WO2021256968A1 - Bifidobacterium bifidum strain 8 for use as a probiotic - Google Patents

Abstract

The invention relates to biotechnology, and more particularly to a strain of Bifidobacterium bifidum that exhibits antagonistic activity towards pathogenic and opportunistic microorganisms, deposited in the National Bioresource Centre of the Kurchatov Institute of Genetics and the Selection of Industrial Microorganisms under collection number АС-2136. The invention broadens the range of probiotics capable of alleviating clinical manifestations of gut dysbacteriosis (dysbiosis) and stomach pain and providing improved functioning of the immune system.

Description

Bifidobacterium bifidum 8 strain used as a probiotic

The technical field to which the invention relates.

The invention relates to biotechnology, medical microbiology, pharmaceutical and food industries and is a strain of bifidobacteria of the species Bifidobacterium bifidum, which can be used in medicines, functional and specialized food products, including biologically active food additives.

State of the art.

Bifidobacteria are the main representatives of human indigenous microflora. The following main functions of bifidobacteria in the human body are described: bifidobacteria form the basis of microflora, as a result of high adhesiveness to the intestinal mucosa and determine the main niches of coexistence for other microorganisms, stabilize the intestinal biofilm; strengthen the intestinal epithelial barrier, necessary for the maturation of epithelial cells; the protein of bifidobacterial cells contains all essential amino acids, including tryptophan (Tereshkova E.A. et al., 2020; Ivanova E.V., 2018).

According to modern studies, the species Bifidobacterium bifidum predominates in the intestinal microbiocenosis and is necessary for the normal functioning of the human body (Ivanova E.V., 2018; Turroni F. et al., 2019). The species Bifidobacterium bifidum is the dominant species in individuals with a normal microflora composition and with initial degrees of dysbiosis of 1 - 2 degrees. With deep disturbances in microsymbiocenosis (grade 3 dysbiosis), the detection rate of the species Bifidobacterium bifidum decreases (Ivanova E.V., 2018).

There is a lot of data in the literature indicating that Bifidobacterium bifidum can interact with humans and with microorganisms of the intestinal microflora using various mechanisms, in particular, activate human immunity, adhere to the intestinal mucosa and metabolize glycans such as mucin (Turroni F. et al., 2019). It is noteworthy that the species Bifidobacterium bifidum is the only representative among all recognized species of bifidobacteria capable of growth through mucin metabolism (Ruas-Madiedo R. et al., 2008, Turroni F. et al., 2011). Moreover, the metabolism of mucin, carried out by Bifidobacterium bifidum, can activate the production of mucin, thereby increasing the depth of the mucus layer that envelops the mucous membrane, and, thus, enhancing the function epithelial barrier (Rokhsefat S. et al., 2016). These properties make it possible to classify representatives of this type of bifidobacteria as probiotic microorganisms.

Strains Bifidobacterium bifidum 1 and Bifidobacterium bifidum 791 in the composition of domestic probiotic preparations are used on a large scale in all age groups from 1972 to the present (Tereshkova E.A. et al., 2020).

In the modern concept of functional nutrition, prebiotics and synbiotics are essential. According to the generally accepted definition in the scientific literature, prebiotics are food ingredients that combine the following features: 1) resistant to gastric juice, hydrolysis by human digestive enzymes, and not absorbed in the small intestine, 2) fermented by the intestinal microflora 3) selectively stimulate growth and / or activity one or a limited number of groups of bacteria of the gut microbiota that positively affect the health of the host. A mixture of synergistically interacting probiotics and prebiotics, in which they mutually enhance the effectiveness of each other, is called synbiotics (Gibson G.R., Roberfroid M.V., 1995). Also, prebiotics are often associated with the so-called bifidogenic effect (Roberfroid M. et al., 2010).

Known strain Bifidobacterium bifidum 792 (patent RU 2215028), which is isolated from the intestinal contents of a healthy infant, actively multiplying in a nutrient medium with the accumulation of production biomass in a short time of cultivation with a high concentration of bifidobacteria. It has acid-forming and inhibitory properties against pathogenic and putrefactive microflora. This makes it possible to obtain fermented, fermented and non-fermented food products, starter cultures, hygienic and cosmetic products, biologically active additives and bacterial preparations that ensure the normalization of microbiocenoses of the human body. Known strains Bifidobacterium bifidum 79-32 (patent RU 2314340) and Bifidobacterium bifidum B-UH VKPM Ac-1875 (patent RU 2453592), isolated from the intestinal contents of a healthy infant, which, in addition to the above properties, ferment or ferment milk.

However, for these strains, information on resistance to antibacterial substances is not provided, which complicates the possibility of their use as an active component of drugs in the complex therapy of a number of diseases. Also, for all these strains, the species is not confirmed by modern molecular genetic methods, which for the isolated from natural sources of microorganisms is now mandatory (Bergey's Manual of Systematic Bacteriology. Volume 5).

Known strain Bifidobacterium bifidum 791 / BAG (patent RU 2165454), obtained by selection of strain Bifidobacterium bifidum 791. The strain is more resistant to damaging factors of cultivation media and aggressive media of the human gastrointestinal tract, provides utilization of a wider range of amino acids, ie, has a higher viability. and colonizing properties, which ensures the production of drugs and products based on it with longer shelf life and higher therapeutic and prophylactic properties. However, for the specified strain, information on resistance to antibacterial substances is also not provided, which complicates the possibility of its use as an active component of drugs in the complex therapy of a number of diseases.

Known strain Bifidobacterium bifidum OV-19 (patent RU 2375444), isolated from the intestinal contents of a healthy infant, which, in addition to the above properties, is resistant to a wide range of antibiotics, and also ferments milk with the formation of an even clot.

The disadvantage of all the above strains is that neither at the genotype level, nor at the phenotype level, the attitude towards mucin and bile salts is not determined, which, as mentioned above, is an important feature that affects the effectiveness of the strain as a probiotic.

Closest to the claimed invention is the Bifidobacterium IATA-ES2 WO2010007198 A1 strain isolated from the feces of a healthy breastfed baby and identified by 16S rRNA sequencing as a representative of the Bifidobacterium bifidum species. The strain is proposed to be used in order to improve protective functions and reduce the risk of diseases. The following mechanisms of its action are shown: 1) regulation of intestinal glycosylation, which favors that in healthy people, 2) modulation of the interaction between epithelial cells and intestinal microbiota, promoting the adhesion of beneficial bifidobacteria, and 3) regulation of the immunological response. However, no antibacterial resistance information is provided for this strain.

For all of the above strains, an assessment of the ability to ferment selectively stimulating the growth of bifidobacteria carbohydrate substrates - prebiotics, is not given, which limits the possibility of their use in these biotic functional and specialized food products. This feature, as indicated above, is the main criterion for development based on the strain synbiotic products. In vivo efficacy studies have either not been conducted or have been carried out in animal models, i.e. the results obtained cannot be fully transferred to humans.

Disclosure of the essence of the invention.

The objective of the present invention is to obtain a strain of microorganisms, confirmed by molecular genetic research methods, belonging to the species Bifidobacterium bifidum, characterized by the indicators necessary for inclusion in the composition of probiotic drugs, functional and specialized food products, including biologically active food additives.

When solving this problem, the following technical results are achieved: the strain is an antagonist of pathogenic and opportunistic microorganisms, ferments milk with the formation of a clot, is distinguished by active growth and formation of organic acids in media containing carbohydrate substrates - prebiotics that selectively stimulate the growth of bifidobacteria, which leads to an increase in antagonism against opportunistic microorganisms. The strain is resistant to antimicrobial substances kanamycin, gentamycin, amikacin. As a result of a genome-wide study, it was shown that the strain is characterized by the presence of genes encoding peptides that are responsible for adhesion to mucin and intestinal mucosa, as well as genes encoding enzymes that hydrolyze mucin. As a result of a genome-wide study, it was shown that the strain is characterized by the presence of genes responsible for the hydrolysis of bile salts, which indicates resistance to bile and participation in the regulation of the transformation of this group of substances in the liver-intestine-liver cycle, promoting digestion. The use of fermented products and bacterial preparations based on the claimed strain leads to the disappearance of clinical manifestations of intestinal dysbiosis (dysbiosis), stomach pain, improved function of the immune system, improved general condition, and increased efficiency.

