Lactic acid bacteria isolated from mother's milk with probiotic activity and inhibitory activity against body weight augmentation

Abstract

The present invention relates to a lactic acid bacterium isolated from human mother's milk, more precisely a Lactobacillus gasseri BNR17 strain that is isolated from Korean mother's milk and has excellent probiotic activity including acid resistance, bile acid resistance and antimicrobial activity and weight gaining inhibitory effect as well. Again, the Lactobacillus gasseri BNR17 of the present invention has excellent acid resistance, bile acid resistance, enteric absorption activity and antimicrobial activity against pathogenic microorganisms, in addition to the weight gaining inhibitory effect by synthesizing indigestible polysaccharides from monosaccharides included in food taken and releasing the synthesized polysaccharides out of the body. Therefore, the strain of the invention, owing to such beneficiary effects, can be effectively used not only for the production of fermented milk, other fermented food products and animal feeds but also for the production of live cell products and food additives for preventing weight gaining.

Claims

.[.1. A Lactobacillus gasseri BNR17 strain of a biologically pure culture deposited at Korean Collection for Type Culture of Korea Research Institute of Biotechnology and Bioscience under the Accession number of KCTC 10902BP..].

.[.2. The Lactobacillus gasseri BNR17 strain according to claim 1, wherein the strain contains 16S rRNA sequence represented by SEQ ID NO: 1..].

.[.3. A composition containing an effective dose of the Lactobacillus gasseri BNR17 of claim 1..].

.[.4. The composition according to claim 3, wherein the composition is selected from the group consisting of food, food additive, animal feed and animal feed additive..].

.[.5. The composition according to claim 4, wherein the animal feed additive contains at least one selected from the group consisting of other non-pathogenic microorganisms, enzymes and a mixture thereof..].

.[.6. A pharmaceutical composition comprising an effective dose of the Lactobacillus gasseri BNR17 of claim 1..].

.[.7. A culture solution of the Lactobacillus gasseri BNR17 of claim 1..].

.[.8. A method for inhibiting weight gain comprising administering to a subject an effective dose of the Lactobacillus gasseri BNR17 of claim 1..].

.Iadd.9. A composition comprising Lactobacillus gasseri BNR17 strain in an enteric coating, wherein the L. gasseri BNR17 strain is the strain deposited at Korean Collection for Type Culture of Korea Research Institute of Biotechnology and Bioscience under the Accession number of KCTC 10902BP..Iaddend.

.Iadd.10. The composition of claim 9, which further comprises (a) one or more selected from the group consisting of acacia gum, calcium phosphate, alginate, tragacanth gum, calcium silicate, silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, and (b) one or more selected from the group consisting of a lactose, dextrose, sucrose, sorbitol, mannitol, starch, gelatin, and water..Iaddend.

.Iadd.11. The composition of claim 9, which is in a form of capsule or tablet..Iaddend.

.Iadd.12. The composition of claim 9, which further comprises one or more additional probiotic microorganism selected from the group consisting of Saccharomyces cerevisiae, Bacillus coagulans, Bacillus licheniformis, Bacillus subtilis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Enterococcus faecium, Enterococcus faecalis, Lactobacillus acidophilus, Lactobacillus alimentarius, Lactobacillus casei, Lactobacillus curvatus, Lactobacillus delbruckii, Lactobacillus johnsonii, Lactobacillus farciminus, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus sakei, Lactococcus lactis and Pediococcus acidilactici..Iaddend.

.Iadd.13. A dry powder comprising live Lactobacillus gasseri cells and a protectant, wherein the live Lactobacillus gasseri cells are of the Lactobacillus gasseri BNR17 strain deposited at Korean Collection for Type Culture of Korea Research Institute of Biotechnology and Bioscience under the Accession number of KCTC 10902BP, and wherein the protectant comprises powdered skim milk, whey, and/or sucrose..Iaddend.

.Iadd.14. A method of producing a product, comprising mixing a dry powder of claim 13 with at least one substance selected from the group a diluent, a high-fiber additive, an encapsulant, a lipid, an enzyme and a non-pathogenic microorganism, wherein the live Lactobacillus gasseri cells are of the Lactobacillus gasseri BNR17 strain deposited at Korean Collection for Type Culture of Korea Research Institute of Biotechnology and Bioscience under the Accession number of KCTC 10902BP; and wherein the protectant comprises powdered skim milk, whey, and/or sucrose..Iaddend.

