KR101768678B1 - Bifidobacterium longum ssp. infantis BI9988 isolated from Korean longevity village and having high nutraceutical activities - Google Patents

Abstract

The novel Bifidobacterium ronggeom subspecies Infante tooth (Bifidobacterium longum ssp. Infantis) BI9988 strain or culture fluid thereof is not a risk of acid and bile is excellent and antibiotic resistance transition, intestinal cell adhesion function, immune increase of the invention (Macrophage activity performance), inhibition of food poisoning bacteria, and cholesterol digestion ability. Therefore, the strain and the culture thereof are useful as a prophylactic or lactobacillus preparation, a fermented milk product, a health supplement, a feed additive, And can be used as a health functional additive for various products such as cosmetics.

Description

Bifidobacterium longomum subspecies BI9988 {Bifidobacterium longum ssp. infantis BI9988 isolated from Korean longevity village and having high nutraceutical activities}

The present invention relates to novel Bifidobacterium longum subspecies ( Bifidobacterium spp.) Isolated from a long-lived elderly person living in a longevity village in Korea, which helps promote health functions such as intestinal function activation, immune enhancement and cholesterol suppression longum ssp. infantis ).

Most of the lactic acid bacteria that have been commercialized and sold in Korea are lactic acid bacteria that have been developed separately from westerners or their foods, and lactic acid bacteria that are not found in the intestines of everyday Korean people. Therefore, we have developed a Korean lactic acid bacterium by isolating the lactic acid bacteria from adults who are living in a typical long belt of Korea and have healthy intestinal activity, and identify the various functions of the lactic acid bacteria as a probiotic, , Which is a problematic antibiotic resistant metastatic disease, has been developed and used as an effective probiotic agent.

Probiotics are defined as single or multiple strains of live microorganisms that have beneficial effects on the host by improving intestinal microbial properties when ingested in modest amounts. However, recently, Salminen et al. (Salminen et al., 1999. Probiotics: how should they be defined , Trends in food Science & Technology, 10, 107-110), a living microbial formulation contained in food and feed or dietary supplements designed to promote the health of humans and animals.

Probiotic lactic acid bacteria are significantly Lactobacillus bacteria (Lactobacillus), Lactococcus (Lactococcus), Streptococcus (Streptococcus), Lou Kono Stock (Leuconostoc), Peddie Oh Caucus (Pediococcus), and separated by Bifidobacterium (Bifidobacterium), ministers and urinary It promotes human health by preventing the microorganism contamination of the reproductive system and maintaining the intestinal health, constipation relief, inhibition of the growth of harmful bacteria, anticancer, immunity enhancement, cholesterol reduction, production of conjugated linoleic acid (CLA) and inhibition of Helicobacter pylori It plays an important role.

Preferable conditions for the probiotics should be safety first, tolerance to acid and bile, adherence to the intestinal epithelium, inhibition against pathogenic bacteria, , Should have an immunostimulating effect and should not cause an antibiotic resistant metastasis.

Bacteria have antibiotic resistance in two ways. The first is tolerance inherent to the patient without being metastasized by the inherent resistance. For example, most lactic acid bacteria, except Lactobacillus acidophilus, exhibit resistance to vancomycin due to a thick cell wall structure and a thick cell wall, and this inherent resistance is not caused by metastasis Do not transfer resistance genes to other bacteria. The second is resistance to extrinsic resistance. Exogenous resistance refers to the tolerance that a resistant gene binds to a plasmid or a transposon and enters into other external bacterium. In this case, the resistance gene of this resistant bacterium easily transmits tolerance to other bacteria.

Previously, resistant lactic acid bacteria were known to be good, and antibiotic-resistant lactic acid bacteria that survived by treating various antibiotics were considered to be good lactic acid bacteria. Now, resistance genes of lactic acid bacteria resistant to various antibiotics are transferred to intestinal bacteria, It is reported that it causes. In the western countries, the antibiotic-resistant lactic acid bacteria have been unable to commercialize all of the resistant genes that have been transferred for a long time (Clinical Laboratory Standard Institute, CLSI, USA).