Implementation of the invention

The specified technical result was achieved by obtaining a new bacterial strain Bifidobacterium bifidum 8, which is isolated from the contents intestines of a healthy person, identified by 16S rRNA, and deposited at the National Bioresource Center All-Russian collection of industrial microorganisms of the National Research Center "Kurchatov Institute" - GosNIIigenetics, collection number AS-2136.

The inventive strain has the following properties.

Full-length sequence of the gene encoding 16S ribosomal RNA, SEQ ID NO 1, is 100% homologous to the type strain of the species Bifidobacterium bifidum DSM 20456 (ATCC 29521).

Cultural and morphological properties.

Obligate anaerobic, optimal growth temperature 36 - 38 ° C.

Gram-positive, immobile, non-spore-forming, straight or slightly curved rods with thickening or bifurcation at one or both ends. They are located one at a time, usually 2 or in clusters. A characteristic feature is the arrangement of paired cells at an angle of 90 ° to each other in the late phases of growth.

When cultivated in Blaurock's medium modified by GI Goncharova at a temperature of 37 - 38 ° C for 36 - 48 hours it forms white colonies in the form of weakly expanding cones up to 10 mm long. When cultivated on the surface of an agar (dense) medium, Bifidobacterium Broth from Himedia under anaerobic conditions (bioMerieux GENbox anaer, temperature 37-38 ° C, duration 72 hours) forms flat grayish colonies of R-form, up to 5 mm in diameter.

Physiological and biochemical properties of the strain

The strain does not liquefy gelatin, does not produce catalase, and ferments milk with the formation of a clot. Utilizes glucose without the formation of gas, with the formation of lactic and acetic acids, lowering the pH of the medium to 3.8 - 4.2. Utilizes sucrose, lactose, mannose, cellobiose. Does not utilize: arabinose, xylose, inulin (highly purified), salicin, sorbitol.

The strain is resistant to kanamycin, gentamicin, amikacin. The strain is sensitive to azithromycin, amoxicillin, clarithromycin, doxycycline, tetracycline, ceftriaxone, vancomycin, cefepime, lincomycin, imipenem. In relation to, levofloxacin, metronidazole, trimethoprim, ciprofloxacin, ofloxacin is intermediate, taking into account the concentration that minimally inhibits growth (see examples). The strain is an active antagonist of pathogenic microorganisms. In the presence of prebiotics, phrutooligosaccharides (FOS), or lactulose as the only carbohydrate in the nutrient medium, it is characterized by the highest degree of suppression of microbial food contaminants and infectious agents, as exemplified by the test strains of Bacillus cereus and Staphylococcus aureus.

Established the harmlessness of the strain on white outbred mice weighing 14-16 g. Suspension of the lyophilized strain was injected at the rate of approximately 1x10 ⁹ CFU. Within 5 days, all the mice remained alive and did not lose weight.

The strain is stored in lyophilized form in a sucrose-gelatinous drying medium for at least 5 years in hermetically sealed vials without losing these properties.

Example 1

To confirm the antagonistic activity of the strain, the suspension of the lyophilisate, reconstituted in physiological saline, was plated on Petri dishes with MRS-5 medium with a stroke along the diameter. The culture was incubated under anaerobic conditions (bioMerieux GENbox anaer) at 37 ° C for 48 hours. After that, test strains were inoculated perpendicular to the growth zone of the strain, but without touching it, using a separate dish for each. After 20 hours, the zone of no growth was determined (Table 1). Strain Bifidobacterium bifidum 8 showed high antagonistic activity against test strains of pathogenic and opportunistic microorganisms.

Table 1 - Antagonistic activity of the Bifidobacterium bifidum 8 strain

Example 2 The lyophilized culture of the Bifidobacterium bifidum 8 strain was resuspended in saline and the previously pasteurized 10% skim milk suspension was inoculated at a rate of approximately 2 x 10 ⁶ CFU / ml. The incubation was carried out at 37 ° C for 48 hours. The results are shown in Table 2. The Bifidobacterium bifidum 8 strain ferments milk to form a curd.

Table 2 - Indicators of milk fermentation by the Bifidobacterium bifidum 8 strain

Example 3

Confirmation of the active growth of the Bifidobacterium bifidum 8 strain and the formation of organic acids in media containing selectively stimulating the growth of bifidobacteria carbohydrate substrates - prebiotics, as well as their synergistic effect against opportunistic microorganisms was carried out according to the previously described method (Karetkin V.A. et al., 2019 ). Representatives of the species B. cereus are food contaminants and causative agents of intestinal infections. B. cereus strain ATCC 9634 was used as a test strain. Fermentation of a mixed culture of bifidobacteria and a test strain was carried out at a temperature of 37 ° C under anaerobic conditions (2% carbon dioxide, 98% nitrogen) with constant stirring in a liquid medium containing as the only carbohydrate 1% of one of the well-known commercial prebiotics: fructooligosaccharides (FOS, Orafti ^® P95, BENEO ORAFTI, Belgium), isomaltulose (Palatinose ™, BENEO Palatinit GmbH, Germany), dry lactulose Lactusan (Felitsata Holding LLC, RF) and D-raffinose (LLC DiaM, RF). At the initial moment of the experiment and after 9 h of growth, the abundance of both microorganisms was assessed by sowing on selective media. Acids were determined by high performance liquid chromatography on an Agilent 1220 chromatograph with a refractometric detector and a Hi-Plex H column (4.6 x 250 mm). The inventive strain Bifidobacterium bifidum 8 is characterized by active growth and the greatest suppression of the test strain in the synbiotic composition with FOS and lactulose (Table 3).

Table 3 - The effect of commercial prebiotics on the activity of the Bifidobacterium bifidum 8 strain against B. cereus ATCC 9634

Staphylococcus aureus is considered one of the main causes of enterocolitis and diarrhea in patients treated with antibiotics in a hospital setting (Lo T.S., Borchardt S.M., 2009). The studies were carried out using a similar technique. The inventive strain Bifidobacterium bifidum 8 is characterized by active growth and the greatest suppression of the test strain S. aureus ATCC 43300 in a synbiotic composition with FOS and lactulose (Table 4).

Table 4 - The effect of commercial prebiotics on the activity of the Bifidobacterium bifidum 8 strain against S. aureus ATCC 43300

Example 4 The sensitivity of the claimed strain to antimicrobial substances was determined by the disk-diffusion method and by the method of serial dilutions in a liquid nutrient medium according to MUK 4.2.1890-04.

The daily culture of the strain obtained by incubation in Blaurock's medium at 37 ° C was standardized with sterile buffered saline to OD560 = 2.1 and sown in 0.1 ml of the resulting suspension on plates with Bifidobacterium Broth medium from Himedia. Disks with antibiotics were placed on the surface of the medium (see Table 5). Incubation was carried out under anaerobic conditions (bioMerieux GENbox anaer) at 37 ° C for 3 days, after which the diameter of the growth inhibition zone was determined (Table 5).

Table 5 - Studies of the sensitivity of the Bifidobacterium bifidum 8 strain to antimicrobial substances by the disk-diffusion method

For the method of serial dilutions, MRS liquid nutrient medium with cysteine and ascorbic acid and antimicrobial substances (see Table 6) were used for biochemical studies. Medicines based on these substances are most often used in modern therapy. For each substance, a series of serial two-fold dilutions was prepared in a sterile medium from 512 to 0.125 μg / ml. Inoculated with 10% daily culture of the strain, previously standardized to OD560 = 1.5. After 10 hours of incubation at 37 ° C under anaerobic conditions (2% carbon dioxide, 98% nitrogen) and with constant stirring the turbidity of the suspension was measured, the curves of the dependence of this value on the concentration of the antimicrobial substance were sigmoidal. The dependences were used to determine the minimum inhibitory concentration (MIC) and the minimum concentration of the antimicrobial substance at which the observed growth inhibition begins (Table 6). The determination of this value is associated with the following considerations. To achieve a therapeutic effect, a certain amount of living cells of bifidobacteria must enter the intestines of a person or animal, which can be ensured only with minimal exposure to antibacterial substances. Therefore, resistance was determined taking into account the indicated value, and not only taking into account the MIC, as is customary for pathogenic microorganisms, when the survival of even a small number of cells can lead to disease.

