the metabolic pathway of the agarose by the intestinal microflora of foil steroid player bieoseu (Bacteroides plebeius) DSM 17135 and Bifidobacterium ronggeom Infante tooth (Bifidobacterium longum subsp. Infantis) ATCC 15697.
Figure 13 shows a schematic diagram of agarotriose production and separation process from agarose (A: agarotriose production process through acid treatment and enzymatic saccharification, B: AHG, D-Gal, agarotriose production through enzymatic saccharification And purification process).
14 shows the results of agarotriose production through a two-step enzymatic reaction from agarose and agarotriose purification results using a size exclusion column (A: TLC result, B: HPLC result).
Figure 15 is a ratio of neo-agarotetraose and neo-agarobiose produced from agarose through an enzymatic reaction of BpGH16A (BACPLE_01670), an endotype beta-agarase enzyme derived from Bacteroides plebeius DSM 17135, FIG. This is the HPLC quantitative analysis result showing.
16 shows the TLC result of analyzing the difference in the degree of separation for each fraction using a size exclusion column.
FIG. 17 shows the results of measuring the yield and purity of agarotriose using an HPLC KS-802 sugar column.

The present inventors produced agarotriose from agarose, which is a major polysaccharide constituting red algae, through endotype beta-agarose and neoagarobiose hydrolase reaction, and used a size-exclusion chromatography technique. Among the enzyme reaction products, only agarotriose was purely separated and purified, and the fermentation ability of the probiotic Bifidobacteria was tested to prove the prebiotic effect of agarotriose. In addition, neo-agarobiose produced through agarotriose fermentation by Bifidobacteria is decomposed into galactose and 3,6-AHG by neo-agarobiose hydrolase of Bacteroides plebeius, an intestinal microorganism. Since AHG is a physiologically active substance that has anti-inflammatory and anti-collectal cancer prevention effects, it has been confirmed that agaotiose can expect not only prebiotic activity but also physiological activities such as anticancer and anti-inflammatory through metabolism by intestinal microbes. I did.

Accordingly, the present invention comprises at least one substrate selected from the group consisting of agar, agarose, neoagarohexaose, and agarotriose; Bacteroides plebeius strain; And it provides a pharmaceutical composition comprising a strain of Bifidobacterium (Bifidobacterium).

The foil steroid player bieoseu (Bacteroides plebeius) strains may comprise a foil steroid player bieoseu (Bacteroides plebeius) DSM 17135 strain.

The Bifidobacterium (Bifidobacterium) strain is Bifidobacterium ronggeom Infante tooth (Bifidobacterium longum subsp. Infantis) ATCC 17930, Bifidobacterium ronggeom Infante tooth (Bifidobacterium longum subsp. Infantis) ATCC 15702, Bifidobacterium bipyridinium B. bifidum DSM 20082 or Bifidobacterium B. kashiwanohense DSM 21854 and the like.

A pharmaceutical composition of the present invention is characterized in that the foil steroid player bieoseu the substrate by (Bacteroides plebeius) strain and the Bifidobacterium (Bifidobacterium) strain is finally decomposed into 3,6-AHG.

More specifically, 3,6-AHG is a beta-agarase of SEQ ID NO: 1 derived from Bacteroides plebeius DSM 17135 strain and a neo-agarobiose of SEQ ID NO: 2 by galactosyl during Days (beta-galactosidase) - 3 or beta of 4: hydrolases (neo-agarobiose hydrolase) and Bifidobacterium ronggeom Infante tooth (. Bifidobacterium longum subsp infantis) ATCC SEQ ID NO of 15 697 strains derived from It can be degraded from the substrate.

The beta-agarase is derived from Bacteroides plebeius DSM 17135, an enzyme that decomposes into neoagarotetraose and neoagarobiose using agar, agarose, or neoagarohexaose as a substrate. And may be represented by the amino acid sequence of SEQ ID NO: 1.

The beta-agarase can be transcribed and translated through a DNA segment, that is, a coding gene, involved in producing a polypeptide including an intervening sequence between individual coding segments as well as regions before and after the coding region of the enzyme. In addition, proteins having hydrolytic activity of the agar, agarose, or neoagarohexaose as mutant proteins such as one or more substitutions, deletions, translocations, additions, etc. of the enzyme are also included in the scope of the rights of the enzymes of the present invention. Specifically, the sequence identity with the amino acid sequence disclosed in SEQ ID NO: 1 is 80% or more, 85% or more, 90% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more And 99% or more of an amino acid sequence.

The beta-agarase is Bacteroides plebeius DSM 17135 It can be isolated and purified from the supernatant of culture, and can be produced and separated by strains other than Bacteroides plebeius DSM 17135 or artificial chemical synthesis using genetic engineering recombination technology. In the case of using the recombinant technology, the culture supernatant or supernatant of the transformed E. coli by transforming into E. coli may be replaced, but is not particularly limited thereto. According to one embodiment, it may be obtained from E. coli or a culture thereof transformed with a recombinant vector comprising a nucleic acid sequence of a gene encoding beta-agarase.

The neo-agarobiose hydrolase is derived from Bacteroides plebeius DSM 17135, and agarotriose, galactose or 3,6- It is an enzyme that decomposes into anhydro-L-galactose, and may be represented by the amino acid sequence of SEQ ID NO: 2.

It can be transcribed and translated through a DNA segment, that is, a coding gene, involved in producing a polypeptide including an intervening sequence between individual coding segments as well as regions before and after the coding region of the neo-agarobiose hydrolase. In addition, proteins having hydrolytic activity of the agar, agarose, or neoagarohexaose as mutant proteins such as one or more substitutions, deletions, translocations, additions, etc. of the enzyme are also included in the scope of the rights of the enzymes of the present invention. Specifically, the sequence identity with the amino acid sequence disclosed in SEQ ID NO: 2 is 80% or more, 85% or more, 90% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more And 99% or more of an amino acid sequence.

The neo-agarobiose hydrolase is Bacteroides plebeius DSM 17135 It can be isolated and purified from the supernatant of culture, and can be produced and separated by strains other than Bacteroides plebeius DSM 17135 or artificial chemical synthesis using genetic engineering recombination technology. In the case of using the recombinant technology, the culture supernatant or supernatant of the transformed E. coli by transforming into E. coli may be replaced, but is not particularly limited thereto. According to one embodiment, it may be obtained from E. coli or a culture thereof transformed with a recombinant vector comprising a nucleic acid sequence of a gene encoding a neo-agarobiose hydrolase.

The beta-galactosidase City Days is the enzyme that breaks down the to be derived from Bifidobacterium ronggeom Infante Tees (. Bifidobacterium longum subsp infantis) ATCC 15697, sweetheart Lot Rios with bios and galactose as neo-agar, from Blon_2016, Blon_2334 gene It is a protein to be produced, and may be represented by the amino acid sequence of SEQ ID NO: 3 or 4.

The beta-galactosidase can be transcribed and translated through a DNA segment, that is, a coding gene, involved in producing a polypeptide including a region before and after the coding region of the enzyme, as well as an intervening sequence between individual coding segments. In addition, proteins having hydrolytic activity of agarotriose as mutant proteins such as one or more substitutions, deletions, translocations, and additions of the enzyme are also included in the scope of the enzyme of the present invention, preferably SEQ ID NO: 3 Or an amino acid having a sequence identity of 80% or more, 85% or more, 90% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, and 99% or more of the amino acid sequence disclosed in 4 It contains the sequence.

The beta-galactosidase City Days is Bifidobacterium ronggeom Infante Tees (. Bifidobacterium longum subsp infantis) ATCC 15697 It can be isolated and purified from the culture supernatant water, and a genetically engineered recombinant techniques may be produced and separated by the like Bifidobacterium ronggeom Infante tooth (Bifidobacterium longum subsp. Infantis) ATCC 15697 strain or other artificial chemical synthesis. In the case of using the recombinant technology, the culture supernatant or supernatant of the transformed E. coli by transforming into E. coli may be replaced, but is not particularly limited thereto. According to one embodiment, it may be obtained from E. coli or a culture thereof transformed with a recombinant vector comprising a nucleic acid sequence of a gene encoding beta-agarase.

In this specification, “protein” and “polypeptide” are used interchangeably herein.