Since Bifidobacterium was isolated by Tisser in 1899, Bifidobacteria are considered to be beneficial to the human body, such as normal functioning of bowel movements and intestinal colonies (Benno, Y., K. Sawada and T. Mitsuoka. 1984 The intestinal microflora of infants, composition of fecal flora in breast-fed and bottle-fed infants. Microbiol. Immunol. 28: 975-986). The physiological characteristics of Lactobacillus bifidus are similar to that of Lactobacillus and are called Lactobacillus bifidus (Weiss, JE and Rettger LF 1938. Lactobacillus bifidus . J. Bacteriology. 28: 501-527) Currently Bifidobacterium It is classified separately.

Bifidobacteria, an anaerobic intestinal microorganism distributed in the intestines of adults, are lactic acid bacteria that exist more than 1,000 times more than Lactobacillus and Streptococcus. Fructose-6-phosphate phosphoketolase (FPPK) ) And the specific metabolic system (Scardovi, V. and LD Trovatelli 1965. The fructose-6-phosphate shunt as a peculiar pattern of hexose degradation in genus Bifidobacterium. Ann. Microbiol. Enzymol. 15: 19-29) (Collins, EB and BJ Hall 1984. Growth of bifidobacteria in milk and preparation of Bifidobacterium infantis for a dietary adjunct J. Dairy Sci. 7: 1376-1380) and produces hydrogen peroxide (H ₂ O ₂ ) and useful antibiotics to prevent intestinal diseases. In addition, it is nutritionally helpful to synthesize vitamins (Shahani, KM and RC Chandan, 1979. Nutritional and healthful aspects of cultured and culture-cintaining dairy foods, J. Dairy Sci. 62: 1685-1694) , It is considered to be a beneficial strain for our health because there is little production of toxic substances such as ammonia, amine and hydrogen sulfide. Recently, many studies have been carried out on the activation of immune functions such as anticancer activity and host immune system (Sasaki, T., S. Fukami, and S. Namioka. 1994. Enhanced resistance of mice to Esherichia coli infection induced administration of peptidoglycan derived from Bifidobacterium thermophilum . J. Vet. Med. Sci. 53: 433-437). Recently, bifidobacteria having major physiological functions have been widely used for dairy products and formal products as well as lactic acid bacteria, lactobacilli and intestinal bacteria, which have been used before as a material for functional foods.

Accordingly, the present inventors have made efforts to find Bifidobacteria which are safe and resistant to acid, biliary, immune and cholesterol. As a result, it has been found that Bifidobacterium Bifidobacterium long submucosal bacterium isolated from the bacterium belonging to the genus Bacillus subtilis, which is known as intestinal toxic enzyme, is capable of binding to the colon epithelium, Bifidobacterium strains with excellent antibiotic resistance and excellent cholesterol digestibility, such as Bifidobacterium longum ssp. detailing infantis BI9988 was by checking that you can use it as a lactic acid bacteria preparation, fermented milk products, dietary supplements, animal feed additives, additives of various products such as pharmaceuticals, cosmetics and probiotics have completed the present invention.

It is an object of the present invention to provide novel Bifidobacterium longum subphylum isolates ( Bifidobacterium longum ssp. infantis ).

It is still another object of the present invention to provide a novel biocidal composition comprising a novel Bifidobacterium longum subspecific strain or a culture thereof as an active ingredient, and a health food for enhancing and enhancing bowel activity.

In order to achieve the above object, the present invention relates to novel Bifidobacterium subspecies ronggeom Infante tooth (Bifidobacterium longum ssp. infantis ).

Also, the present invention provides a novel biocidal composition comprising Bifidobacterium longanum subsp. Botulinum BI9988 or a culture thereof as an active ingredient.

In addition, the present invention relates to novel Bifidobacterium longum subsp. Botulinum toxin BI9988, or a health functional food for enhancing bowel activity and inhibiting harmful bacteria in the intestines, containing the active ingredient thereof as an active ingredient, for improving the health of the bowel, enhancing immunity and inhibiting cholesterol .