Table 6 - Study of the sensitivity of the strain to antimicrobial substances by the method of serial dilutions in a liquid nutrient medium

The strain is resistant (U) to kanamycin, gentamicin, amikacin. Sensitive (H) to amoxicillin, ceftriaxone, cefepime, imipenem, tetracycline, doxycycline, azithromycin, clarithromycin, lincomycin, vancomycin. For ofloxacin, ciprofloxacin, levofloxacin, metronidazole, nifuroxazide, trimethoprim is intermediate (P) taking into account the minimum concentration of the beginning of the observed growth suppression.

The results of a genome-wide study showed the absence of genes with tetracycline resistance markers tet (W) (SEQ ID NO: 2) and trp-tet (W) (SEQ ID NO: 3), which was also phenotypically confirmed.

Example 5

Molecular biological characteristics of the strain were determined on the basis of a genome-wide study.

Analysis of the resulting genome showed that the Bifidobacterium bifidum 8 strain does not contain plasmids.

Analysis of the resulting genome showed the presence of genes encoding cell wall peptides responsible for specific adhesion. Collagen binding protein is involved in adhesion to the epithelium. The homology of the detected structure (SEQ ID NO: 4) with the peptide of fimbrinal pili Bifidobacterium bifidum (NCBI GenBank SEQ ID NO: BBA48285.1) is 99%.

The protein encoded by the OrpA gene is responsible for binding Caco-2 to intestinal epithelial cells (Guglielmetti S. et al., 2008). The homology of the detected structure (SEQ ID NO: 5) with the oligopeptide binding protein OrpA of Bifidobacterium bifidum (NCBI GenBank SEQ ID NO: KLN80891.1) is 100%.

The formation of transaldolase significantly increases the degree of binding of bacterial cells, representatives of the intestinal microbiota, to mucin (Gonzalez-Rodriguez I. et al., 2012). The homology of the detected structure (SEQ ID NO: 6) with the Bifidobacterium bifidum transaldolase (NCBI GenBank SEQ ID NO: WP 003813255.1) is 100%.

Endo alpha-N-acetylgalactosaminidase is involved in the hydrolytic degradation of mucin (cleavage of carbohydrate residues from the peptide chain). The homology of the detected structure (SEQ ID NO: 7) with endo alpha-N-acetylgalactosaminidase Bifidobacterium bifidum ATCC 29521 (NCBI GenBank SEQ ID NO: BAQ97477.1) is 99%.

One of the main mechanisms that determine the resistance of intestinal bacteria to bile salts secreted by the liver is their cleavage by enzymes (Ruiz L. et al., 2013). The homology of the detected structure (SEQ ID NO: 8) with Bifidobacterium bifidum bile salt hydrolase (NCBI GenBank SEQ ID NO: AAR39435.1) is 99%. Phosphopyruvate hydrolase is also involved in the breakdown of bile salts. The homology of the detected structure (SEQ ID NO: 9) with Bifidobacterium bifidum phosphopyruvate hydrolase (NCBI GenBank SEQ ID NO: WP 003812660.1) is 100%.

Example 6.

Use of the Bifidobacterium bifidum 8 strain in medicines. On the basis of the Bifidobacterium bifidum 8 strain, a lyophilized biomass of bifidobacteria was obtained. A mother culture (inoculum) in Blaurock's medium was obtained from a pure culture by carrying out 2-3 passages according to one of the known methods. Incubation was carried out at 37 ° C. Further, cultivation was carried out while maintaining the pH in the range of 5.5 - 7.0. The resulting culture, containing at least 2x 10 ⁹ CFU / ml, was separated from the culture medium using centrifugation, separation, or other methods used for bacterial cell suspensions. A sucrose-gelatin drying medium was added to the resulting concentrate, frozen in vials or on drying trays made of food alloy steel to a temperature of no higher than minus 40 - 45 ° C and subjected to freeze drying. The residual moisture was no more than 5%. The resulting lyophilized biomass, containing at least 5 ^c 10 ¹⁰ CFU / g, is used as an active component of drugs, standardizing with a filler at the rate of a single dose (5-10) ^c 10 ⁸ CFU.

Example 7.

Use of the Bifidobacterium bifidum 8 strain in functional and specialized food products, including biologically active food supplements. The lyophilized biomass obtained analogously to the method described in example 6 is used as the main ingredient of probiotic biologically active food supplements in various marketable forms (powders, capsules, etc.) at the rate of 1x10 ⁸ - P10 ⁹ CFU in a single dose. Lyophilized biomass of bifidobacteria of the Bifidobacterium bifidum 8 strain is introduced into liquid fermented products for fermentation and fermentation, or for enrichment into fermented or non-fermented products at the rate of 10 ⁶ - 10 ⁷ CFU in 1 g of the finished product. Due to this, the resulting products can be classified as functional food products.

Example 8. Obtained as described in example 6, a cell suspension of the Bifidobacterium bifidum 8 strain in a drying medium was dosed into penicillin vials at the rate of a single dose (5-10) x10 ⁸ CFU and lyophilized. The use of lyophilized biomass in the specified single dose 4 times a day orally in courses from 8 to 32 days in male and female volunteers aged 45 years, 61 years, 66 years, 72 years, 93 years led to the disappearance of severe pain in the stomach that developed against the background exacerbation of chronic gastritis (without taking medications); the disappearance of the manifestations of intestinal dysbiosis (dysbiosis) (flatulence, abdominal pain, irregular stools, loose stools, stool retention); rapid relief of ARVI symptoms (within 1 - 3 days), especially when taken in the early stages of the disease; the disappearance of labial herpes; improving the general condition, increasing physical, mental activity, working capacity, improving the emotional state (the disappearance of low mood, irritability, emotional lability).

Example 9.

Woman, 57 years old. Multiple courses of chemotherapy for ovarian cancer. Against this background, pronounced dysbiosis (dysbiosis) of the intestine, abdominal pain, the intake of any food is accompanied by flatulence, diarrheal syndrome with watery stools. Drink yogurt enriched with bifidobacteria strain Bifidobacterium bifidum in an amount of 8 June ¹⁰ CFU in 1 ml for 4 days, the first two days of 300 ml per day, the following day 200 ml per day. The total volume of kefir is 1 liter. Diarrhea completely disappeared on the second day of taking kefir, on the fourth day the stool acquired a formed consistency, abdominal pain disappeared, flatulence significantly decreased, and the general condition improved.

Bibliography:

1. Tereshkova E.A., Karetkin B.A., Doroshenko E.O., Lanskikh A.G. Sorbed probiotics. Mechanism of action. - M .: TD DeLi, 2020 - 36 p.

2. Ivanova E.V. The role of bifidoflora in the associative symbiosis of the human intestinal myrobiota. Diss. d.m.s. Orenburg. 2018: 295.

3. Turroni F., Duranti S., Milani C., Lugli G. A., van Sinderen D., Ventura M. Bifidobacterium bifidum: A Key Member of the Early Human Gut Microbiota. // Microorganisms. 2019, 9; 7 (11).