In the present invention, the fact that the polypeptide has a specific ratio (e.g., 80%, 85%, 90%, 95%, or 99%) of sequence identity to another sequence means that when aligning the two sequences, the sequence It means that the amino acid residues in the ratio are the same when comparing them. The alignment and percent homology or identity can be determined by any suitable software program known in the art, for example those described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al. (eds) 1987 Supplement 30 section 7.7.18). Can be used to determine. Preferred programs include the GCG Pileup program, FASTA (Pearson et al . 1988 Proc. Natl Acad. Sci USA 85:2444-2448), and BLAST (BLAST Manual, Altschul et al., Natl. Cent. Biotechnol. Inf., Natl Lib. Med. (NCIB NLM NIH), Bethesda, MD, and Altschul et al. 1997 NAR25:3389-3402). Another preferred alignment program is ALIGN Plus (Scientific and Educational Software, PA), which preferably uses basic parameters. Another sequence software program that can be used is the TFASTA Data Searching Program available in Sequence Software Package Version 6.0 (Genetics Computer Group, University of Wisconsin, Madison, WI).

The term "recombinant" when used in connection with a cell, nucleic acid, protein or vector in the present invention means that the cell, nucleic acid, protein or vector is modified by the introduction of a heterologous nucleic acid or protein or alteration of the original nucleic acid or protein, or It refers to that the cell is derived from such a modified cell. That is, for example, a recombinant cell expresses a gene that is not found within the original (non-recombinant) form of the cell, or otherwise expresses an intrinsic gene that is abnormally expressed or not expressed at all. Manifest.

As used herein, "nucleic acid" encompasses single-stranded or double-stranded DNA, RNA, and chemical modifications thereof. “Nucleic acid” and “polynucleotide” may be used interchangeably herein. Since the genetic code is degenerate, one or more codons can be used to encode a specific amino acid, and the present invention encompasses polynucleotides encoding a specific amino acid sequence.

The term "introduction" for inserting a nucleic acid sequence into a cell means "transfection", or "transformation" or "transduction", and refers to the integration of a nucleic acid sequence into a eukaryotic or prokaryotic cell. Mention is included, wherein the nucleic acid sequence is integrated into the genome of the cell (eg, chromosome, plasmid, plastid, or mitochondrial DNA) and converted into an autonomous replicon, or transiently expressed.

The pharmaceutical composition of the present invention metabolizes agar, agarose, neo-agarohexaose, or agarotriose to 3,6-AHG having anti-inflammatory and anti-cancer activity, so it can be used for the prevention or treatment of cancer or inflammatory diseases.

The term "prevention" used in the present invention refers to any action that suppresses or delays the onset of cancer or inflammatory disease by administering the pharmaceutical composition of the present invention to an individual.

The term "treatment" used in the present invention refers to any action to improve or benefit the symptoms of cancer or inflammatory disease by administering the pharmaceutical composition of the present invention to an individual.

In the present invention, the term "effective amount" means an amount of a compound capable of exhibiting an anticancer effect or suppressing inflammation.

Examples of the cancer include colon cancer, cervical cancer, breast cancer, gastric cancer, and liver cancer.

In the present invention, the term'anti-inflammatory effect' or'anti-inflammatory activity' refers to suppressing inflammation, and the inflammation is one of the defense reactions of living tissues against certain stimuli, tissue deterioration, circulatory disorders and effusion, tissue It refers to a complex lesion that involves three types of proliferation. More specifically, inflammation is part of innate immunity, and, as in other animals, innate immunity in humans recognizes patterns on the cell surface that are specific to pathogens. Phagocytes recognize cells with such surfaces as non-magnetic and attack pathogens. If pathogens break through the body's physical barriers, an inflammatory reaction occurs. The inflammatory reaction is a non-specific defense action that creates a hostile environment for microorganisms invading the wound. In the inflammatory reaction, when a wound or an external infectious agent enters the body, the white blood cells responsible for the initial stage immune response flock to express cytokines. Therefore, the amount of expression of cytokines in cells becomes an indicator of activation of the inflammatory response.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier includes carriers and vehicles commonly used in the field of medicine, and specifically, ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances (e.g., Various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g. protamine sulfate, disodium hydrogen phosphate, camium hydrogen phosphate, sodium chloride and zinc salts), colloidal Silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substrate, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene glycol or wool paper, etc., but are not limited thereto.

In addition, the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, an emulsifying agent, a suspending agent, or a preservative in addition to the above components.

In one aspect, the pharmaceutical composition of the present invention may be formulated and used in various formulations suitable for oral administration or parenteral administration.

Non-limiting examples of formulations for oral administration include troches, lozenges, tablets, aqueous suspensions, oily suspensions, powders, granules, emulsions, hard capsules, soft capsules, syrups or elixirs, etc. Can be mentioned.

In order to formulate the pharmaceutical composition of the present invention for oral administration, a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose, or gelatin; Excipients such as dicalcium phosphate; Disintegrants such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax can be used, and sweeteners, fragrances, and syrups can also be used. I can.

Furthermore, in the case of capsules, in addition to the above-mentioned substances, a liquid carrier such as fatty oil may be used.

Non-limiting examples of the parenteral preparation include injections, suppositories, respiratory inhalation powders, aerosols for sprays, oral sprays, oral detergents, toothpastes, ointments, powders for application, oils, creams, etc. have.

In order to formulate the pharmaceutical composition of the present invention for parenteral administration, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, external preparations, etc. can be used. , Vegetable oils such as olive oil, injectable esters such as ethyloleate, etc. may be used.

In addition, more specifically, when the pharmaceutical composition of the present invention is formulated as an injection solution, the pharmaceutical composition of the present invention is prepared as a solution or suspension by mixing in water with a stabilizer or buffer, and the unit of the ampoule or vial It can be formulated for administration. In addition, when the pharmaceutical composition of the present invention is formulated with an aerosol, a propellant or the like may be blended together with an additive so that the aqueous concentrate or wet powder is dispersed.

In addition, when formulating the pharmaceutical composition of the present invention into an ointment, cream, etc., animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. Can be formulated using as a carrier.

The pharmaceutically effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, administration mode, administration time and/or route of administration of the composition, and the response to be achieved by administration of the pharmaceutical composition of the present invention Type and degree, type of subject to be administered, age, weight, general health condition, symptom or degree of disease, sex, diet, excretion, drugs used simultaneously with the subject or at different times, components of other compositions, etc. It may be varied according to various factors including factors and similar factors well known in the medical field, and a person of ordinary skill in the art can easily determine and prescribe an effective dosage for a desired treatment. The administration of the pharmaceutical composition of the present invention may be administered once a day, or may be administered several times. Therefore, the above dosage does not limit the scope of the present invention in any way.

The route of administration and the method of administration of the pharmaceutical composition of the present invention may each be independent, and the method is not particularly limited, and any route of administration and administration method may be followed as long as the pharmaceutical composition can reach the desired site. I can. The pharmaceutical composition may be administered orally or parenterally.

As the parenteral administration method, for example, intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration or subcutaneous administration, etc. may be used, and a method of applying, spraying, or inhaling the pharmaceutical composition to a diseased site It can be used, but is not limited to these.

The pharmaceutical composition of the present invention may be preferably administered orally or administered by injection.

The present invention also provides a method of treating cancer or an inflammatory disease by administering an effective amount of the pharmaceutical composition to a subject.

The term "individual" used in the present invention means all animals including mammals including mice, livestock, humans, and the like.

In the method of treating cancer or inflammatory disease of the present invention, the description of the dosage, route of administration, mode of administration, etc. of the pharmaceutical composition is the same as described above with respect to the pharmaceutical composition.

The present invention also includes at least one substrate selected from the group consisting of agar, agarose, neoagarohexaose and agarotriose; Bacteroides plebeius strain; And it provides a food composition comprising the Bifidobacterium (Bifidobacterium) strain.

The food composition may be prepared as a food formulation prepared by encapsulation, powdering, suspension, or the like.

Since the food formulation can be consumed on a daily basis, it is very useful because it can be expected to prevent or improve cancer or inflammatory diseases.

There is no particular limitation on the type of food, and may include, for example, dairy products, health foods in the usual sense, and the like.

The present invention also provides a reaction product obtained after reacting any one of agar, agarose, or neo-agarohexaose with the beta-agarase of SEQ ID NO: 1 or 5, and the neo- Reacting with agarobiose hydrolase; And it provides a method for producing agarotriose comprising the step of purifying agarotriose from the reaction product to a size exclusion column.

Since the conventional process of performing enzymatic hydrolysis after weak acid pretreatment (FIG. 13A) generates a large amount of salt in the neutralization process after pretreatment, and if a low concentration neutral buffer is used, the pretreatment reaction must be carried out at high temperature (170°C). A high-temperature, high-pressure reactor is required, and the focus is on improving the production yield of 3,6-AHG, so the production yield of agarotriose is quite low. In particular, acetic acid used for pretreatment causes an unpleasant odor, so agarotriose is free. It can be a problem if it is used as a biotic material. Accordingly, the method of manufacturing agarotrios according to the present invention solves the above-described problems in the following manner.