The novel Bifidobacterium ronggeom subspecies Infante tooth (Bifidobacterium longum ssp. Infantis) BI9988 strain or culture fluid thereof is not a risk of acid and bile is excellent and antibiotic resistance transition, intestinal cell adhesion function, immune increase of the invention (Macrophage activity performance), inhibition of mutation, inhibition of food poisoning bacteria, and cholesterol digestion ability. Therefore, the strain and the culture thereof are useful as a prophylactic or lactic acid bacterial preparation, a dairy product, a health supplement, a feed additive , Pharmaceuticals, cosmetics, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. longum ssp. Photo of a Gram stain infantis BI9988.
Figure 2 shows that Bifidobacterium longum ssp. an electron micrograph infantis BI9988.
Figure 3 shows that Bifidobacterium longum ssp. After Gram staining that the infantis BI9988 is bonded to the sheet cell line, HT29-MTX cells is a photograph observed by an optical microscope.
Fig. 4 shows the results of the analysis of Bifidobacterium longum ssp. is a diagram showing the sequence of the 16S rRNA infantis BI9988.

Hereinafter, the present invention will be described in detail.

The present invention provides a novel Bifidobacterium subspecies ronggeom Infante tooth (Bifidobacterium longum ssp. Infantis) strain.

This strain has a 16S rRNA sequence represented by SEQ ID NO: 1, deposited as accession number of KCTC 12747BP Bifidobacterium subspecies ronggeom Infante tooth (Bifidobacterium longum ssp. that the infantis) BI9988 is preferred.

In a specific embodiment of the present invention, the present inventors have found that in order to isolate and identify bifidobacteria with excellent acid and bile resistance, the elderly people aged 80 years and over who live in the longevity village of the experimental subject, The feces were collected and sequentially diluted with sterilized saline at ten times the volume, and the strains of Bifidobacterium were isolated through anaerobic culture. Bifidobacterium produces acetic acid and lactic acid at a ratio of 1.5: 1 from glucose via a specific metabolic system of fructose-6-phosphate phosphoketolase (F-6-PPK) 6-PPK-positive strains were identified as Bifidobacterium. The fermentation patterns of the selected strains were determined by using API 50 CHL identification kit (bioMerieux Vitex, Inc, France) Bacteriology and Roy and Ward (Roy, D. and P. Ward, 1990. Evaluation of Rapid Methods for Differentiation of Bifidobacterium Species, J. Appl. Bacteriol. 69: 739-749). Finally, the nucleotide sequence of the 16S rRNA gene was identified by analyzing the Microseq 16S rRNA gene kit (Perkin Elmer Applied Biosystem). The strain was stained with Gram stain, and the morphology was confirmed by an optical microscope and an electron microscope. Typical examples of Bifidobacterium (Fig. 1 to Fig. 2).

Further, the BLAST search results of 16S rRNA (http://www.ncbi.nlm.nih.gov/blast) and the selected strains having the 16S rRNA sequence represented by SEQ ID NO: 1 are respectively the Bifidobacterium longum subsp. Bifidobacterium longum subsp. infantis) ATCC 15697 (T) and showed a homology of 99.67%, and thus a bipyridinium the strain isolated by the present invention gambling Te ronggeom Solarium spp Infante tooth (Bifidobacterium longum ssp. infantis) were named BI9988 (hereinafter, BI9988), the strain KCTC 12747BP (Bifidobacterium longum ssp. infantis BI9988) with the international deposit was the Korea Research Institute of Bioscience and Biotechnology Microbial Resources Center (Korean Collection of Type Culture, KCTC ).

In addition, the present invention relates to novel Bifidobacterium gambling deposited as accession number of KCTC 12747BP Te ronggeom Solarium spp Infante tooth (Bifidobacterium longum ssp. infantis) BI9988, or provides a probiotic composition containing a culture thereof as an active ingredient.

The novel Bifidobacterium longule subsp. Botulinum strain BI9988 has the 16S rRNA nucleotide sequence shown in SEQ ID NO: 1.

The culture medium of the microorganism contains various antimicrobial organic acids and non-proteinic antimicrobial substances produced by microorganisms, and includes a cholesterol inhibitor.

In addition, the composition according to the present invention is suitable for ingesting together with the lactic acid bacteria of the present invention, further inhibiting the growth of harmful microorganisms upon ingestion, and further adding other known microorganisms having an activity of improving intestinal microflora .