4. Ruas-Madiedo P., Gueimonde M., Femandez-Garcia M., de los Reyes-Gavilan CG, Margolles A. Mucin degradation by Bifidobacterium strains isolated from the human intestinal microbiota. // Appl. Environ. Microbiol. 2008, 74, 1936-1940. Turroni F., Milani C., van Sinderen, D., Ventura, M. Genetic strategies for mucin metabolism in Bifidobacterium bifidum PRL2010: An example of possible human-microbe co-evolution. Gut Microbes 2011, 2, 183-189. Rokhsefat S., Lin AF, Comelli EM Mucin-Microbiota Interaction During Postnatal Maturation of the Intestinal Ecosystem: Clinical Implications. Dig. Dis. Sci. 2016, 61, 1473-1486. Gibson GR, Roberfroid MB Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics // The Journal of Nutrition. 1995, Vol. 125, p. 1401-1412 Roberfroid M., Gibson GR, Hoyles L., et al. Prebiotic effects: metabolic and health benefits. // Br J Nutr. 2010, Vol. 104, S. 2, S1-S63 Bergey's Manual of Systematic Bacteriology. Volume 5: The Actinobacteria Editors: Whitman, W., Goodfellow, M., Kampfer, P., Busse, H.-J., Trujillo, M., Ludwig, W., Suzuki, K.-L, Parte, A . (Eds.) 2012 Karetkin BA, Guseva EV, Evdokimova SA et al. A quantitative model of Bacillus cereus ATCC 9634 growth inhibition by bifidobacteria for synbiotic effect evaluation // World Journal of Microbiology and Biotechnology. 2019. Vol. 35. P. 89 Lo TS, Borchardt SM Antibiotic-associated diarrhea due to methicillin-resistant Staphylococcus aureus // Diagnostic microbiology and infectious disease. 2009. Vol. 63 (4), P. 388-389 Guglielmetti S., Tamagnini L, Mora D., Minuzzo M., Scarafoni A., Arioli S., Heilman J., Karp M., Parini C. Implication of an outer surface lipoprotein in adhesion of Bifidobacterium bifidum to Caco-2 cells. Applied and environmental microbiology, 2008, 74 (15), 4695-4702 Gonzalez-Rodriguez I., Sanchez B., Ruiz L., Turroni F., Ventura M., Ruas-Madiedo P., Gueimonde M., Margolles A. Role of extracellular transaldolase from Bifidobacterium bifidum in mucin adhesion and aggregation. Applied and environmental microbiology 2012, 78 (ll), 3992-3998. Ruiz L., Margolles A., Sanchez B. Bile resistance mechanisms in Lactobacillus and Bifidobacterium. // Frontiers in microbiology, 2013, 4, 396 Sequence Listing <160> 9

<210> 1

<211> 1534

<212> DNA

<213> Bifidobacterium bifidum

<400> 1 ttttttgtgg agggttcgat tctggctcag gatgaacgct ggcggcgtgc ttaacacatg 60 caagtcgaac gggatccatc aagcttgctt ggtggtgaga gtggcgaacg ggtgagtaat 120 gcgtgaccga cctgccccat gctccggaat agctcctgga aacgggtggt aatgccggat 180 gttccacatg atcgcatgtg attgtgggaa agattctatc ggcgtgggat ggggtcgcgt 240 cctatcagct tgttggtgag gtaacggctc accaaggctt cgacgggtag ccggcctgag 300 agggcgaccg gccacattgg gactgagata cggcccagac tcctacggga ggcagcagtg 360 gggaatattg cacaatgggc gcaagcctga tgcagcgacg ccgcgtgagg gatggaggcc 420 ttcgggttgt aaacctcttt tgtttgggag caagccttcg ggtgagtgta cctttcgaat 480 aagcgccggc taactacgtg ccagcagccg cggtaatacg tagggcgcaa gcgttatccg 540 gatttattgg gcgtaaaggg ctcgtaggcg gctcgtcgcg tccggtgtga aagtccatcg 600 cttaacggtg gatctgcgcc gggtacgggc gggctggagt gcggtagggg agactggaat 660 tcccggtgta acggtggaat gtgtagatat cgggaagaac accgatggcg aaggcaggtc 720 tctgggccgt cactgacgct gaggagcgaa agcgtgggga gcgaacagga ttagataccc 780 tggtagtcca cgccgtaaac ggtggacgct ggatgtgggg cacgttccac gtgttccgtg 840 tcggagctaa cg cgttaagc gtcccgcctg gggagtacgg ccgcaaggct aaaactcaaa 900 gaaattgacg ggggcccgca caagcggcgg agcatgcgga ttaattcgat gcaacgcgaa 960 gaaccttacc tgggcttgac atgttcccga cgacgccaga gatggcgttt cccttcgggg 1020 cgggttcaca ggtggtgcat ggtcgtcgtc agctcgtgtc gtgagatgtt gggttaagtc 1080 ccgcaacgag cgcaaccctc gccccgtgtt gccagcacgt tatggtggga actcacgggg 1140 gaccgccggg gttaactcgg aggaaggtgg ggatgacgtc agatcatcat gccccttacg 1200 tccagggctt cacgcatgct acaatggccg gtacagcggg atgcgacatg gcgacatgga 1260 gcggatccct gaaaaccggt ctcagttcgg atcggagcct gcaacccggc tccgtgaagg 1320 cggagtcgct agtaatcgcg gatcagcaac gccgcggtga atgcgttccc gggccttgta 1380 cacaccgccc gtcaagtcat gaaagtgggc agcacccgaa gccggtggcc taaccccttg 1440 tgggatggag ccgtctaagg tgaggctcgt gattgggact aagtcgtaac aaggtagccg 1500 taccggaagg tgcggctgga tcacctcctt tcta 1534 <210> 2 <211> 23 <212> DNA