.Iadd.15. A culture comprising a culture medium and Lactobacillus gasseri BNR17, wherein the culture is obtained by culturing the Lactobacillus gasseri BNR17 in a culture medium suitable for Lactobacillus gasseri BNR17; wherein the L. gasseri BNR17 strain is the strain deposited at Korean Collection for Type Culture of Korea Research Institute of Biotechnology and Bioscience under the Accession number of KCTC 10902BP; wherein the culture comprises an extracellular polysaccharide produced by the Lactobacillus gasseri BNR17; and wherein the culturing is carried out for 12-36 hours..Iaddend.

.Iadd.16. The culture of claim 15, wherein the culture medium is an MRS medium supplemented with 2% glucose..Iaddend.

.Iadd.17. A method for inhibiting growth of a pathogenic bacterium in intestine of a subject in need thereof comprising administering Lactobacillus gasseri BNR17 strain deposited at Korean Collection for Type Culture of Korea Research Institute of Biotechnology and Bioscience under the Accession number of KCTC 10902BP, to the subject, wherein the pathogenic bacterium is selected from the group consisting of E. coli, B. cereus, L. monocytogenes, P. mirabilis, S. aureus, and S. typhimurium..Iaddend.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:

(2) FIG. 1 is a diagram showing the sequence comparison between Lactobacillus gasseri BNR17 of the invention (upper sequence in figure, SEQ. ID. NO: 1) and Lactobacillus gasseri KC26 (NCBI GENBANK Accession No: AF243156, lower sequence in figure, SEQ. ID. NO: 2) 16s rRNAs.

(3) FIG. 2 is a microphotograph showing the enteric adherence of Lactobacillus gasseri BNR17 of the invention.

(4) FIG. 3 is a set of photographs showing that Lactobacillus gasseri BNR17 of the invention has antimicrobial activity against various pathogenic bacteria.

(5) FIG. 4 is a graph showing the glucose consumption and polysaccharide production according to the growth of Lactobacillus gasseri BNR17 of the invention. ; cell growth, .diamond-solid.; glucose concentration, ; EPS (polysaccharide) concentration

(6) FIG. 5 is a graph showing the amount of feces of a rat taking Lactobacillus gasseri BNR17 of the invention.

(7) FIG. 6 is a graph showing the EPS (polysaccharide) concentration in the feces of a rat taking Lactobacillus gasseri BNR17 of the invention.

(8) FIG. 7 is a set of electrophoresis photographs showing the RAPD-PCR profiles of colonies isolated from other organs than the small intestine of a rat taking Lactobacillus gasseri BNR17 of the invention, in which primers represented by SEQ. ID. NO: 7 (A), NO: 8 (B) and NO: 9 (C) were used.

(9) Lane 1; Lb. gasseri BNR17, Lanes 2-5; colonies isolated from other organs than the small intestine of a rat taking Lb. gasseri BNR17,

(10) M; DNA size marker.

EXAMPLES

(11) Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

(12) However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1

Lactic Acid Bacterium Isolation from Human Mother's Milk

(13) Human mother's milk was taken from a woman delivered of a baby not more than two weeks ago. Then, the mother's milk was diluted with PBS and the undiluted milk and diluted milk were distributed on a lactobacillus selection medium respectively. The medium was cultured at 37° C. for 2-3 days and the colonies generated therein were sorted by morphology and color. The isolated colonies were Gram-stained and observed under a microscope to select those colonies that were Gram-positive and had rod-shaped structure. The selected colonies were cultured in MRS liquid medium (pH 6.8) at 37° C. for 24 hours. Colonies in the culture solution under the pH lower than 4.5 were selected. The colonies were cultured in MRS medium (pH 2.0) for 2 hours, followed by further culture in MRS medium supplemented with 0.3% oxgall for 9 hours. The survived lactobacillus strain that exhibited acid resistance and bile acid resistance was isolated and identified by 16S rRNA sequencing. As a result, the strain was confirmed to belong Lactobacillus gasseri species (SEQ. ID. NO: 1, FIG. 1) and named as “Lactobacillus gasseri BNR17”.