The strain may be selected from the group consisting of gentamicin, streptomycin, tetracycline, erythromycin, clindamycin, chloramphenicol, ampicillin, and vancomycin. There is no extrinsic resistance (transposability) to antibiotics and no activity of the carcinogenic beta-glucosidase.

The strain has excellent intestinal cell adhesion, acid resistance, biliary and antimicrobial activity, and has immunity enhancing activity by increasing secretion of TNF-a, IL-6, or IL-l [beta] in immune cells.

In a particular embodiment of the present invention, the inventors have found that the above-described strain BI9988 separation to separate a bipyridinium gambling measuring the acid resistance of Te Solarium strain cultured for 24 hours in TP broth (broth) after a cell density of 10 ⁸ -10 ¹⁰ (PH 7.0) and acidic TP medium (pH 5.0), and cultured in an anaerobic jar system at 37 ° C for 48 hours. The most probable number method (MPN) was used to determine the acidity of BI9988, which was isolated in the present invention, as compared with the reference strain (ATCC15697) (Conway, PL, SL Gorback, and BR Goldin, 1987. Survival of lactic acid bacteria in the human stomach and adhesion to intestinal cells (J. Dairy Sci. 70: 1-12).

In order to measure the bile resistance of the isolated Bifidobacterium strains, the present inventors measured the percentage of bacteria grown on a TP plate medium containing 0.25% of OX-BILE against the number of bacteria grown on a TP plate medium as the survival rate As a result, it was confirmed that the bile bile resistance of the strain BI9988 of the present invention exhibits a high biliary effect as compared with the reference strain (see Table 3).

In order to measure the antibiotic resistance of the isolated Bifidobacterium strains, the inventors of the present invention conducted a study on the antibiotic resistance of the isolated Bifidobacterium strains according to the ISO guidelines, using gentamicin, streptomycin, tetracycline, erythromycin, Clindamycin, chloramphenicol, ampicillin, and vancomycin were used in a concentration range of 0.5 to 256 占 퐂 / ml. After 2 days, the growth of Bifidobacterium strain BI9988 of the present invention was confirmed to establish a minimal inhibitory concentration (MIC). As a result, BI9988, a strain of the present invention, showed sensitivity to all antibiotics presented in the ISO guidelines , Indicating that there is no risk of antibiotic-resistant metastasis due to lack of antibiotic resistance (see Table 5).

In order to confirm the stability of the Bifidobacterium strain, the present inventors measured the activity of the carcinogenic beta-glucosidase, which Bifidobacterium may have. As a result, the strain BI9988, which is a strain of the present invention, It was confirmed that the activity of β-glucosidase was not detected.

Further, in order to measure the inhibitory effect of the isolated Bifidobacterium strains on the food poisoning bacteria, the inventors of the present invention used Escherichia coli coli O157: H7, CCARM 0012) , Salmonella typhimurium (Salmonella typhimurium, CCARM 8001), Salmonella Entebbe utility disk (Salmonella enteritidis, CCARM 8010), L. monocytogenes cytokines jeneseu (Listeria monocytogenes, CCARM 0019), Staphylococcus aureus (Staphylococcus aureus B, CCARM 0045), Vibrio parahaemolyticus , CCARM 7001) was inoculated into a liquid medium to measure the number of viable cells of the food poisoning strain against the culture filtrate of the strain BI9988 of the present invention and expressed as a log value. As a result, the survival rate of the food poisoning bacteria cultured with the strain of the present invention was lowered, and it was confirmed that the strain BI9988 of the present invention produced substances inhibiting food poisoning bacteria and inhibited the growth of food poisoning bacteria (see Table 6). Thus, it was confirmed that the strain of the present invention produces substances inhibiting the growth of a strain causing food poisoning.

The present inventors measured the ability of the isolated strain BI9988 to inhibit Clostridium difficile, which is an anaerobic bacterium. When the strain was cultured with the strain of the present invention, the survival rate of Clostridium difficile was lowered. As a result, the strain BI9988 It was confirmed that the growth of Clostridium difficile was inhibited (see Table 7).