<213> Artificial Sequence <400> 2 tggaattctt gcccatgtag acg 23

<210> 3 <211> 22 <212> DNA

<213> Artificial Sequence <400> 3 attcagcgac gaactggcac ag 22

<210> 4 <211> 534 <212> PRT

<213> Bifidobacterium bifidum <400> 4

Met Lys Phe Lys Lys Leu Phe Ala Gly Val Ala Ala Ala Ala Thr Leu 1 5 10 15

Leu Ala Gly Met Ala Phe Gly Thr Gly Ala Ala Asn Ala Ala Glu Thr 20 2 5 30

Thr Val Asp Thr Ala Ala Thr Val Thr Phe Lys Ala Ser Lys Glu Lys

35 40 45

Gin Leu Thr Ser Ala Gin Leu Ser Ala Tyr Lys lie Ala Asp Tyr Val

50 55 60

Asn Tyr Gly Thr Ala Lys Thr Pro Val Tyr Gly Val Lys Thr Ala Ala 65 70 75 80

Gly Ala Asn Arg Thr Lys Leu Ala Ala Ala Leu Thr Ala Ala Gly Phe

85 90 95

Gin Asn Val Pro lie Asp Gly Thr Thr Asp Leu Met Ala Trp Ala Met

100 105 110

Asn Gin Lys Thr Thr Thr Asp Thr Asp Gly Thr Val Thr Gin Val Gin

115 120 1 25 Phe Asp Gin Ser Glu Thr Arg Pro Trp Asn Asn Pro Ser Val Thr Arg

130 135 140

Lys Phe Ala Asp Ala Leu Gin Ala Gly Asn Pro Phe Thr Ala Thr Ala

145 150 155 160

Asp Pro Phe Val Leu Asn Lys Ala Thr Gly Asn Asp Thr Asp Gly Trp

165 170 175

Thr Ala Thr Asn Lys Thr Ala Leu Thr Ala Gly Val Tyr Leu Phe Leu

180 185 190

Asp Gly Asn Ala Ser Thr Asp Thr Leu Thr Gin Ala Val Pro Met lie

195 200 205

Val Ser Thr Gly Ser Val Asp Ala Glu Gly Val Leu Ser Leu Gly Asp

210 215 220

Ser Thr Ala Glu Val Asp Met Lys Ser Thr Val Ser Gly Thr Gin Thr

225 230 235 240

Lys Ser Thr Thr Ser Lys Ser Ala Ser Val Gly Glu Thr Val Pro Phe

245 250 255

Glu Leu Gly Tyr Thr lie Pro Asn Pro Val Pro Thr Asp Phe Thr Leu

260 265 270

Gin Phe Lys Asp Val Pro Ser Lys Gly Leu Thr Val Asn Phe Ala Ser

275 280 285

Leu Thr Val Lys Ala Gly Asp Lys Val Leu Thr Asp Thr Asp Tyr Thr

290 295 300

Val Glu Asn Asn Leu Thr Asp Asn Lys Gly Asp Gly Thr Asn Thr Phe

305 310 315 320

Val Val Lys He Thr Asp Pro Ala Lys Tyr Ala Gly Lys Gin He Thr

325 330 335

He Thr Tyr Asn Ala Thr Val Asn Asp Glu Ala Glu Thr Val Glu Gly

340 345 350

Gin Asp Tyr His Ala Val Thr Asn Lys Leu Val Gly Asn Asp Gly Thr

355 360 365

Pro He Pro Gly Thr Glu Thr Leu Thr Lys He Phe Gly Phe Lys Phe

370 375 380

Thr Lys Val Asn Ala Gin Gly Glu Ala Val Glu Gly Ala Lys Phe Thr

385 390 395 400 Leu Ser Val Ala Lys Asp Gin Asn Gly Val Leu Pro Asn Ser Asp Lys

405 410 415

Tyr Pro Leu Glu Val Thr Ser Gly Ala Asn Gly Val Val Lys Phe Asp 420 425 430

Gly Leu Lys Ala Gly Ser Tyr Thr Val Thr Glu Thr Ala Val Ala Asp 435 440 445

Gly Tyr Gin Asp Phe Lys Ala Ser Phe Thr Val Ala lie Asp Glu Asn 450 455 460

Gly Lys Val Thr Phe Ala Gly Thr Asp Ser Trp Gly Leu Ala Pro Lys 465 470 475 480

Gly Ser Ala Asp Asp Tyr Lys Val Thr Asn Val Lys Ser Val Phe Glu

485 490 495

Leu Pro Lys Thr Gly Ala Ala Gly lie Ala Leu Phe Val Val He Ala 500 505 510

Ala Leu Leu Gly Gly Ala Ala Ala Thr Val Tyr Ala Lys Ser Arg Arg 515 520 525

Thr Ser Arg Ala Leu Arg 53 0

<210> 5 <211> 596 <212> PRT

<213> Bifidobacterium bifidum <400> 5

Met Gin Ser Met Ser Phe Ala Ser Thr Ala Arg Lys Val Thr Ala Phe

1 5 10 15

Ala Ala Ala Ala Ala Thr Leu Leu Ala Leu Gly Ala Cys Gly Asn Ser

20 25 30

Asn Asn Gly Ser Ala Gly Asn Asn Gly Ala Lys Ser Thr Ser Glu Pro

35 40 45

Gin Glu Gly Val Pro Thr Asn Tyr Thr Gly Ser Phe Pro Met Pro Asp 50 5 5 60

Pro Gly Lys Ala Tyr Asn Asn Pro Lys Asp Arg Gly Glu Leu Lys Gin 65 70 75 80 Gly Gly Thr Leu Thr Leu Pro lie Thr Glu lie Gly Pro Asn Trp Asn

85 90 95

Ala He Asp Val Asn Gly Asn Thr Val Tyr Met Ser Gly Leu Trp Arg

100 105 110

Phe Tyr Met Pro Cys He Trp Glu Tyr Pro Ala Asp Gly Ser Leu Glu

115 12 0 125

Asn He Lys Pro Asn Thr Asn Tyr Val Thr Lys Val Glu Gin Thr Ser

130 135 140

Asp Ser Pro Glu Thr Val Val Phe Thr He Asn Asp Lys Ala Ser Trp

145 150 155 160

Asn Asp Gly Thr Pro He Thr Trp Lys Asp Phe Glu Ser Thr Trp Lys

165 170 175

Val Gin Ser Gly Lys Ser Asp Lys Phe Thr Pro Ala Leu Thr Thr Gly

180 185 190

Tyr Asp Gin He Lys Ser Val Thr Lys Gly Asp Thr Asp Lys Gin Ala

195 200 205

Val Val Thr Phe Glu Lys Asp Phe Tyr Pro Tyr Gin Ser Leu Phe Asn

210 215 220

Leu Leu Tyr Pro Ser Glu Ala Leu Thr Gly Asp Asp Thr Lys Asp Gly

225 230 235 240

Asp Thr Phe Ser Lys Gly Trp Ser Asn Asp Ser His Ser Asp Lys Trp

245 250 255

Gly Ala Gly Pro Phe Val Val Ser Lys Ser Ser Asp Ser Glu Val Thr

260 265 270

Phe Thr Pro Asn Pro Lys Trp Trp Gly Asp Lys Pro Leu Leu Asp Lys

275 280 285

Val Val Leu Lys Gin Met Glu Pro Ser Ala Ala Met Asn Ala Phe Lys

290 295 300

Asn Gly Glu He Asp Ala Val Gly Ala Ser Asp Ala Glu Ser Met Thr

305 310 315 320

Ser Ala Lys Thr Val Lys Asp Ala Tyr Leu Arg Arg Ser Tyr Asp Ser

325 330 335

Gly Val Tyr Thr Leu Thr He Asn Thr Lys Asn He Pro Leu Glu Val

340 34 5 3 50 Arg Lys Ala Phe Val Gin Gly Val Asp Arg Ser Gin Leu Gin Lys lie 355 360 365

Asp Phe Gin Gly lie Glu Trp Thr Glu Lys Thr Pro Gly Ser Leu He 370 375 380

Leu Pro Gin Phe Gin Asp Gly Tyr Glu Asp Asn Met Pro Ala Glu Ser 385 390 395 400

Lys Tyr Ser Thr Ala Asn Ala Thr Lys Thr Leu Glu Asp Ala Gly Tyr

405 410 415

Lys Lys Asn Ser Asp Gly Tyr Tyr Glu Lys Gly Gly Lys Val Ala Ala 420 425 430

He Thr Tyr Thr Thr Phe Ser Asp Ser Ala Ser Thr Lys Ala Lys Ala 435 440 445

Thr Ala He Gin Lys Met Ala Lys Thr Val Gly He Lys Val Ser He 450 455 460

Asp He Lys Ala Ser Asn Thr Phe Ser Asp Thr Val Ser Ser Gly Asn 465 470 475 480

Trp Asp Met He Gly Leu Gly Trp Ser Ala Ser Asp Pro Phe Gly Tyr

485 490 495

Ala Ser Ser Ala Tyr Gin Leu Tyr Gly Ser Lys Ser Glu Ser Asn Phe 500 505 510

Ser Phe Thr Gly Thr Asp Glu He Asp Lys Glu Leu Glu Ser He Pro 515 520 525

Gly He Lys Asp Ser Thr Lys Ala He Ala Gin Phe Asn Lys Ala Glu 530 535 540

Lys Glu Ala Gin Lys Leu Tyr Ala Gin He Pro Phe Glu Asn Gly Gin 545 550 555 560

He Thr Val Ala Val Lys Lys Gly Leu Ala Asn Tyr Gly Pro Ala Gly

565 570 575

Tyr Ser Ser Gly Ala Arg Val Met Val Thr Val His Pro Glu Asn Leu 580 585 590

Gly Trp Glu Lys 595

<210> 6 <211> 367 <212> PRT

<213> Bifidobacterium bifidum <400> 6

Met Thr Glu Ala Thr Gin Arg Thr Ser Asp Asn Gly Val Ser lie Trp

1 5 10 15

Leu Asp Asp Leu Ser Arg Thr Arg lie Glu Ser Gly Ser Leu Gin Glu 20 25 30

Leu He Lys Asp Lys Asn Val Val Gly Val Thr Thr Asn Pro Ser He 35 40 45

Phe Gin Lys Ala Leu Ser Gin Val Gly Pro Tyr Asp Ala Gin Leu Lys 50 55 60

Glu Leu Gly Lys Val Asp Val Glu Thr Ala Val Arg Glu Leu Thr Thr 65 70 75 80

Thr Asp Val Arg Asn Ala Thr Asp He Phe Arg Glu He Ala Glu Ala

85 90 95

Thr Asp Phe Val Asp Gly Arg Val Ser He Glu Val Asp Pro Arg Leu

100 105 110

Ala His Asp Thr Glu Asn Thr Glu Lys Gin Ala Val Glu Leu Trp Glu

115 120 125

Lys Val Asn Arg Pro Asn Ala Met He Lys He Pro Ala Thr Leu Glu

130 135 140

Gly Leu Pro Ala He Thr Ala Thr Leu Ala Lys Gly He Ser Val Asn 145 150 155 160

Val Thr Leu He Phe Ser Leu Glu Arg Tyr Glu Gin Val He Asp Ala

165 170 175

Tyr He Glu Gly He Ala Gin Ala Ala Ala Asn Gly His Asp Leu Lys

180 185 190

His He Gly Ser Val Ala Ser Phe Phe Val Ser Arg Val Asp Thr Ala 195 200 205

Val Asp Lys Leu Leu Glu Ala Asn Gly Ser Asp Glu Ala Lys Ala Leu

210 215 220

Glu Gly Lys Ala Ala Val Ala Asn Ala Arg Leu Ala Tyr Glu Leu Phe

225 230 235 240

Glu Lys Lys Phe Ala Ala Asp Pro Arg Trp Ala Asp Leu Ala Ala Lys

245 250 255 Gly Ala Lys Val Gin Arg Pro Leu Trp Ala Ser Thr Gly Thr Lys Asn 260 265 270