First, in the production of agarotriose, beta-agarase, which mainly produces neoagarotetraose, which is a precursor of agarotriose, is applied, omitting the pretreatment process, and two-step enzymatic reaction (i.e., endotype beta-) under mild conditions. Agarase, Neo-Agarobiose Hydrolase) yields high yields of agarotriose, 3,6-AHG, and D-Gal.

Second, when purifying agalottriose, a size exclusion chromatography technique is used to separate monosaccharides and trisaccharides through a size exclusion bio-P2 Gel column using the difference in polymerization degree to obtain high-purity agarotriose (Fig. 13B). ). This purification process does not use an organic solvent harmful to the human body, uses water as a mobile phase, and has the advantage of obtaining a purified high yield of agarotriose since there is little amount of agarotriose lost during the purification process.

The beta-agarase decomposes any one of agar, agarose, or neo-agarohexaose into neo-agaro-oligosaccharides, neo-agarobiose and neo-agarotetraose, and the above-described Bacteroides plebeius (Bacteroides plebeius). ) Heat resistance of SEQ ID NO: 5 that decomposes into neoagarotetraose and neoagarohexaose using the beta-agarase of SEQ ID NO: 1 derived from DSM 17135 strain, or using agar or agarose as a substrate Agar degrading enzyme can be used.

The heat-resistant agar-degrading enzyme may be derived from Saccharophagus degradans 2-40 ^T , but is not particularly limited thereto.

The heat-resistant agar decomposition enzyme with saccharide wave Gus de gras thiooxidans (Saccharophagus degradans) 2-40 ^T can be isolated and purified from the culture supernatant of water, oil in Saccharomyces using the engineered recombinant techniques wave Gus de gras thiooxidans (Saccharophagusdegradans) 2 It can be produced and separated by strains other than -40 ^{T or by artificial chemical synthesis.} In the case of using the recombinant technology, it may be used by replacing the culture supernatant or supernatant of the transformed E. coli by transforming E. coli, but is not particularly limited thereto.

The reaction of the agar, agarose, or neo-agarohexase with any one of the substrates and beta-agarase may be performed for 5 minutes to 12 hours at 0 to 200 rpm at a temperature of 30 to 60°C.

The neo-agarobiose hydrolase decomposes neo-agarobiose and neo-agarotetraose into 3,6-AHG, D-Gal, and agarotriose, and the above-described bacteroid flaviers (Bacteroides plebeius ) DSM 17135 derived Using the neo-agarobiose hydrolase of SEQ ID NO: 2, or the alpha-neo agarobiose number of SEQ ID NO: 6 derived from Saccharophagus degradans 2-40 ^T Degrading enzymes can be used.

Wherein the saccharide having a wave Gus Gras thiooxidans (Saccharophagus degradans) derived from the alpha 2-40 ^T - neo agar decomposed into BIOS hydrolysis enzyme in Saccharomyces wave Gus de gras thiooxidans (Saccharophagus degradans) 2-40 ^T culture supernatant or the supernatant of the water It can be isolated and purified from, and can be produced and separated by strains other than Saccharophagus degradans 2-40 ^{T or artificial chemical synthesis using genetic engineering recombination technology.}

The reaction of the reaction product of the beta-agarase and the neo-agarobiose hydrolase may be performed for 30 minutes to 12 hours at 0 to 200 rpm under a temperature condition of 25 to 45°C.

After obtaining the monosaccharide, 3,6-AHG and D-Gal produced by the neo-agarobiose hydrolase and the trisaccharide, agarotriose, a size exclusion column was used to prepare purified agarotriose with high purity and high yield. You can get it.

Hereinafter, the present invention is described in more detail through examples according to the present invention, but the scope of the present invention is not limited by the examples presented below.

<Example 1> Decomposition experiment of agarose by beta-agarase

Enzymatic reaction was carried out to produce oligosaccharides derived from agar having various degrees of polymerization including agarotriose from agarose, which is a major carbohydrate constituting red algae. First, using agarose at a concentration of 1% (w/v) as a substrate, S. degradans The enzyme reaction of Aga16B, an endo-type agarase derived from ^{2-40 T, was performed.} At this time, the enzyme reaction of Aga16B was carried out at 50° C. and 200 rpm for 2 hours.

As a result of the enzymatic reaction, neoagarotetraose (DP4) and neoagarohexaose (DP6) were produced (FIG. 2B).

Using this reaction product as a substrate, the next enzyme, S. degradans Agarotriose (DP3), agaropentaose (DP5), and 3,6-AHG were produced through an enzymatic reaction of 2-40 ^T- derived neo-agarobiose hydrolase ( Sd NABH). The Sd NABH enzymatic reaction was carried out at 30° C. and 200 rpm for 2 hours.

In addition, using the Aga16B reaction product as a substrate, S. degradans Neoagarobiose (DP2), a disaccharide, was produced through the enzyme reaction of Aga50D, an exo-type agarase derived from 2-40 ^T. The Aga50D enzyme reaction was carried out at 30° C. and 200 rpm for 2 hours.

Next, the enzymatic reaction conditions of BpGH16A (BACPLE_01670), an endotype beta-agarase enzyme derived from DSM 17135, Bacteroides plebeius , an intestinal microorganism, are as follows: Enzyme loading amount 8 mg BpGH16A/g agarose, buffer 20 mM Tris-HCl (pH 7.0), reaction temperature was carried out for 2 hours at 40 ℃.

The enzymatic reaction conditions of the neoagarobiose hydrolase BpGH117 (BACPLE_01671) are as follows: Enzyme loading amount 4 mg BpGH117/g Neoagarobiose, buffer 20 mM Tris-HCl (pH 7.0), at a reaction temperature of 40°C. It proceeded for 2 hours.

As shown in FIG. 2C, agarotriose and 3,6-AHG could be produced from agarose through a combination of enzyme reactions related to agalysis derived from intestinal microorganisms, Bacteroides plebeius, DSM 17135. As a result of analyzing the reaction product through thin layer chromatography (TLC), the agarose substrate (lane 1) was produced as a major product of neoagarotetraose (DP4) through the enzymatic reaction of BpGH16A (BACPLE_01670) (lane 2). After that, agarotriose and 3,6-AHG were produced through the enzymatic reaction of BpGH117 (BACPLE_01671) (lane 2). When the two enzymes were reacted at the same time, agarotriose and 3,6-AHG were mainly produced as in the case of sequential reactions (lane 3).

<Example 2> Recombination of BpGH16A (BACPLE_01670), BpGH50 (BACPLE_01683), and BpGH117 (BACPLE_01671), which are endotype beta-agarase enzymes derived from Bacteroides plebeius DSM 17135, and neo-agarobiose hydrolase BpGH117 (BACPLE_01671) and Enzyme reaction experiment

As shown in FIG. 3, in the case of BpGH50 (BACPLE_01683), a GH50 family enzyme derived from Bacteroides plebeius DSM 17135, beta-agarase activity did not appear.

<Example 3> Enzymatic reaction of BpGH16A (BACPLE_01670), an endotype beta-agarase enzyme derived from Bacteroides plebeius DSM 17135 from an agarose substrate, and BpGH117 (BACPLE_01671), a neoagarobiose hydrolase Production experiment of agarotriose and AHG through

Each of the purified recombinant enzymes, Aga16B, Aga50D, Sd , oligosaccharides of various degrees of polymerization, produced through the reaction of NABH, neoagarobiose, and 3,6-AHG were purified through size exclusion column chromatography. At this time, sephadex G-10 was used as a column resin for size-exclusion column chromatography.

As shown in FIG. 4, agarotriose and 3,6-AHG were produced from agarose through the reaction of BpGH16A (BACPLE_01670), which is an endotype beta-agarase enzyme, and BpGH117 (BACPLE_01671), which is a neoagarobiose hydrolase. .