The present inventors measured IL-1, IL-6, and TNF-α of macrophage cell line RAW 264.7 cells by the strain to measure the immunoreactivity of BI9988, the isolated Bifidobacterium strain of the present invention (TNF-α, IL-1 and IL-6) from the macrophages compared to the cultured filtrate of the reference strains, when the culture filtrate of the strain of the present invention was added. Secretion was activated, it was confirmed that the strain BI9988 of the present invention significantly increased TNF-α, IL-1 and IL-6 production of macrophages and increased macrophage immune activity (see Table 8).

The present inventors measured the ability of the isolated strain to adhere to Caco-2 cells. As a result, it was confirmed that the strain BI9988 of the present invention had much more bacteria attached to the colon cells than the reference strain, It was confirmed that the BI9988 strain of the present invention was well bound to the colon cell line Caco-2 cells (see FIGS. 3 and 9).

In order to confirm the ability of the isolated Bifidobacterium isolate to inhibit cholesterol (resolution), the Bifidobacterium strain of the present invention was cultured in MRS medium containing soluble cholesterol (7.2 mg), and the supernatant was separated The amount of residual cholesterol was shown. As a result, when the strain of the present invention was added, the content of cholesterol remaining in the culture medium was reduced compared with the case where the reference strain was added, and thus the BI9988 strain of the present invention was found to have excellent cholesterol-inhibiting ability 10).

Thus, the novel Bifidobacterium ronggeom subspecies of the present invention Infante tooth (Bifidobacterium longum ssp. infantis) BI9988 strain or culture fluid thereof is acid-resistant and in biliary which makes it a superior and there is no risk of antibiotic resistance transition, excellent in such as intestinal cell adhesion function, immune increase available (macro phages bow performance), sikjungdokgyun inhibitory ability and cholesterol digestive function The strain and its culture can be used as an additive to various products such as a prophylactic or lactic acid bacterial preparation, a fermented milk product, a health supplement, a feed additive, a medicine, a cosmetic, etc. for improving bowel activity and intestinal health.

The composition of the present invention may be prepared according to a conventional method for producing a prod- uct composition, and may be generally in the form of a culture suspension or a dry powder. The effective amount of the above-mentioned Bifidobacterium longum subsp. Botulinum toxin strain BI9988, or a culture thereof, may be selected from one or two or more kinds of pharmaceutically acceptable conventional carriers or one or more additives, . ≪ / RTI >

The carrier may be selected from one or more selected from among diluents, lubricants, binders, disintegrants, sweeteners, stabilizers and preservatives. Examples of the additives include one or more of flavorings, vitamins and antioxidants Can be used.

In the present invention, the carrier and additives may be any pharmaceutically acceptable ones. Specific examples of the diluent include lactose monohydrate, trehalose, cornstarch, soybean oil, Microcrystalline cellulose or mannitol may be preferably used and the lubricant may be magnesium stearate or talc. The binder may be polyvinylpyrrolidone (PVP) or hydroxypropyl It is preferable to select from hydroxypropylcellulose (HPC). The disintegrant is preferably selected from carboxymethylcellulose calcium (Ca-CMC), sodium starch glycollate, polacrylin potassium or cross-linked polyvinylpyrrolidone And the sweetening agent is selected from white sugar, fructose, sorbitol or aspartame. Examples of the stabilizer include sodium carboxymethylcellulose sodium (Na-CMC), beta-cyclodextrin (beta -cyclodextrin) white bee's wax or xanthan gum and the preservative is selected from the group consisting of methyl p-hydroxy benzoate, methlparaben, propyl p-hydroxybenzoate, propylparaben, or potassium sorbate potassium sorbate).

In addition, the present invention provides a health food containing Bifidobacterium longum subsp. Rhinotracis BI9988 deposited under accession number KCTC 12747BP, or a culture thereof as an active ingredient.

The novel Bifidobacterium longule subsp. Botulinum strain BI9988 has the 16S rRNA nucleotide sequence shown in SEQ ID NO: 1.

The culture medium of the microorganism contains various antimicrobial organic acids and non-proteinic antimicrobial substances produced by microorganisms, and includes a cholesterol inhibitor.

In addition, the composition according to the present invention is suitable for ingesting together with the lactic acid bacteria of the present invention, further inhibiting the growth of harmful microorganisms upon ingestion, and further adding other known microorganisms having an activity of improving intestinal microflora .