Example 2

Sugar Utilization of the Isolated Lactic Acid Bacterium

(14) Sugar utilization of Lactobacillus gasseri BNR17 of the invention isolated above was investigated by comparing with other standard strains using API50CHL kit (Biomerieux, France) and the results are shown in Table 1. In Table 1, 5314 indicates Lactobacillus gasseri CECT5714; 5315 indicates Lactobacillus gasseri CECT5715; 11413 indicates Lactobacillus gasseri LMG11413; 18194 indicates Lactobacillus gasseri LMG18194; 4479 indicates Lactobacillus gasseri CECT4479; 18176 indicates Lactobacillus gasseri LMG 18176; and 13047 indicates Lactobacillus gasseri LMG13047.

(15) TABLE-US-00001 TABLE 1 BNR17 5714 5715 11413 18194 4479 18176 13047 Glycerol 0 0 0 0 0 0 0 0 Erythritol 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 4 0 0 0 0 0 Ribose 0 0 0 0 0 0 0 0 D-Xylose 0 0 0 0 0 0 0 0 L-Xylose 0 0 0 0 0 0 0 0 Adonitol 0 0 0 0 0 0 0 0 β-Methyl- 0 0 0 0 0 0 0 0 xyloside Galactose 5 5 5 5 5 5 5 5 D-Glucose 5 5 5 5 5 5 5 5 D-Fructose 5 5 5 5 5 5 5 5 D-mannose 5 5 5 5 5 5 5 5 0 0 0 5 0 0 0 0 Rhamnose 0 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 Inositol 0 0 0 0 0 0 0 0 0 0 3 5 0 0 0 0 Sorbitol 0 0 0 0 0 0 0 0 α-Methyl-D- 0 0 0 0 0 0 0 0 mannoside α-Methyl-D- 0 0 0 0 0 0 0 0 glucoside N-Acetlyglu- 5 4 5 5 5 5 5 5 cosamine 5 5 5 0 0 5 5 0 5 5 5 1 1 5 5 4 Esculine 5 5 5 5 5 5 5 5 Salicine 5 5 5 5 5 5 5 3 Cellobiose 5 5 5 5 5 5 5 5 Maltose 5 5 5 5 5 5 5 5 5 0 4 5 5 5 5 0 0 0 0 0 0 0 5 0 Saccharose 5 5 5 5 5 5 5 5 Trehalose 5 5 5 5 5 5 5 5 Inuline 0 0 0 0 0 0 0 0 Melezilose 0 0 0 0 0 0 0 0 D-Raffmose 0 0 0 0 0 0 5 0 5 3 5 1 1 5 3 3 0 0 0 4 4 0 0 0 Xylitol 0 0 0 0 0 0 0 0 b-Gentobiose 5 5 5 5 5 5 5 5 5 0 0 0 0 0 0 0 D-Lycose 0 0 0 0 0 0 0 0 5 5 5 3 3 5 5 5 D-Fucose 0 0 0 0 0 0 0 0 L-Fucose 0 0 0 0 0 0 0 0 D-Arabinol 0 0 0 0 0 0 0 0

(16) As shown in Table 1, compared with other lactobacillus strains, the Lactobacillus gasseri BNR17 of the invention was distinguishable in sugar utilization from others (shown in thick Italics).

Example 3

Enzyme Activity of the Isolated Lactic Acid Bacterium

(17) The enzyme activity of the Lactobacillus gasseri BNR17 isolated in Example 1 was compared with those of other standard strains using APIZYM kit (Biomerieux, France) and the results are shown in Table 2. In Table 2, 13134 indicates Lactobacillus gasseri LMG13134.