Ala Ala Tyr Ser Asp Cys Lys Tyr Val Asp Glu Leu Val Ala Lys His 275 280 285 lie Val Asn Thr Met Pro Glu Lys Thr Leu Asn Ala Leu Ala Asp His 290 295 300

Gly Asn Gly Ala Pro Ser lie Glu Gly Thr Tyr Glu Glu Ser His Ala 305 310 315 320

Val He Asp Lys Leu Ala Glu Leu Gly He Asn Leu Lys Asp Val Thr

325 330 335

Asp Lys Leu Glu Ala Asp Gly Val Ala Ala Phe He Lys Ser Trp Asp 340 345 350

Ser Val Leu Ala Asp Val Gin Ser Gly He Asp Arg Val Asn Ala 355 360 365

<210> 7 <211> 1931 <212> PRT

<213> Bifidobacterium bifidum <400> 7

Met Arg Lys Arg Val Val Ser Val Ala Leu Ala Val Ala Leu Ala Val 1 5 10 15

Ala Pro Leu Gly Val Gly Ser Thr Ala Ser Ala Ala Pro Leu Ser Ala

20 25 30

Ser Asp Leu Gin Thr Leu Ala Leu Arg Ser Ala Ala Ser Ser Asn Asp 35 40 45

Ala Asn Ala Asn Asp Val Ala Thr Val Ala Asp Asp Ala Ala Val Asn

50 55 60

Gly Trp Thr He Asp Arg Asn Thr Ala Lys Gly Gly Glu He Leu Ala 65 70 75 80

Ala Gly Thr Gly Asp Tyr Ala Gly Trp Thr His Phe Lys Ser Thr Ser

85 90 95

Ala Asn Gly Asn Ala Thr Ser Ser Ser Gly Tyr Pro Ala Val Ala He

100 105 110 Ser Gly Lys Thr lie Asp Leu Thr Arg Ala Gly Glu Phe Ser lie Lys 115 120 125

Val Lys Ser Pro Gin Ala Gly Ser Ala Asn Arg Phe Gly Phe Tyr Leu 130 135 140

Gly Tyr Lys Asp Pro Gly Asn Ala Leu Phe Leu Gly Tyr Asp Lys Gly

145 150 155 160

Gly Trp Phe Trp Gin Lys Tyr Val Asp Gly Asn Gly Asp Trp Tyr Asn

165 170 175

Gly Thr Arg Val Ala Ala Pro Ala Ala Asn Ala Glu He Thr Val Asn 180 185 190

Val Ser Trp Thr Ala Ala Lys Val Ala Thr Leu Thr He Asp Gly Gin 195 200 205

Lys Ala Phe Asp Val Asp Tyr Ser Ser Met Thr Ala Leu Thr Asn Lys 210 215 220

Leu Ala Met Lys Ala Gly Ser Tyr Ser Gly Thr Ser Glu Val Thr Asp 225 230 235 240

Val Tyr Phe Lys Asn Phe Thr Val Gly Glu Val Ala Lys His Asn Val

245 250 255

Thr Gly Lys Val Val Asp Ala Ser Gly Ala Ala He Ala Gly Ala Glu 260 265 270

Val Val Thr Gly Lys Asn Ser Ala Thr Thr Ala Ala Asp Gly Thr Phe 275 280 285

Thr Leu Thr Gly Leu Ala Ala Gly Asp Tyr Thr Leu Thr Val Ser Ala

290 295 300

Glu Gly Tyr Asp Asp Ala Thr Lys Thr Val Thr Val Ala Asp Gly Asn

305 310 315 320

Ala Ser Val Gly Asn He Thr Leu Asn Lys Ser Ala Glu Val Ala Thr

325 330 335

Glu Thr Leu Ser Thr Ala Ala Met Asp Val Arg Val Lys Lys Asn Phe 340 345 350

Pro Ser Val Tyr Asp Tyr Thr Met Lys Lys Phe Gly Gly Lys He Met 355 360 365

Tyr Gly Gin Pro Lys Asp Val Arg Val He Thr He Asn Gly Thr Asp 370 375 380 Val Thr Leu Lys Asp Ser Asp Val Thr Phe Lys Lys Val Ser Ala Thr