<Example 4> using a Bioscreen C and the per each tablet of a carbon source, the probiotic microorganism is Bifidobacterium ronggeom Infante tooth (Bifidobacterium longum subsp. Infantis) culture experiments of ATCC 15697 strain

In order to verify the prebiotic effect of sugar agar derived from bifidobacteria using each purified sugars, including agar lot Rios as a sole carbon source also bakteo Bifidobacterium ronggeom Infante tooth (Bifidobacterium longum subsp. Infantis) of ATCC 15697 strain Cell growth was monitored. At this time, the medium composition is BactoPeptone 10 g/L, yeast extract 5 g/L, K ₂ HPO ₄ anhydride 2 g/L, Na acetate anhydride 5 g/L, NH ₄ citrate tribasic 2 g/L, Mg sulfate heptahydrate 0.2 g/L, 0.05 g/L of Mn sulfate, 1 mL/L of polysorbate 80, 0.5 g/L of cysteine, and 5 g/L of each purified sugar were used, and cultured at 37°C.

As shown in Figure 5, Bifidobacterium longum infantis (Bifidobacterium longum subsp. infantis ) It was confirmed that ATCC 15697 strain selectively fermented only agarotriose among various purified sugars.

<Example 5> Analysis of Bifidobacterium ronggeom Infante tooth (Bifidobacterium longum subsp. Infantis) ATCC 15697 strain agar lot Rios fermentation profile of

Leeum Bifidobacterium conditions in the test tube to monitor the fermentation products ronggeom Infante Tees (Bifidobacterium longum subsp. Infantis) ATCC 15697 strain was cultured.

In, agar lot Rios 5 g / L concentration of the carbon source condition as shown in Figure 6. Bifidobacterium ronggeom Infante tooth (Bifidobacterium longum subsp. Infantis) ATCC 15697 has decomposed agar lot Rios the BIOS to galactose and neo agar galactose is It was fermented in cells to produce acetic acid. In the case of neo-agarobiose, it was no longer degraded within the cell, but was secreted and accumulated outside the cell.

<Example 6> Bifidobacterium ronggeom Infante tooth (Bifidobacterium longum subsp. Infantis) agar lot Rios and BIOS resolution experiments with neo-agar using the crude enzyme solution of the ATCC 15697 strain

To determine the metabolic pathways of agar lot Rios Bifidobacterium ronggeom Infante tooth (Bifidobacterium longum subsp. Infantis) were tested for crude enzyme solution of the ATCC 15697 strain. For this, dear one lot Rios Bifidobacterium ronggeom cultured under conditions Infante Tees (Bifidobacterium longum subsp. Infantis) The culture medium of ATCC 15697 was centrifuged (14,000 rpm, 5 minutes, 4° C.) to separate the cells and the medium. Extracellular coenzyme was obtained by precipitation of ammonium sulfate in the supernatant. In addition, the cell-free extract containing intracellular coenzyme was resuspended in 20 mM Tris-HCl buffer, sonicated to disrupt the cells, and centrifuged the lysate to obtain a supernatant coenzyme. In the coenzyme experiment, 2 mg/mL coenzyme and 2 mg/mL agarotriose were used as substrates and reacted in 20 mM Tris-HCl buffer (pH 7.0) at 30° C. and 200 rpm under enzymatic reaction conditions for 2 hours.

As shown in FIG. 7, it was confirmed that the beta-galactosidase reaction that decomposes agarotriose into galactose and neoagarobiose occurs in a cell-free extract including intracellular coenzymes. In addition, it was confirmed that the activity of the coenzyme did not appear with respect to the neo-agarobiose.

<Example 7> Bifidobacterium ronggeom Infante tooth (. Bifidobacterium longum subsp infantis) ATCC four of 15 697 isolates the type of beta-galactosidase when Days encoding recombinant protein production and enzymatic reaction of the gene (Blon_2016, Blon_2123, Blon_2334 and Blon_2416) Experiment

In order to determine whether having a certain enzyme activity of genes disassemble the Aga lot Rios Bifidobacterium ronggeom Infante Tees (Bifidobacterium longum subsp infantis.) ATCC 15697 strain has four kinds of beta in-cloned all galactosidase City Days After overexpression in E. coli, each enzyme protein was purified to test the enzyme activity.

As shown in FIG. 8, it was confirmed that the proteins of the two enzyme genes Blon_2016 and Blon_2334 exhibited activity.

<Example 8> Bifidobacterium ronggeom Infante tooth (Bifidobacterium longum subsp. Infantis) different strains agar lot Rios to efficacy experiments belonging to

Aga lot Rios is Bifidobacterium ronggeom Infante Tees (Bifidobacterium longum subsp. Infantis) ATCC 15697 strain in addition to other probiotic to about prebiotic Bifidobacterium for the effect to make sure ronggeom Infante Tees (Bifidobacterium longum subsp microorganisms . infantis) ATCC 17930, Bifidobacterium ronggeom Infante tooth (Bifidobacterium longum subsp. infantis) ATCC 15702, Bifidobacterium Stadium bifidobacteria (B. bifidum) DSM 20082 and Bifidobacterium Kashiwanohense ( B. kashiwanohense ) DSM 21854 was cultured in the agarotriose single carbon source condition.

As it is shown in Figs. 9 and 10, the four kinds of the probiotic micro-organisms of all metabolic agar lot Rios and Bifidobacterium ronggeom Infante tooth (Bifidobacterium longum subsp. Infantis) ATCC 15697 and, like agar lot Rios galactose and neo-agar After decomposition with lobiose, galactose was fermented to produce acetic acid.

<Example 9> Stability test of agarotriose against artificial gastric juice

In order to test whether agarotriose can reach the intestine without being decomposed, it was confirmed whether it was decomposed by TLC and HPLC after reacting in artificial gastric juice for each hour.

As shown in FIG. 11, it was confirmed that 80% or more of agarotriose remained after 3 hours reaction at 37°C.

From the above results, it was confirmed for the first time that agarotriose is a novel prebiotic derived from seaweed, which is selectively fermented by probiotics. Agarotriose can be produced by the action of agarose and NABH enzymes possessed by marine-derived S. degradans 2-40 ^T or Bacteroides plebeius derived from intestinal microorganisms. (Fig. 12). Agarotriose moves into cells by the ABC transporter-related gene possessed by the probiotic Bifidobacter, and is decomposed into galactose and neo-agarobiose by beta-galactosidase in the cell, and galactose is used for acetic acid fermentation. In addition, neo-agarobiose can be decomposed into galactose and 3,6-AHG in the intestine by the neo-agarobiose hydrolase possessed by the intestinal microorganism Bacteroides plebeius. AHG is known as a physiologically active substance that has anti-inflammatory properties and prevents colon cancer.Agaotriose is produced through metabolism by intestinal microbes such as Bifidobacter and Bacteroides plebeius, as well as prebiotic activity. Due to the AHG, it can exhibit colorectal cancer prevention and anti-inflammatory physiological activity (FIG. 12).

<Example 10> BACPLE_01670 and NABH recombinant enzyme production

Bacteroides plebeius derived beta-agarase hydrolase BACPLE_01670 gene was introduced into E. coli BL21 (DE3) using pET21a vector. In order to pre-culture the recombinant E. coli into which the gene was introduced, it was cultured at 37° C. for 9 hours in 10 mL of LB broth containing 100 μg/mL of ampicillin in a 50 mL-conical tube. Thereafter, 10 mL of the entire culture medium is inoculated into 1 L of the main culture medium of the same medium composition, and then 0.1 if the optical density value grows to the mid exponential stage (OD 0.4 to 0.6) using an optical density spectroscope. Mm isopropyl-beta-di-thiogalactopyranoside (IPTG) was added and induced at 16° C. for 16 hours to express intracellular protein. Thereafter, the cell culture solution was transferred to a 500 mL-tube and centrifuged at 10,000 rpm for 30 minutes at 4°C to obtain cells. To prevent protein denaturation, cells collected in 30 mL of Tris buffer (20 mM Tris-HCl, pH 7.0) were released again, and cells were lysed using a sonicator. Then, it was centrifuged at 16,000 rpm for 1 hour at 4°C. After the protein was purified using a histrap column (5ml GE health care), the size of each purified protein was confirmed using an SDS-PAGE gel. A salt (Imidazole) used for protein purification was removed using a desalting column. The concentration of the recombinant protein from which the salt was removed was quantified by the BCA analysis method.

Next, the alpha-neo agarobiose hydrolase NABH gene derived from Saccharophagus degradans 2-40 ^T was introduced into E. coli BL21 (DE3) using the pET21a vector. A recombinant protein was prepared as described above.

<Example 11> Enzymatic reaction of BACPLE_01670 and NABH

BACPLE_01670 In the enzyme reaction, agarose at a concentration of 1% (w/v) was used as the substrate, and the reaction was carried out for 10 hours at 50° C. and 100 rpm in 20 mM Tris-HCl buffer (pH 7.0).