(18) TABLE-US-00002 TABLE 2 BNR17 11413 13047 13134 18176 18194 4479 5714 5715 Control − − − − − − − − − Alkaline phosphates − − − − − − − − − Esterase (C4) 1 2 1 1 1 1 1 1 1 Esterase lipase (C8) − − − 1 − − − − − Lipase (C4) − − − − − − − − − Leucine + + + + + + + + + arylamidase 1 1 1 3 2 − − − 1 1 1 1 4 2 3 4 1 1 Trypsin − − − − − − − − − α-chymotrypsin − − 1 − − − − − − + 1 2 + − 1 2 1 1 + 1 + + + − + + + + + − − + − + + + + 4 1 − + + − − − − − − 1 1 + − 1 2 1 − 1 3 + + 3 4 + 1 + 1 − + − − − 1 α-mannosidase − − − − − − − − α-fiteesidase − − − − − − − − −

(19) As shown in Table 2, the Lactobacillus gasseri BNR17 of the invention was distinguishable in enzyme activity from other strains (shown in thick Italics).

Example 4

Acid Resistance and Bile Acid Resistance

(20) To investigate acid resistance and bile acid resistance of the strain of the invention, Lactobacillus gasseri BNR17 was inoculated in 4 ml of MRS liquid medium and cultured at 37° C. for 18-20 hours. Some of the culture solution was reinoculated in another MRS medium with regulated pH of 2.0 at the concentration of 10.sup.7 CFU/ml and cultured at 37° C. for 2 hours. The number of live cells was counted using MRS agar plate. The culture solution tested for acid resistance was used again for centrifugation. The cells were recovered and inoculated in MRS liquid medium (pH 6.8) supplemented with 0.3% oxgall, followed by culture at 37° C. for 9 hours. The number of live cells was also counted using MRS agar plate. The results are shown in Table 3.

(21) TABLE-US-00003 TABLE 3 Before Treatment at 0.3% expall treatment pH 2.0 treatment BNR17 3.1 × 10.sup.7 2.1 × 10.sup.7 1.5 × 10.sup.7

(22) As a result, even after the treatment with strong acid (pH 2.0), the Lactobacillus gasseri BNR17 exhibited high survival rate, and so did in the medium supplemented with 0.3% oxgall.

Example 5

Enteric Adherence

(23) The strain of the invention was inoculated on the plate on which a human intestinal epithelial cell line CaCo-2 was cultured in PRMI1640 (Gibco) at the concentration of 10.sup.7 CFU/ml. The strain was cultured at 37° C. for one hour, followed by washing three times with PBS to eliminate non-adhered cells. The sample was fixed with methanol and then stained with crystal violet, followed by observation under a microscope. As a result, the Lactobacillus gasseri BNR17 of the invention was confirmed to be very well adhered on CaCo-2 cells (FIG. 2).

Example 6

Antimicrobial Activity Against Pathogenic Bacteria

(24) E. coli, S. aureus, S. typhimurium, B. cereus, L. monocytogenes and P. mirabilis were cultured at 37° C. for 18 hours in BHI liquid medium (Difco) and then inoculated in 6 of 5 ml BHI agar media (agar content: 0.7%) respectively at the concentration of 10.sup.5 CFU/ml. These media were overlapped on 6 plates with BHI agar media (agar content: 1.5%) fixed thereon. After hardening those 6 plate media, a well of 4 mm in diameter was made in each medium, in which 40 μl of the supernatant (2×) of lactic acid bacterium culture solution cultured at 37° C. for 24 was added, followed by culture at 37° C. for 5 hours.

(25) As a result, a clear growth inhibition ring was observed around the well, suggesting that the strain of the invention has antimicrobial activity against various pathogenic bacteria (Table 4 and FIG. 3).

(26) TABLE-US-00004 TABLE 4 Diameter of growth Pathogenic bacteria inhibition ring (mm) E. coli KCTC1039 16 B. cereus KCTC1526 16 L. monocytogenes KCTC3710 12 P mirabilis KCTC2510 14 S. aureus KCTC1928  6 S. typhimurium KCTC2421 18

Example 7

Antibiotic Resistance

(27) Lactobacillus gasseri BNR17 culture solution was smeared on MRS agar plate by using a swab, on which a disc containing erythromycin, penicillin, gentamycin, kanamycin, streptomycin, bacitracin, chloramphenicol, vancomycin, tetracycline, ampicillin, cefoxitin, rifampin, neomycin, nalidixic acid, ciprofloxacin, polymixcin B or trimethoprim was placed, followed by culture at 37° C. for 24 hours.