385 390 395 400

Glu Ala Gin Tyr Thr Leu Asn Val Lys Ser Gly Asn Lys lie Asn Ala

405 410 415

Val Val Thr Val Gin lie Lys Val Val Asp Asn Thr Leu Lys Leu Asn

420 425 430

Val Thr Lys He Val Asn Lys Ala Asp Asp Ala Lys Thr Glu Ala Glu

435 440 445

Glu Asn Pro Val Gin Thr He Ala Phe Pro Asn Gin Ser Leu He Ser

450 455 460

Val Arg Ser Gly Gin Asp Gly Ala Gin Phe Thr Gly Ala Arg Met Ser

465 470 475 480

Ser Asp Thr Ala Arg Pro Gly Asp Thr Asn Phe Gly He Thr Ala Asp

485 490 495

Thr Thr Val Gly Asn Ala Asn Asp Tyr Thr Tyr Gly Phe Val Ser Gly

500 505 510

Asn Gly Leu Ser Ala Gly Leu Trp Ser Asn Ser Glu His Asp Gly Thr

515 520 525

Thr Val Gly Asn Thr Val Ala Gly Gly Ala Arg Asn Thr Arg Val Leu

530 535 540

Thr Ser Thr Gin Lys Val Gly Lys Ala Thr Ser Phe Gly Leu Gly Thr

545 550 555 560

Ala Pro Trp Tyr Tyr His Arg Val Val Thr Asp Thr Lys Lys Arg Thr

565 570 575

Tyr Thr Val Glu Glu Thr Asp Met Pro Lys Met Ala Val Ala He Ala

580 585 590

Gly Asp Glu Asn Glu Asp Gly Thr Val Asn Trp Glu Asp Gly Ala He

595 600 605

Ala Tyr Arg Asp He Met Asn Asn Pro Tyr Lys Ser Glu Glu Val Pro

610 615 620

Glu Leu Val Ala Trp Arg He Ala Met Asn Phe Gly Ser Gin Ala Gin

625 630 635 640

Asn Pro Phe Leu Thr Thr Leu Asp Asn Val Lys Lys Val Ala Leu Asn

645 650 655 Thr Asp Gly Leu Gly Gin Ser Val Leu Leu Lys Gly Tyr Gly Asn Glu

660 665 670

Gly His Asp Ser Gly His Pro Asp Tyr Gly Asp lie Asn Thr Arg Ala

675 680 685

Gly Gly Ala Ala Asp Met Asn Thr Leu Met Glu Lys Gly Thr Glu Tyr

690 695 700

Gly Ala Arg Phe Gly Val His Val Asn Ala Ser Glu Met Tyr Pro Glu

705 710 715 720

Ala Lys Ala Phe Ser Glu Asp Met Val Arg Arg Asn Ser Ser Gly Gly

725 730 735

Leu Ser Tyr Gly Trp Asn Trp Leu Asp Gin Gly lie Gly He Asp Gly

740 745 750

He Tyr Asp Leu Ala Ser Gly Met Arg Lys Ser Arg Phe Ala Asp Leu

755 760 765

Lys Ser Lys Val Gly Asp Asn Met Asp Phe He Tyr Leu Asp Val Trp

770 775 780

Gly Asn Asn Thr Ser Gly Ala Glu Asp Ser Trp Glu Thr Arg Lys Met

785 790 795 800

Ser Gin Met He Asn Gin Asn Gly Trp Arg Met Thr Thr Glu Trp Gly

805 810 815

Ala Gly Asn Glu Tyr Asp Ala Thr Phe Gin His Trp Ala Ala Asp Leu

820 825 830

Thr Tyr Gly Gly Ser Gly Met Lys Gly Glu Asn Ser Gin Val Met Arg

835 840 845

Phe Leu Arg Asn His Gin Lys Asp Ser Trp Val Gly Asp Tyr Pro Ser

850 855 860

Tyr Gly Gin Ala Ala Asn Ala Pro Leu Leu Gly Gly Tyr Ser Met Lys

865 870 875 880

Asp Phe Glu Gly Trp Gin Gly Arg Asn Asp Tyr Ala Ala Tyr He Arg

885 890 895

Asn Leu Tyr Thr His Asp Val Ser Thr Lys Phe He Gin His Phe Lys

900 905 910

Val Val Arg Trp Val Asn Ser Pro Leu Asp Ala Thr Ser Val Lys Asp

915 92 0 9 25 Ala Ser Val Asn Asn Gly Asn Glu Gin lie Thr Leu Lys Asp Asp His

930 935 940

Gly Asn Val Val Val Leu Ser Arg Gly Ser Asn Asp Thr Asn Asn Gly

945 950 955 960

Ala Tyr Arg Asn Arg Thr lie Thr Leu Asn Gly He Thr Val Ala Ser

965 970 975

Gly Ala Val Ser Pro Gly Asn Ser Asn Thr Val Lys Gly Thr Glu Ser

980 985 990

Tyr Leu Leu Pro Trp Leu Trp Asp Val Asn Thr Gly Lys Leu Val Lys

995 1000 1005

Ser Ser Asp Glu Lys Leu Tyr His Trp Asn Thr Gin Gly Gly Thr Thr

1010 1015 1020

Glu Trp Thr Leu Pro Lys Asp Trp Gin Asn Leu Ala Ser Val Lys Val

1025 1030 1035 1040

Tyr Gin Leu Thr Asp Gin Gly Lys Thr Asn Glu Lys Thr Val Ala Val

1045 1050 1055

Ser Gly Gly Lys He Ser Leu Thr Ala Glu Ala Glu Thr Pro Tyr Val

1060 1065 1070

Val Thr Lys Gly Ser Glu Lys Gin He Ser Val Lys Trp Ser Glu Gly

1075 1080 1085

Met His Val Val Asp Ala Gly Phe Asn Gly Gly Gin Asn Thr Leu Lys

1090 1095 1100

Asp Asn Trp Ala Val Ser Gly Thr Gly Lys Ala Glu Val Glu Gly Thr

1105 1110 1115 1120

Asn Asn Ala Met Leu Arg Leu Thr Gly Asp Val Lys Val Ser Gin Lys

1125 1130 1135

Leu Thr Asp Leu Thr Ala Gly Lys Arg Tyr Ala He Tyr Val Gly Val

1140 1145 1150

Asp Asn Arg Thr Asn Ser Pro Ala Lys He Thr Val Thr Asn Gly Thr

1155 1160 1165

Lys Val Leu Ala Thr Asn Glu Thr Gly Lys Ser He Ala Lys Asn Tyr

1170 1175 1180

He Lys Ala Tyr Gly His Asn Thr Tyr Ser Asn Thr Glu Gly Gly Ser

1185 1190 119 5 1200 Ser Tug Phe Gin Asn Met Tyr Val Trp Phe Val Ala Pro Glu Ser Gly 1205 1210 1215

Asp Val Lys Val Thr Leu Ser His Ser Gly Ala Cys Asp Asn Thr Asp 1220 1225 1230

His Val Tyr Phe Asp Asp Val Arg Val Leu Glu Asn Gly Tyr Lys Gly 1235 1240 1245

Leu Thr Leu Asn Ala Asp Gly Thr Leu Lys Thr Leu Thr Asn Asp Phe 1250 1255 1260

Glu Asp Asn Ala Gin Gly lie Trp Pro Phe Val Val Ser Gly Ser Glu 1265 1270 1275 1280

Gly Val Glu Asp Asn Arg lie His Leu Ser Glu Leu His Ala Pro Phe 1285 1290 1295

Thr Gin Ala Gly Trp Asp Val Lys Lys Met Asp Asp Val Leu Asp Gly 1300 1305 1310

Lys Trp Ser Val Lys Ala Asn Gly Leu He Gin Lys Gly Thr Leu He 1315 1320 1325

Tyr Gin Thr He Pro Gin Asn Val Lys Leu Glu Pro Gly Glu Thr Tyr 1330 1335 1340

Lys Val Ser Phe Lys Tyr Gin Ser Gly Ser Asp Asp He Tyr Ala He 1345 1350 1355 1360

Ala Thr Gly Asp Gly Glu Tyr Asn Ala Ser Thr Val Lys Leu Thr Asn 1365 1370 1375

Leu Lys Lys Ala Leu Gly Glu Asp Gly Thr Ala Glu Phe Glu He Thr 1380 1385 1390

Gly Ser He Thr Gly Asp Ser Trp Phe Gly He Tyr Ser Thr Ser Thr 1395 1400 1405

Ala Pro Asp Leu Gin Asn Thr Ser Asp Ser Ala Ala Ala Phe Gly Gly 1410 1415 1420

Tyr Lys Asp Phe Val Leu Asp Asp Leu Lys Val Glu His Val Ala Ser 1425 1430 1435 1440

Ala Glu Arg Thr Lys Ala Asp Ala Glu Ala Lys Leu Lys Glu Val Lys 1445 1450 1455

Asp Thr Tyr Asn Gly Lys Ser Gly Asp Tyr Ser Ala Glu Val Trp Thr 1460 1465 1470 Thr Tug Val Asn Thr Val Ala Glu lie Glu Ala Leu lie Ala Lys Asp

1475 1480 1485

Lys Pro Asp Tyr Thr Thr Ala Tyr Asn Lys Ala Val Ala Leu Ala Glu

1490 1495 1500

Tyr Met Lys Asn Ala Pro Gly Asp Asp Ser Asn Asp Ala Tyr Asp Val

1505 1510 1515 1520

Ala Thr Asp Ala Tyr Thr Val Glu Ala Gly Ser Gin Gin Ala Leu Ser

1525 1530 1535

Gly Gly Asn Glu Gly Pro Ala Ser Leu Ala Gin Asp Gly Asn Ala Gly

1540 1545 1550

Thr His Trp His Thr Ser Trp Ser Ala Asn Ala Val Ser Ala Gly Thr

1555 1560 1565

Ala Trp Tyr Gin Phe Asn Leu Asn Glu Pro Thr Thr He Asp Gly Leu

1570 1575 1580

Arg Tyr Met Ala Arg Ser Gly Ser Ala Asn Ala Asn Gly Lys He Lys

1585 1590 1595 1600

Lys Tyr Lys He Thr Leu Thr Leu Ser Asp Gly Thr Thr Lys Asp Val

1605 1610 1615

Val Thr Asn Gly Thr Phe Thr Thr Thr Ser Gly Val Trp Gin Lys Val

1620 1625 1630

Lys Phe Asp Ala Val Lys Asn Val Thr Lys Val Arg He Thr Ala Leu

1635 1640 1645

Glu Thr Ala Gly Gin Ser Ala Gly Glu Val Asn Thr Tyr Ala Ser Ala

1650 1655 1660

Ala Glu Leu Arg Val Thr Thr Val Arg Asp Val Pro Ser Thr Glu Val

1665 1670 1675 1680

Lys Val Asn Lys Cys Asp Leu Gin Asn Leu Tyr Asp Asp Ala Ser Ala

1685 1690 1695

Leu Thr Glu Ala Thr Tyr Thr Ala Asp Thr Trp Lys Val Leu Val Ala

1700 1705 1710

Lys Arg Asp Ala Ala Lys Lys Val Leu Asp Asp Glu Asn Ala Thr Ala

1715 1720 1725

Tyr Asp Val Ala Leu Ala Tyr Gin Asn Leu Lys Asp Ala He Ala Ala

1730 1735 1740 Leu Glu Glu Arg Val Asp Thr Ser Lys Leu Ala Gly Leu Val Ala Asp 1745 1750 1755 1760