BACPLE_01670 The enzyme reaction product of the enzyme reaction, neoagarotetraose and neoagarobiose were used as substrates for the NABH enzymatic reaction, and the enzyme reaction was carried out at 37°C and 100 rpm for 10 hours.

After each step of the enzymatic reaction, the reaction product was analyzed through TLC. TLC analysis conditions were 1 μl of the enzyme reaction product loaded on a silica gel plate as a stationary phase, and n-butanol: ethanol: water, 3:1:1 (v/v/v) as a mobile phase solvent, and developed for 1 hour. After that, color development was performed using 10% sulfuric acid in ethanol and 0.2% 1,3-dihydroxynaphthalene in ethanol.

As shown in FIG. 14A, agarose was decomposed into neoagaroligosaccharide through the enzymatic reaction of BACPLE_01670, an endotype beta-agarase, and the main products at this time correspond to the degree of polymerization (DP) DP2 and DP4. They were Neo-Agarobiose and Neo-Agarotetraose. After that, the trisaccharide agarotriose and 3,6-AHG were produced from DP4 through the enzymatic reaction of the alpha-agaraase neoagarobiose hydrolase (NABH), and D-galactose and 3,6-AHG were produced from DP2. Was created.

<Example 12> BACPLE_01670 Enzyme reaction product HPLC analysis

BACPLE_01670 The substances produced during the enzymatic reaction are neoagarotetraose (DP4) and neoagarobiose (DP2) (Fig. 14B). Among these, the precursor to make agarotriose is neoagarotetraose, and the more products of DP4, the more agarotriose can be obtained. In order to find out the ratio of DP4 generation from BACPLE_01670, it was calculated using the HPLC KS-802 size exclusion column.

As shown in FIG. 15, the reaction product of BACPLE_01670 is from 1 g of agarose to 0.795 g of neoagarotetraose (0.795 g neoagarotetraose/g agarose) and 0.205 g of neoagarobiose (0.205 g neoagarobiose/g agarose). ) To obtain the results.

<Example 13> DP3 (Agarotriose) and DP1 (3,6-AHG, D-Gal) separation using a size-exclusion column (Bio Gel-P2 column)

The final reaction products obtained from Examples 10 and 11 were 3,6-AHG, D-Gal, and agarotriose, of which 3,6-AHG and D-Gal were separated from agarotriose by using a sugar separation column. Was used. The machine used AKTA prime (GE healthcare). The mobile phase used to perform the sugar separation was distilled water and the flow was 0.3 mL/min for 10 minutes. After stabilizing the column, 2 mL of the reaction product was injected, and 1 mL of the solution coming out through the column was added to a 2 mL-Eppendorf tube and analyzed by TLC. TLC analysis conditions were the same as in Example 11.

As shown in FIG. 16, as a result of analyzing the samples collected for each fraction by TLC, it was confirmed that DP3 (agarotriose) and DP1 (3,6-AHG, D-Gal) were separated.

<Example 14> HPLC quantitative analysis of yield and purity of agarotriose separated through enzymatic saccharification and size exclusion column from agarose

After confirming the agarotriose obtained through the sugar separation column in Example 13 by TLC, fractions of agarotriose with high purity were collected. Fractions from No. 55 to No. 61 were collected in a 15 mL-conical tube and stirred to mix well. After that, some of the 7 mL was sampled and the yield and purity were analyzed through an HPLC KS-802 column.

As shown in FIG. 17, 0.4 g of agarotriose was obtained from 1 g of agarose (0.4 g NAB/g agarose), and it was confirmed that about 90.2% of the sample was agarotriose.