(28) As a result, Lactobacillus gasseri BNR17 of the invention was confirmed to have resistance against gentamycin, streptomycin and trimethoprim.

Example 8

Detection of the Antimicrobial Peptide Gene

(29) Bacteriocin gene was investigated by PCR performed by using the Lactobacillus gasseri BNR17 genomic DNA as a template and primers represented by SEQ. ID. NO: 3 and NO: 4 which were specific to the nucleotide sequence of a gene of bacteriocin known as an antimicrobial peptide produced by Lactobacillus gasseri species.

(30) As a result, the PCR product corresponding to gassericin was confirmed and represented by SEQ. ID. NO: 5. The nucleotide sequence was compared with that of gassericin T (NCBI Blast Search No. AB029612) represented by SEQ. ID. NO: 6 and confirmed to have approximately 98% homology.

Example 9

β-glucuronidase Activity

(31) β-glucuronidase produced by enterobacteria has been known as one of oncogenic enzymes and thus the strain that has this enzyme activity is considered as a harmful strain. To investigate whether the Lactobacillus gasseri BNR17 of the invention has β-glucuronidase activity or not, the enzyme activity of Lactobacillus gasseri BNR17 was tested using API ZYM kit (Biomerieux, France).

(32) As a result, the strain of the invention was confirmed not to have β-glucuronidase activity, suggesting that the strain was safe (Table 5).

(33) TABLE-US-00005 TABLE 5 Enzyme Activity α-galactosidase Positive β-galactosidase Positive β-glucuronidase Negative α-glucosidase Negative β-glucosidase Positive

Example 10

Glucose Consumption and Polysaccharide Production

(34) Lactobacillus gasseri BNR17 was inoculated in MRS medium (Difco) prepared by adding 2% glucose (w/v) at the concentration of 10.sup.6 cfu/ml and cultured for 96 hours, during which the cell number was measured stepwise and at the same time glucose consumption and extracellular polysaccharide (EPS) production were measured as well.

(35) As a result, the highest level of Lactobacillus gasseri BNR17 was observed on the 12.sup.th hour of culture and since then the level had been decreased. Glucose concentration was rapidly reduced after 7 hours of culture and no changes of glucose concentration were detected after 36 hours, suggesting that most of glucose was consumed within 36 hours from the culture started. EPS production was maximized on the 24.sup.th hour (the highest level: 520 mg/k) and was slightly reduced on the 36.sup.th hour but increased again thereafter. It was presumed to be attributed to autolysis of the cells causing various polysaccharides in the cells to be released into the culture solution (FIG. 4).

Example 11

Decomposition of the Polysaccharide Produced by Lactobacillus Gasseri BNR17 by a Digestive Enzyme

(36) 100 mg of each α-amylase (Sigma) and pancreatin (Sigma) was dissolved in 0.05 M phosphate buffer (pH 7.0). 50 μl of the above enzyme solution and 150 μl of 0.05 M phosphate buffer (pH 7.0) were added to 200 μl of polysaccharide (EPS) solution extracted from the supernatant of Lactobacillus gasseri BNR17 culture solution, followed by reaction at 37° C. for one hour. The reaction mixture was heated at 100° C. for 15 minutes to inactivate enzymes therein, followed by cooling at room temperature. Glucose concentration was measured with a glucose kit (Sigma).

(37) As a result, glucose was not detected in the polysaccharide solution before the treatment of each digestive enzyme, while 3.70 mg/l and 19.1 mg/l of glucose were respectively detected after the treatment of pancreatin and α-amylase. This result indicates that the polysaccharide produced by Lactobacillus gasseri BNR17 was hardly decomposed by a digestive enzyme.