Ala Glu Lys Leu Lys Glu Ser Ala Tyr Thr Lys Asp Ser Trp Ala Ala 1765 1770 1775

Phe Lys Lys Ala Leu Asp Ala Ala Lys Ala Val Leu Asn Asn Ala Asn 1780 1785 1790

Ala Thr Lys Ala Asp Val Asp Ala Ala Tyr Asn Ala Leu Asn Ala Ala 1795 1800 1805

Met Lys Ala Leu Lys Pro Ala Ser Ser Lys Pro Thr Pro Asn Pro Glu 1810 1815 1820

Thr Thr Asp Lys Ser Lys Leu Gin Ala Thr lie Asp Gin Ala Lys Ala 1825 1830 1835 1840

Leu Asp Leu Ser Gly Tyr Thr Lys Lys Ser Val Gin Ala Val Arg Asp 1845 1850 1855

Ala Leu Ala Lys Ala Gin Ser Val Leu Ala Asp Asp Asn Ala Thr Gin 1860 1865 1870

Ala Asp lie Asp Ala Ala Gin Lys Ala Leu Ala Asp Ala He Ala Ala 1875 1880 1885

Leu Glu Lys Ala Asp Ala Asn Gly Asn Ala He Ser Lys Thr Gly Ala 189 0 189 5 1900

Asn Val Ala Val He Gly Met Ala Gly Met Met Leu Val Ala Ala Ala 1905 1910 1915 1920

Gly Ala Val Phe He Ala Arg Lys Arg Ala Glu

1925 1930

<210> 8 <211> 316 <212> PRT

<213> Bifidobacterium bifidum <400> 8

Met Cys Thr Gly Val Arg Phe Ser Asp Asp Glu Gly Asn Met Tyr Phe 1 5 10 15

Gly Arg Asn Leu Asp Trp Ser Phe Ser Tyr Gly Glu Thr He Leu Val

20 25 30

Thr Pro Arg Gly Tyr Gin Tyr Asp Tyr Val Tyr Gly Ala Glu Gly Lys 35 4 0 45

Ser Glu Pro Asn Ala Val lie Gly Val Gly Val Val Met Val Asp Arg

50 55 60

Pro Met Tyr Phe Asp Cys Ala Asn Glu His Gly Leu Ala lie Ala Gly

65 70 75 80

Leu Asn Phe Pro Gly Tyr Ala Ser Phe Ala His Glu Pro Val Glu Gly

85 90 95

Thr Glu Asn Val Ala Thr Phe Glu Phe Pro Leu Trp Val Ala Arg Asn

100 105 110

Phe Asp Ser Val Asp Glu Val Glu Glu Ala Leu Lys Asn Val Thr Leu

115 120 125

Val Ser Gin Val Val Pro Gly Gin Gin Glu Ser Leu Leu His Trp Phe

130 135 140

He Gly Asp Gly Thr Arg Ser He Val Val Glu Gin Met Ala Asp Gly

145 150 155 160

Met His Val His His Asp Asp Val Asp Val Leu Thr Asn Gin Pro Thr

165 170 175

Phe Asp Phe His Met Glu Asn Leu Arg Asn Tyr Met Cys Val Ser Asn

180 185 190

Glu Met Ala Glu Pro Thr Thr Trp Gly Lys Ala Glu Leu Ser Ala Trp

195 200 205

Gly Ala Gly Val Ser Met His Gly He Pro Gly Asp Val Ser Ser Pro

210 215 220

Ser Arg Phe Val Arg Val Ala Tyr Thr Asn Thr His Tyr Pro Gin Gin

225 230 235 240

Asn Asn Glu Ala Ala Asn Val Ser Arg Leu Phe His Thr Leu Val Ser

245 250 255

Val Gin Met Val Asp Gly Met Ser Lys Met Gly Asn Gly Gin Phe Glu

260 265 270

Arg Thr Leu Phe Thr Ser Gly Tyr Ser Gly Lys Thr Asn Thr Tyr Tyr

275 280 285

Met Asn Thr Tyr Glu Asp Pro Ala He Arg Ser Phe Ala Met Ser Asp

290 295 300

Phe Asp Met Asp Ser Ser Glu Leu He Thr Ala Asp

305 310 315 <210> 9 <211> 432 <212> PRT

<213> Bifidobacterium bifidum

<400> 9

Met Ala Ala lie Glu Ser Val Tyr Ala Arg Gin lie Leu Asp Ser Arg

1 5 10 15

Gly Asn Pro Thr Val Glu Val He Leu Asp Thr Glu Asp Gly Ala Glu 20 25 30

Gly Arg Gly Leu Val Pro Ser Gly Ala Ser Thr Gly Glu Ala Glu Ala 35 40 45

Trp Glu Arg Arg Asp Gly Asp Lys Ser Val Tyr Gly Gly Lys Gly Val 50 5 5 60

Leu Asn Ala Val Lys Ala Val Asn Glu Val He Ala Pro Lys Val He 65 70 75 80

Gly Met Asp Ala Thr Asp Gin Arg Ala Leu Asp Asp Leu Met He Glu

85 90 95

Leu Asp Gly Thr Pro Asn Lys Gly Lys Leu Gly Ala Asn Ala He Leu

100 105 110

Gly Val Ser Leu Ala Ala Leu Tyr Ala Ser Ala Glu Ser Ala Glu Leu

115 120 125

Pro Leu Tyr Arg Tyr He Gly Gly Thr Asn Gly His He Leu Pro Val

130 135 140

Pro Asn Met Asn He Met Asn Gly Gly Ala His Ala Asp Phe Ala Thr 145 150 155 160

Asp He Gin Glu Tyr Met He Ser Pro Tyr Gly Phe Asp Thr Tyr Ser

165 170 175

Glu Ala Leu Arg Ala Gly Val Glu Val Tyr His Thr Leu Lys Asn Val

180 185 190

Leu Lys Lys Glu Gly Leu Asn Thr Gly Leu Gly Asp Glu Gly Gly Phe 195 200 205

Ala Pro Lys Met Lys Ser Asn Glu Asp Ser Leu Lys Tyr He Met Asp

210 215 220

Ala He Ser Ala Ala Gly Tyr Glu Pro Gly Lys Gin He Gly He Cys

22 5 2 30 235 24 0 Leu Asp Val Ala Ser Ser Glu Phe Tyr Asn Lys Glu Thr Gly Lys Tyr

245 250 255

Arg Phe Asp Gly Glu Glu Arg Asp Ser Ala Tyr Met Leu Asp Tyr Tyr

260 265 270

Glu Asn Leu lie Asn Glu Tyr Pro lie Val Ser He Glu Asp Pro Phe

275 280 285

Asn Glu Glu Gly Trp Glu Asp Trp Ala Glu He Thr Arg Arg Leu Gly

290 295 300

Asp Arg Leu Gin Phe Val Gly Asp Asp Leu Leu Val Thr Asn Pro Ala

305 310 315 320

Arg Leu Arg Lys Ala He Asp Met Gly Ala Ala Asn Ser Leu Leu Val

325 330 335

Lys Leu Asn Gin He Gly Ser Val Thr Glu Thr Leu Asp Ala He Glu

340 345 350

Leu Ala Thr Glu Asn Gly Tyr Thr Ser Met Val Ser His Arg Ser Gly

355 360 365

Glu Thr Pro Asp Thr Thr He Ser Asp Leu Ala Val Ala Lys Asn Thr

370 375 380

Arg Gin He Lys Thr Gly Ala Pro Ala Arg Gly Glu Arg Val Ala Lys

385 390 395 400

Tyr Asn Arg Leu Leu Glu He Glu Glu Glu Leu Gly Ser Thr Ala Gin

405 410 415

Tyr Ala Gly Tyr Ser Ala Phe Lys Ala Cys Lys Lys Tyr He Ala Lys 420 425 4 30

Claims

CLAIM

1. Strain Bifidobacterium bifidum 8, collection number VKPM AC-2136 used as a probiotic.