<110> KOREA UNIVERSITY RESEARCH AND BUSINESS FOUNDATION <120> Method for producing agarotriose and prebiotics use thereof <130> P19U13C1071 <160> 6 <170> KopatentIn 2.0 <210> 1 <211> 316 <212> PRT <213> Bacteroides plebeius DSM 17135 <400> 1 Met Lys Arg Lys Leu Phe Thr Ile Cys Leu Ala Ser Leu Gln Phe Ala 1 5 10 15 Cys Ala Ala Glu Asn Leu Asn Asn Lys Ser Tyr Glu Trp Asp Ile Tyr 20 25 30 Pro Val Pro Ala Asn Ala Gly Asp Gly Met Val Trp Lys Leu His Pro 35 40 45 Gln Ser Asp Asp Phe Asn Tyr Ile Ala Asp Glu Lys Asp Lys Gly Lys 50 55 60 Glu Phe Tyr Ala Lys Trp Thr Asp Phe Tyr His Asn His Trp Thr Gly 65 70 75 80 Pro Ala Pro Thr Ile Trp Gln Arg Asp His Val Ser Val Ser Asp Gly 85 90 95 Phe Leu Lys Ile Arg Ala Ser Arg Pro Glu Asp Val Pro Leu Lys Lys 100 105 110 Val Val Ser Gly Pro Asn Thr Lys Glu Leu Pro Gly Thr Tyr Thr Gly 115 120 125 Cys Ile Thr Ser Lys Thr Arg Val Lys Tyr Pro Val Tyr Val Glu Ala 130 135 140 Tyr Ala Lys Leu Ser Asn Ser Thr Met Ala Ser Asp Val Trp Met Leu 145 150 155 160 Ser Pro Asp Asp Thr Gln Glu Ile Asp Ile Ile Glu Ala Tyr Gly Gly 165 170 175 Asp Arg Asp Gly Gly Gly Tyr Gly Ala Asp Arg Leu His Leu Ser His 180 185 190 His Ile Phe Ile Arg Gln Pro Phe Lys Asp Tyr Gln Pro Lys Asp Ser 195 200 205 Gly Ser Trp Tyr Lys Asp Asp Lys Gly Thr Leu Trp Arg Asp Asp Phe 210 215 220 His Arg Val Gly Val Phe Trp Lys Asp Pro Phe Thr Leu Glu Tyr Tyr 225 230 235 240 Val Asp Gly Glu Leu Val Arg Thr Ile Ser Gly Lys Asp Ile Ile Asp 245 250 255 Pro Asn Asn Tyr Thr Gly Gly Thr Gly Leu Val Lys Asp Met Asp Ile 260 265 270 Ile Ile Asn Met Glu Asp Gln Ser Trp Arg Ala Val Lys Gly Leu Ser 275 280 285 Pro Thr Asp Glu Glu Leu Lys Asn Val Glu Asp His Thr Phe Leu Val 290 295 300 Asp Trp Ile Arg Val Tyr Thr Leu Val Pro Glu Glu 305 310 315 <210> 2 <211> 402 <212> PRT <213> Bacteroides plebeius DSM 17135 <400> 2 Met Arg Lys Ile Ile Phe Ala Ala Gly Met Met Ser Leu Leu Ala Ala 1 5 10 15 Cys Gly Asn Thr Gly Asn Thr Gln Thr Ile Ala Val Asp Asp Thr Gln 20 25 30 Asn Tyr Asp Glu Arg Lys Ala Asp Ser Leu Gly Ile Pro Lys Gly Asn 35 40 45 Lys Leu Ser Ala Ala Met Lys Arg Ala Met Lys Trp Glu Asn His Asp 50 55 60 Asn Lys Trp Phe Phe Glu Tyr Lys Met Glu Pro Leu Lys Gly Asp Leu 65 70 75 80 Ala Tyr Glu Glu Gly Val Val Arg Arg Asp Pro Ser Ala Met Leu Lys 85 90 95 Ile Gly Asp Thr Tyr Tyr Val Trp Tyr Ser Lys Ser Tyr Gly Pro Thr 100 105 110 Gln Gly Phe Ala Gly Asp Ile Glu Lys Asp Lys Val Phe Pro Trp Asp 115 120 125 Arg Cys Asp Ile Trp Tyr Ala Thr Ser Lys Asp Gly Leu Thr Trp Lys 130 135 140 Glu Gln Gly Ile Ala Val Lys Arg Gly Glu Lys Gly Ala Tyr Asp Asp 145 150 155 160 Arg Ser Val Phe Thr Pro Glu Val Met Glu Trp Lys Gly Lys Tyr Tyr 165 170 175 Leu Cys Tyr Gln Ala Val Lys Ser Pro Tyr Thr Val Arg Val Lys Asn 180 185 190 Thr Ile Gly Met Ala Cys Ala Asp Ser Pro Glu Gly Leu Trp Thr Lys 195 200 205 Thr Asp Lys Pro Val Leu Glu Pro Ser Asp Thr Gly Glu Trp Glu Gly 210 215 220 Asp Glu Asp Asn Arg Phe Lys Val Val Ser Lys Gly Asp Phe Asp Ser 225 230 235 240 His Lys Val His Asp Pro Cys Ile Ile Pro Tyr Asn Gly Lys Phe Tyr 245 250 255 Met Tyr Tyr Lys Gly Glu Arg Met Gly Glu Glu Ile Thr Trp Gly Gly 260 265 270 Arg Glu Ile Lys His Gly Val Ala Ile Ala Glu Asn Pro Met Gly Pro 275 280 285 Tyr Val Lys Ser Glu Tyr Asn Pro Ile Ser Asn Ser Gly His Glu Val 290 295 300 Cys Val Trp Pro Tyr Lys Gly Gly Ile Ala Ser Leu Ile Thr Thr Asp 305 310 315 320 Gly Pro Glu Lys Asn Thr Leu Gln Trp Ser Pro Asp Gly Ile Asn Phe 325 330 335 Glu Ile Met Ser Val Val Lys Gly Ala Pro His Ala Ile Gly Leu Asn 340 345 350 Arg Ser Ala Asp Ala Glu Lys Glu Pro Thr Glu Ile Leu Arg Trp Gly 355 360 365 Leu Thr His Ile Tyr Asn Ser Ser Asp Tyr Gln Ser Ile Met Arg Phe 370 375 380 Ser Thr Trp Thr Leu Gln Thr His Thr Ala Lys Gly Glu Ser Lys Glu 385 390 395 400 Arg Lys <210> 3 <211> 691 <212> PRT <213> Bifidobacterium longum subsp. infantis ATCC 15697 <400> 3 Met Glu His Arg Ala Phe Lys Trp Pro Gln Pro Leu Ala Gly Asn Lys 1 5 10 15 Pro Arg Ile Trp Tyr Gly Gly Asp Tyr Asn Pro Asp Gln Trp Pro Glu 20 25 30 Glu Val Trp Asp Glu Asp Val Ala Leu Met Gln Gln Ala Gly Val Asn 35 40 45 Leu Val Ser Val Ala Ile Phe Ser Trp Ala Lys Leu Glu Pro Glu Glu 50 55 60 Gly Val Tyr Asp Phe Asp Trp Leu Asp Arg Val Ile Asp Lys Leu Gly 65 70 75 80 Lys Ala Gly Ile Ala Val Asp Leu Ala Ser Gly Thr Ala Ser Pro Pro 85 90 95 Met Trp Met Thr Gln Ala His Pro Glu Ile Leu Trp Val Asp Tyr Arg 100 105 110 Gly Asp Val Cys Gln Pro Gly Ala Arg Gln His Trp Arg Ala Thr Ser 115 120 125 Pro Val Phe Leu Asp Tyr Ala Leu Asn Leu Cys Arg Lys Met Ala Glu 130 135 140 His Tyr Lys Asp Asn Pro Tyr Val Val Ser Trp His Val Ser Asn Glu 145 150 155 160 Tyr Gly Cys His Asn Arg Phe Asp Tyr Ser Glu Asp Ala Glu Arg Ala 165 170 175 Phe Gln Lys Trp Cys Glu Lys Lys Tyr Gly Thr Ile Asp Ala Val Asn 180 185 190 Asp Ala Trp Gly Thr Ala Phe Trp Ala Gln Arg Met Asn Asn Phe Ser 195 200 205 Glu Ile Ile Pro Pro Arg Phe Ile Gly Asp Gly Asn Phe Met Asn Pro 210 215 220 Gly Lys Leu Leu Asp Trp Lys Arg Phe Ser Ser Asp Ala Leu Leu Asp 225 230 235 240 Phe Tyr Lys Ala Glu Arg Asp Ala Leu Leu Glu Ile Ala Pro Lys Pro 245 250 255 Gln Thr Thr Asn Phe Met Val Ser Ala Gly Cys Thr Val Leu Asp Tyr 260 265 270 Asp Lys Trp Gly His Asp Val Asp Phe Val Ser Asn Asp His Tyr Phe 275 280 285 Ser Pro Gly Glu Ala His Phe Asp Glu Met Ala Tyr Ala Ala Cys Leu 290 295 300 Thr Asp Gly Ile Ala Arg Lys Asn Pro Trp Phe Leu Met Glu His Ser 305 310 315 320 Thr Ser Ala Val Asn Trp Arg Pro Thr Asn Tyr Arg Leu Glu Pro Gly 325 330 335 Glu Leu Val Arg Asp Ser Leu Ala His Leu Ala Met Gly Ala Asp Ala 340 345 350 Ile Cys Tyr Phe Gln Trp Arg Gln Ser Lys Ala Gly Ala Glu Lys Trp 355 360 365 His Ser Ala Met Val Pro His Ala Gly Pro Asp Ser Gln Ile Phe Arg 370 375 380 Asp Val Cys Glu Leu Gly Ala Asp Leu Asn Lys Leu Ala Asp Glu Gly 385 390 395 400 Leu Leu Ser Thr Lys Leu Val Lys Ser Lys Val Ala Ile Val Phe Asp 405 410 415 Tyr Glu Ser Gln Trp Ala Thr Glu His Thr Ala Thr Pro Thr Gln Glu 420 425 430 Val Arg His Trp Thr Glu Pro Leu Asp Trp Phe Arg Ala Leu Ala Asp 435 440 445 Asn Gly Leu Thr Ala Asp Val Val Pro Val Arg Gly Pro Trp Asp Glu 450 455 460 Tyr Glu Ala Val Val Leu Pro Ser Leu Ala Ile Leu Ser Glu Gln Thr 465 470 475 480 Thr Arg Arg Val Arg Glu Tyr Val Ala Asn Gly Gly Lys Leu Phe Val 485 490 495 Thr Tyr Tyr Thr Gly Leu Val Asp Asp Arg Asp His Val Trp Leu Gly 500 505 510 Gly Tyr Pro Gly Ser Ile Arg Asp Val Val Gly Val Arg Val Glu Glu 515 520 525 Phe Ala Pro Met Gly Thr Asp Ala Pro Gly Thr Met Asp His Leu Asp 530 535 540 Leu Asp Asn Gly Thr Val Ala His Asp Phe Ala Asp Val Ile Thr Ser 545 550 555 560 Val Ala Asp Thr Ala His Val Val Ala Ser Phe Lys Ala Asp Lys Trp 565 570 575 Thr Gly Phe Asp Gly Ala Pro Ala Ile Thr Val Asn Asp Phe Gly Asp 580 585 590 Gly Lys Ala Ala Tyr Val Gly Ala Arg Leu Gly Arg Glu Gly Leu Ala 595 600 605 Lys Ser Leu Pro Ala Leu Leu Glu Glu Leu Gly Ile Glu Thr Ser Ala 610 615 620 Glu Asp Asp Arg Gly Glu Val Leu Arg Val Glu Arg Ala Asp Glu Thr 625 630 635 640 Gly Glu Asn His Phe Val Phe Leu Phe Asn Arg Thr His Asp Val Ala 645 650 655 Val Val Asp Val Glu Gly Glu Pro Leu Val Ala Ser Leu Ala Gln Val 660 665 670 Asn Glu Ser Glu His Thr Ala Ala Ile Gln Pro Asn Gly Val Leu Val 675 680 685 Val Lys Leu 690 <210> 4 <211> 1023 <212> PRT <213> Bifidobacterium longum subsp. infantis ATCC 15697 <400> 4 Met Thr Asp Val Thr His Val Asp Arg Ala Ser Gln Ala Trp Leu Thr 1 5 10 15 Asp Pro Thr Val Phe Glu Val Asn Arg Thr Pro Ala His Ser Ser His 20 25 30 Lys Trp Tyr Ala Arg Asp Pro Gln Ser Gly Gln Trp Ser Asp Leu Lys 35 40 45 Gln Ser Leu Asp Gly Glu Trp Arg Val Glu Val Val Gln Ala Ala Asp 50 55 60 Ile Asn Leu Glu Glu Glu Pro Ala Thr Ala Glu Ser Phe Asp Asp Ser 65 70 75 80 Ser Phe Glu Arg Ile Gln Val Pro Gly His Leu Gln Thr Ala Gly Leu 85 90 95 Met Asn His Lys Tyr Val Asn Val Gln Tyr Pro Trp Asp Gly His Glu 100 105 110 Asn Pro Leu Glu Pro Asn Ile Pro Glu Asn Asn His Val Ala Leu Tyr 115 120 125 Arg Arg Lys Phe Thr Val Ser Ala Pro Val Ala Asn Ala Lys Gln Ala 130 135 140 Gly Gly Ser Val Ser Ile Val Phe His Gly Met Ala Thr Ala Ile Tyr 145 150 155 160 Val Trp Val Asn Gly Ala Phe Val Gly Tyr Gly Glu Asp Gly Phe Thr 165 170 175 Pro Asn Glu Phe Asp Ile Thr Gly Leu Leu His Asp Gly Glu Asn Val 180 185 190 Val Ala Val Ala Cys Tyr Glu Tyr Ser Ser Ala Ser Trp Leu Glu Asp 195 200 205 Gln Asp Phe Trp Arg Leu His Gly Leu Phe Arg Ser Val Glu Leu Ala 210 215 220 Ala Arg Pro His Val His Ile Glu Asn Thr Gln Ile Glu Ala Asp Trp 225 230 235 240 Asp Pro Glu Ala Gly Thr Ala Ser Leu Asp Ala Ala Leu Thr Val Leu 245 250 255 Asn