Example 12

Weight Gaining Inhibitory Effect of Lactobacillus Gasseri BNR17

(38) 8 week old male SD rats were grouped into two. One group was orally administered with PBS only (pH 7.4) and the other group was orally administered with PBS suspended with 10.sup.9 CFU/ml of Lactobacillus gasseri BNR17, everyday for 8 weeks. Changes of weights, food intakes, and blood chemical values such as cholesterol level were measured once a week. The amounts of feces and EPS in feces were also measured to investigate the relation of weight gaining inhibitory effect of Lactobacillus gasseri BNR17 and polysaccharide production capacity thereof. 8 weeks later, all the test animals were sacrificed and dissected to extract the liver, kidney, spleen, MLN (mesenteric lymph node), which were measured their weights. Some of each organ extracted was homogenized and smeared on LBS agar, a lactobacillus selection medium, which was then cultured and RAPD (random amplified polymorphic DNA)-PCR profiles of the generated colonies were investigated. The result was compared with the RAPD-PCR profile of Lactobacillus gasseri BNR17 to investigate whether the strain was transferred to other organs.

(39) As a result, approximately 179.1% weight increase was observed for 8 weeks in the control group orally administered with PBS only, while approximately 171.6% weight increase was observed in the experimental group orally administered with Lactobacillus gasseri BNR17 (Table 6). The experimental group also exhibited lower rates of one-day weight increase and food efficiency ratio than the control.

(40) TABLE-US-00006 TABLE 6 Weight Food Weight (g) gaining efficiency Group Initial weight Final weight* (g/day) ratio** Control 221.20 ± 3.759  393.73 ± 4.860  3.081 0.131 ± 0.078 BNR17 223.66 ± 10.077 380.85 ± 21.517 2.807 0.115 ± 0.067

(41) In Table 6, food efficiency ratio (FER) indicates weight gaining (g day)/food intake (g day). *P<0.05, **P<0.05.

(42) The results of measuring the amounts of feces and EPS in feces of both the control and the experimental groups are shown in FIG. 5 and FIG. 6. The amounts of feces were not much different between the control and the experimental groups, but the ESP amount was significantly increased in the experimental group administered with Lactobacillus gasseri BNR17. This result indicates that Lactobacillus gasseri BNR17 converts sugar components taken inside body into indigestible polysaccharides so as to release the polysaccharides out of the body, resulting in the decrease of in vivo absorption rate and inhibition of weight gaining.

(43) To examine safety of the strain for human administration, blood chemical values and organ weights were measured. Each levels and values were similar in the control and the experimental groups, suggesting that the strain did not cause side effects (Table 7 and Table 8).

(44) TABLE-US-00007 TABLE 7 Cholesterol Glucose HDL LDL Total protein Triglyceride Group (mg/dL) (mg/dL) (mg/dL) (mg/dL) (OL) (mg/dL) Control 99.7 ± 11.7 88.9 ± 12.1 38.5 ± 3.5 21.5 ± 1.6 8.6 ± 0.3 115.9 ± 11.0 BNR17 96.3 ± 6.3  79.2 ± 4. 1 40.8 ± 1.8 20.8 ± 3.1 9.0 ± 0.3 132.6 ± 5.5 

(45) TABLE-US-00008 TABLE 8 Group Liver Kidney Spleen Control 0.029 ± 0.001 0.007 ± 0.000 0.002 ± 0.000 BNR17 0.027 ± 0.003 0.007 ± 0.001 0.002 ± 0.000

(46) In Table 8, each number indicates organ weight (g)/rat weight (g).

(47) To investigate whether the strain was transferred to other organs, RAPD-PCR profiles of colonies of each organ tissue cultured on LBS agar plate were investigated by using primers p1, p2 and OPL5 respectively represented by SEQ. ID. NO: 7-NO: 9. PCR using the primers p1 and p2 was performed as follows; 94° C. (2 minutes), 36° C. (5 minutes), 72° C. (5 minutes)—4 cycles/94° C. (1 minute), 36° C. (1 minute), 72° C. (2 minutes)—36 cycles. PCR using the primer OPL5 was performed as follows; 94° C. (2 minutes)—1 cycle/94° C. (40 seconds), 45° C. (1 minute), 72° C. (1 minute)—2 cycles/94° C. (40 seconds), 52° C. (1 minute), 72° C. (3 minutes)—30 cycles/70° C. (5 minutes)—1 cycle.

(48) As a result, no colonies exhibited similar profiles to BNR17 (FIG. 7). Thus, BNR17 was confirmed to be safe strain which is not transferred to other organs except the small intestine when it is taken.

Manufacturing Example 1

Preparation of Fermented Milk

(49) Raw milk in which milk solid non fat content was regulated by 8-20% using powdered skim milk was sterilized at 72-75° C. for 15 seconds. The sterilized raw milk was cooled down to the proper temperature, to which Lactobacillus gasseri BNR17 of the invention was inoculated at the concentration of 10.sup.6 cfu/ml, followed by culture until pH reached 4-5. Upon completion of the culture, the culture solution was cooled down. In the meantime, 0.1-50 weight % of fruit juice concentrate, 0.1-20 weight % of dietary fiber, 0.5-30 weight % of glucose, 0.1-15 weight % of oligosaccharide, 0.01-10 weight % of calcium and 0.001-5 weight % of vitamin were all dissolved to prepare syrup. The syrup was sterilized, cooled down, and mixed with the above culture solution, followed by stirring for homogenization. The resultant mixture was packed, resulting in the preparation of fermented milk. Flavor, physical property, and taste of the fermented milk product were tested, and the results were satisfactory.

Manufacturing Example 2

Preparation of Lactic Acid Bacteria Powder

(50) Lactobacillus gasseri BNR17 of the invention was inoculated into MRS liquid medium at the concentration of 10.sup.6 cfu/ml, followed by pH-control fermentation at 37° C. for 18-24 hours. pH-control was performed by using 30 volume % NaOH as a neutralizing agent to pH 5.7±0.2. Upon completion of the culture, centrifugation was performed at 4° C. with 10,000×g to recover cells. A protectant supplemented with 5 weight % of skim milk, 2.5 weight % of whey, and 5 weight % of sucrose (for the total weight of the composition) was prepared. Equal amounts of the recovered cells and the protectant were mixed, followed by pulverization by using a freeze dryer. The produced Lactobacillus gasseri BNR17 dried powder contained over 1×10.sup.11 cfu/g live cells. The protectant can additionally include 10 weight % of trehalose, 10 weight % of maltodextrine and 7.5 weight % of lactose.

Manufacturing Example 3

Preparation of Lactic Acid Bacteria Products

(51) Lactic acid bacteria products such as lactic acid bacteria foods and digestives were prepared from the lactic acid bacteria powder produced in Manufacturing Example 2. 10 weight % of oligosaccharide, 20 weight % of anhydrous glucose, 5 weight % of crystalline fructose, 2 weight % of vitamin C, 5 weight % of fruit powder flavor, 5 weight % of aloe, 15 weight % of dietary fiber, and 18 weight % of Psyllium Husk were added to 20 weight % of Lactobacillus gasseri BNR17 dried powder, and the mixture was packed in sticks or bottles. The live cells in the lactic acid bacteria product prepared thereby were more than 5×10.sup.8 cfu/g.

Manufacturing Example 4

Preparation of a Composition for Feed Additive

(52) A composition for feed additive containing Lactobacillus gasseri BNR17 was prepared by the following compositions shown in Table 9.

(53) TABLE-US-00009 TABLE 9 Component ratio of the composition for feed additive (weight %) Lacto- Non- bacillus Enzyme pathogenic gasseri prepa- micro- Amino BNR17 ration organism acid Others Manufacturing 100 — — — — Example <4-1> Manufacturing  90 10 — — — Example <4-2> Manufacturing  80 10 10 — — Example <4-3> Manufacturing  70 10 10 10 — Example <4-4> Manufacturing  60 15 15  8 2 Example <4-5> Manufacturing  50 20 15  8 2 Example <4-6>

(54) The enzyme preparation used herein was a mixture of phytase, cellulase, xylase, maltase and invertase, and the non-pathogenic microorganism was Aspergillus oryzae.

INDUSTRIAL APPLICABILITY

(55) As explained hereinbefore, the Lactobacillus gasseri BNR17 of the invention has wide growth temperature and pH ranges allowed. And, the strain of the invention not only has excellent acid resistance, bile acid resistance and enteric adsorption capacity but also strong antimicrobial activity against pathogenic microorganisms, in addition to weight gaining inhibitory effect. Therefore, the strain of the invention can be effectively used for the production of fermented milk and other fermented products and be very useful as an additive for animal feed as well.

SEQUENCE LISTING

(56) Sequence listing is attached herewith.