Ala Thr Asp Ala Ala Thr Val Arg Ala Thr Leu Lys Asp Ala Asp 260 265 270 Gly Asn Thr Val Trp Gln Thr Thr Gly Asp Ala Glu Ala Gln Thr Ala 275 280 285 Leu Ser Ser Gly Pro Leu Gln Gly Ile Glu Pro Trp Ser Ala Glu Ser 290 295 300 Pro Thr Leu Tyr Glu Leu Asp Val Asp Val Ile Asp Gln Ala Gly Asp 305 310 315 320 Val Ile Glu Cys Thr Ser Gln Lys Val Gly Phe Arg Arg Phe Arg Ile 325 330 335 Glu Asp Gly Ile Leu Thr Ile Asn Gly Lys Arg Ile Val Phe Lys Gly 340 345 350 Ala Asp Arg His Glu Phe Asp Ala Glu Arg Gly Arg Ala Ile Thr Glu 355 360 365 Gln Asp Met Ile Asp Asp Val Val Phe Cys Lys Arg His Asn Ile Asn 370 375 380 Ser Ile Arg Thr Ser His Tyr Pro Asn Gln Glu Arg Trp Tyr Glu Leu 385 390 395 400 Cys Asp Glu Tyr Gly Ile Tyr Leu Ile Asp Glu Thr Asn Leu Glu Ala 405 410 415 His Gly Ser Trp Ser Leu Pro Gly Asp Val Leu Thr Glu Asp Thr Ile 420 425 430 Val Pro Gly Ser Lys Arg Glu Trp Glu Gly Ala Cys Val Asp Arg Val 435 440 445 Asn Ser Met Met Arg Arg Asp Tyr Asn His Pro Ser Val Leu Ile Trp 450 455 460 Ser Leu Gly Asn Glu Ser Tyr Val Gly Asp Val Phe Arg Ala Met Tyr 465 470 475 480 Lys His Val His Asp Ile Asp Pro Asn Arg Pro Val His Tyr Glu Gly 485 490 495 Val Thr His Asn Arg Asp Tyr Asp Asp Val Thr Asp Ile Glu Thr Arg 500 505 510 Met Tyr Ser His Ala Asp Glu Ile Glu Lys Tyr Leu Lys Asp Asp Pro 515 520 525 Lys Lys Pro Tyr Leu Ser Cys Glu Tyr Met His Ala Met Gly Asn Ser 530 535 540 Val Gly Asn Met Asp Glu Tyr Thr Ala Leu Glu Arg Tyr Pro Lys Tyr 545 550 555 560 Gln Gly Gly Phe Ile Trp Asp Phe Ile Asp Gln Ala Ile Tyr Ala Thr 565 570 575 Gln Pro Asp Gly Thr Arg Ser Leu Arg Tyr Gly Gly Asp Phe Gly Asp 580 585 590 Arg Pro Ser Asp Tyr Glu Phe Ser Gly Asp Gly Leu Leu Phe Ala Asp 595 600 605 Arg Lys Pro Ser Pro Lys Ala Gln Glu Val Lys Gln Leu Tyr Ser Asn 610 615 620 Val His Ile Asp Val Thr Lys Asp Ser Val Ser Val Lys Asn Asp Asn 625 630 635 640 Leu Phe Thr Ala Thr Gly Asp Tyr Val Phe Val Leu Ser Val Leu Ala 645 650 655 Asp Gly Lys Pro Val Trp Gln Ser Thr Arg Arg Phe Asp Val Pro Ala 660 665 670 Gly Glu Thr Arg Thr Phe Asp Val Ala Trp Pro Val Ala Ala Tyr Arg 675 680 685 Ala Asp Ala Arg Glu Leu Val Leu Gln Val Ser Gln Arg Leu Ala Lys 690 695 700 Ala Thr Asp Trp Ala Glu Ser Gly Tyr Glu Leu Ala Phe Gly Gln Ala 705 710 715 720 Val Val Pro Ala Asp Ala Thr Ala Thr Pro Asp Thr Lys Pro Ala Asp 725 730 735 Gly Thr Ile Thr Val Gly Arg Trp Asn Ala Gly Val Arg Gly Ala Gly 740 745 750 Arg Glu Val Leu Leu Ser Arg Thr Gln Gly Gly Met Val Ser Tyr Thr 755 760 765 Phe Ala Gly Asn Glu Phe Val Leu Arg Arg Pro Ala Ile Thr Thr Phe 770 775 780 Arg Pro Leu Thr Asp Asn Asp Arg Gly Ala Gly His Gly Phe Glu Arg 785 790 795 800 Val Gln Trp Leu Gly Ala Gly Arg Tyr Ala Arg Cys Val Asp Asn Val 805 810 815 Leu Glu Gln Ile Asp Asp Ser Thr Leu Lys Gly Thr Tyr Thr Tyr Glu 820 825 830 Leu Ala Thr Ala Gln Arg Thr Lys Val Thr Val Ser Tyr Thr Ala His 835 840 845 Thr Asp Gly Arg Val Asn Leu His Val Glu Tyr Pro Gly Glu Gln Gly 850 855 860 Asp Leu Pro Thr Ile Pro Ala Phe Gly Ile Glu Trp Thr Leu Pro Val 865 870 875 880 Gln Tyr Thr Asn Leu Arg Phe Phe Gly Thr Gly Pro Glu Glu Thr Tyr 885 890 895 Leu Asp Arg Lys His Ala Lys Leu Gly Val Trp Asn Thr Asn Ala Phe 900 905 910 Ala Asp His Ala Pro Tyr Leu Met Pro Gln Glu Thr Gly Asn His Glu 915 920 925 Asp Val Arg Trp Ala Glu Ile Thr Asp Asp His Gly His Gly Met Arg 930 935 940 Val Ser Arg Ala Asp Gly Ala Ala Pro Phe Ala Val Ser Leu Leu Pro 945 950 955 960 Tyr Ser Ser Phe Met Leu Glu Glu Ala Gln His Gln Asp Glu Leu Pro 965 970 975 Lys Pro Lys His Met Phe Leu Arg Val Leu Ala Ala Gln Met Gly Val 980 985 990 Gly Gly Asp Asp Ser Trp Met Ser Pro Val His Pro Gln Tyr His Ile 995 1000 1005 Pro Ala Asp Lys Pro Ile Ser Leu Asp Val Asp Leu Glu Leu Ile 1010 1015 1020 <210> 5 <211> 598 <212> PRT <213> Saccharophagus degradans 2-40 <400> 5 Met Lys Thr Thr Lys Cys Ala Leu Ala Ala Leu Phe Phe Ser Thr Pro 1 5 10 15 Leu Met Ala Ala Asp Trp Asp Gly Ile Pro Val Pro Ala Asp Pro Gly 20 25 30 Asn Gly Asn Thr Trp Glu Leu Gln Ser Leu Ser Asp Asp Phe Asn Tyr 35 40 45 Ala Ala Pro Ala Asn Gly Lys Ser Thr Thr Phe Tyr Ser Arg Trp Ser 50 55 60 Glu Gly Phe Ile Asn Ala Trp Leu Gly Pro Gly Gln Thr Glu Phe Tyr 65 70 75 80 Gly Pro Asn Ala Ser Val Glu Gly Gly His Leu Ile Ile Lys Ala Thr 85 90 95 Arg Lys Pro Gly Thr Thr Gln Ile Tyr Thr Gly Ala Ile His Ser Asn 100 105 110 Glu Ser Phe Thr Tyr Pro Leu Tyr Leu Glu Ala Arg Thr Lys Ile Thr 115 120 125 Asn Leu Thr Leu Ala Asn Ala Phe Trp Leu Leu Ser Ser Asp Ser Thr 130 135 140 Glu Glu Ile Asp Val Leu Glu Ser Tyr Gly Ser Asp Arg Ala Thr Glu 145 150 155 160 Thr Trp Phe Asp Glu Arg Leu His Leu Ser His His Val Phe Ile Arg 165 170 175 Gln Pro Phe Gln Asp Tyr Gln Pro Lys Asp Ala Gly Ser Trp Tyr Pro 180 185 190 Asn Pro Asp Gly Gly Thr Trp Arg Asp Gln Phe Phe Arg Ile Gly Val 195 200 205 Tyr Trp Ile Asp Pro Trp Thr Leu Glu Tyr Tyr Val Asn Gly Glu Leu 210 215 220 Val Arg Thr Val Ser Gly Pro Glu Met Ile Asp Pro Tyr Gly Tyr Thr 225 230 235 240 Asn Gly Thr Gly Leu Ser Lys Pro Met Gln Val Ile Phe Asp Ala Glu 245 250 255 His Gln Pro Trp Arg Asp Glu Gln Gly Thr Ala Pro Pro Thr Asp Ala 260 265 270 Glu Leu Ala Asp Ser Ser Arg Asn Gln Phe Leu Ile Asp Trp Val Arg 275 280 285 Phe Tyr Lys Pro Val Ala Ser Asn Asn Gly Gly Gly Asp Pro Gly Asn 290 295 300 Gly Gly Thr Pro Gly Asn Gly Gly Ser Gly Asp Thr Val Val Val Glu 305 310 315 320 Met Ala Asn Phe Ser Ala Thr Gly Lys Glu Gly Ser Ala Val Ala Gly 325 330 335 Asp Thr Phe Thr Gly Phe Asn Pro Ser Gly Ala Asn Asn Ile Asn Tyr 340 345 350 Asn Thr Leu Gly Asp Trp Ala Asp Tyr Thr Val Asn Phe Pro Ala Ala 355 360 365 Gly Asn Tyr Thr Val Asn Leu Ile Ala Ala Ser Pro Val Thr Ser Gly 370 375 380 Leu Gly Ala Asp Ile Leu Val Asp Ser Ser Tyr Ala Gly Thr Ile Pro 385 390 395 400 Val Ser Ser Thr Gly Ala Trp Glu Ile Tyr Asn Thr Phe Ser Leu Pro 405 410 415 Ser Ser Ile Tyr Ile Ala Ser Ala Gly Asn His Thr Ile Arg Val Gln 420 425 430 Ser Ser Gly Gly Ser Ala Trp Gln Trp Asn Gly Asp Glu Leu Arg Phe 435 440 445 Thr Gln Thr Asp Ala Asp Thr Gly Thr Asn Pro Pro Ser Thr Ala Ser 450 455 460 Ile Ala Val Glu Ala Glu Asn Phe Asn Ala Val Gly Gly Thr Phe Ser 465 470 475 480 Asp Gly Gln Ala Gln Pro Val Ser Val Tyr Thr Val Asn Gly Asn Thr 485 490 495 Ala Ile Asn Tyr Val Asn Gln Gly Asp Tyr Ala Asp Tyr Thr Ile Ala 500 505 510 Val Ala Gln Ala Gly Asn Tyr Thr Ile Ser Tyr Gln Ala Gly Ser Gly 515 520 525 Val Thr Gly Gly Ser Ile Glu Phe Leu Val Asn Glu Asn Gly Ser Trp 530 535 540 Ala Ser Lys Thr Val Thr Ala Val Pro Asn Gln Gly Trp Asp Asn Phe 545 550 555 560 Gln Pro Leu Asn Gly Gly Ser Val Tyr Leu Ser Ala Gly Thr His Gln 565 570 575 Val Arg Leu His Gly Ala Gly Ser Asn Asn Trp Gln Trp Asn Leu Asp 580 585 590 Lys Phe Thr Leu Ser Asn 595 <210> 6 <211> 368 <212> PRT <213> Saccharophagus degradans 2-40 <400> 6 Met Ser Asp Ser Lys Val Asn Lys Lys Leu Ser Lys Ala Ser Leu Arg 1 5 10 15 Ala Ile Glu Arg Gly Tyr Asp Glu Lys Gly Pro Glu Trp Leu Phe Glu 20 25 30 Phe Asp Ile Thr Pro Leu Lys Gly Asp Leu Ala Tyr Glu Glu Gly Val 35 40 45 Ile Arg Arg Asp Pro Ser Ala Val Leu Lys Val Asp Asp Glu Tyr His 50 55 60 Val Trp Tyr Thr Lys Gly Glu Gly Glu Thr Val Gly Phe Gly Ser Asp 65 70 75 80 Asn Pro Glu Asp Lys Val Phe Pro Trp Asp Lys Thr Glu Val Trp His 85 90 95 Ala Thr Ser Lys Asp Lys Ile Thr Trp Lys Glu Ile Gly Pro Ala Ile 100 105 110 Gln Arg Gly Ala Ala Gly Ala Tyr Asp Asp Arg Ala Val Phe Thr Pro 115 120 125 Glu Val Leu Arg His Asn Gly Thr Tyr Tyr Leu Val Tyr Gln Thr Val 130 135 140 Lys Ala Pro Tyr Leu Asn Arg Ser Leu Glu His Ile Ala Ile Ala Tyr 145 150 155 160 Ser Asp Ser Pro Phe Gly Pro Trp Thr Lys Ser Asp Ala Pro Ile Leu 165 170 175 Ser Pro Glu Asn Asp Gly Val Trp Asp Thr Asp Glu Asp Asn Arg Phe 180 185 190 Leu Val Lys Glu Lys Gly Ser Phe Asp Ser His Lys Val His Asp Pro 195 200 205 Cys Leu Met Phe Phe Asn Asn Arg Phe Tyr Leu Tyr Tyr Lys Gly Glu 210 215 220 Thr Met Gly Glu Ser Met Asn Met Gly Gly Arg Glu Ile Lys His Gly 225 230 235 240 Val Ala Ile Ala Asp Ser Pro Leu Gly Pro Tyr Thr Lys Ser Glu Tyr 245 250 255 Asn Pro Ile Thr Asn Ser Gly His Glu Val Ala Val Trp Pro Tyr Lys 260 265 270 Gly Gly Met Ala Thr Met Leu Thr Thr Asp Gly Pro Glu Lys Asn Thr 275 280 285 Cys Gln Trp Ala Glu Asp Gly Ile Asn Phe Asp Ile Met Ser His Ile 290 295 300 Lys Gly Ala Pro Glu Ala Val Gly Phe Phe Arg Pro Glu Ser Asp Ser 305 310 315 320 Asp Asp Pro Ile Ser Gly Ile Glu Trp Gly Leu Ser His Lys Tyr Asp 325 330 335 Ala Ser Trp Asn Trp Asn Tyr Leu Cys Phe Phe Lys Thr Arg Arg Gln 340 345 350 Val Leu Asp Ala Gly Ser Tyr Gln Gln Thr Gly Asp Ser Gly Ala Val 355 360 365

Claims (4)

Bacteroides plebeius strains expressing beta-agarase and neo-agarobiose hydrolase; And
Probiotic composition comprising a Bifidobacterium strain having agarotrios metabolism.
The method of claim 1,
Bacteroides plebeius strain comprises a Bacteroides plebeius DSM 17135 strain, a probiotic composition.
The method of claim 1,
Bifidobacterium (Bifidobacterium) strain is Bifidobacterium ronggeom Infante tooth (Bifidobacterium longum subsp. Infantis) ATCC 15697, Bifidobacterium ronggeom Infante tooth (Bifidobacterium longum subsp. Infantis) ATCC 17930, Bifidobacterium ronggeom Infante Bifidobacterium longum subsp. Infantis ATCC 15702, B. bifidum DSM 20082 and Bifidobacterium A probiotic composition selected from the group consisting of B. kashiwanohense DSM 21854.
The method of claim 1,
Probiotic compositions were prepared by agar, agarose and neo by beta-agarase and neo-agarobiose hydrolase from the Bacteroides plebeius DSM 17135 strain. this in agar at least one substrate selected from the group consisting of hexamethylene agarose decomposes generate agar lot Rios, and the agar lot Rios Bifidobacterium ronggeom Infante tooth (Bifidobacterium longum subsp infantis.) ATCC 15697 strain derived from beta-galactosidase A probiotic composition characterized by degradation of 3,6-anhydro-L-galactose by beta-galactosidase.

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