The strain may be selected from the group consisting of gentamicin, streptomycin, tetracycline, erythromycin, clindamycin, chloramphenicol, ampicillin, and vancomycin. There is no extrinsic resistance (transposability) to antibiotics and no activity of the carcinogenic beta-glucosidase.

The strain has excellent intestinal cell adhesion, acid resistance, biliary and antimicrobial activity, and has immunity enhancing activity by increasing secretion of TNF-a, IL-6, or IL-l [beta] in immune cells.

It is preferable that the strain or the culture solution thereof promotes intestinal health activity by inhibiting intestinal pathogenic microorganism growth.

The food may be selected from the group consisting of ice cream, milk, soy milk, yoghurt, dairy products including cheese, and soy milk products, meat, sausage, bread, chocolate candy, snacks, confectionery, pizza, ramen, other noodles, , A drink, an alcoholic beverage, a capsule, or a stick pack.

Accordingly, the novel Bifidobacterium longum subsp. Botulinum toxin strain BI9988 or its culture is excellent in acid resistance and biliary cholesterol, is free of the risk of antibiotic resistance metastasis, is capable of adhering to the intestinal cells, And the cholesterol digestibility, the strain and its culture can be used for various products such as probiotics or lactic acid bacteria, fermented milk products, health supplements, feed additives, medicines and cosmetics for improving bowel activity and intestinal health Can be used as an additive.

Examples of the health food of the present invention include folk remedies for the purpose of improving bowel function such as tea, jellies, juice, extracts and beverages containing the strain of the genus Bifidobacterium or a culture thereof as an active ingredient. The health food for preventing diseases according to the present invention, which has been processed into various forms as described above, is easy to take without any adverse effects on the human body and can be stored for a long period of time.

When the strain of the genus Bifidobacterium or the culture thereof of the present invention is used as a food additive, the strain or the culture thereof may be directly added, used together with other food or food ingredients, and suitably used according to a conventional method . The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). In general, when the food or beverage is produced, it is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on 100 parts by weight of the Bifidobacterium genus strain or the culture medium raw material of the present invention. However, in the case of long-term consumption intended for health and hygiene purposes or health control purposes, the amount may be less than the above range, and there is no problem in terms of safety. Therefore, the active ingredient may be used in an amount of more than the above range.

There is no particular limitation on the kind of the food. Examples of the food to which the Bifidobacterium sp. Strain or the culture thereof of the present invention can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice confection, ice cream , Dairy products including milk, milk substitutes, cream, butter, buttermilk, yogurt, yoghurt, cheese, various soups, drinks, tea, drinks, alcoholic beverages, lactic acid bacteria and vitamin complexes. It includes all health foods.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates are sugar saccharides such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau Martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the Bifidobacterium sp. Strain of the present invention or the culture thereof.

The Bifidobacterium sp. Strain of the present invention or the culture solution thereof according to the present invention may be used in various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, Stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the probiotics of the present invention may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the ratio of such additives is not critical, the strain of the present invention or its culture is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight.

Hereinafter, the present invention will be described in detail with reference to Examples and Production Examples.

However, the following examples and preparation examples illustrate the present invention specifically, and the content of the present invention is not limited by the examples and the production examples.

< Example  1> Bifidobacterium ( Bifidobacterium )

In order to isolate and identify bifidobacteria having excellent acid and biliary properties, the present inventors collected feces of elderly people aged 80 years or older who are living in the longevity village of Jangsu village and have a healthy and regular intestinal activity, The strains were sequentially diluted with water and the strains were isolated using TP medium (Table 1), a selective medium of Bifidobacterium.

Specifically, for anaerobic cultivation, anaerobic cultivation was carried out at 37 ° C for 3 days using a cold catalyst system (Palladium coated alumina pellets: BBL) using gas-pak. Among the colonies produced in the TP medium, Gram positive bacilli of Y or V shape or club type were selected first, and the recombinant anaerobic bacteria were confirmed.

Bifidobacterium produces acetic acid and lactic acid at a ratio of 1.5: 1 from glucose via a specific metabolic system of fructose-6-phosphate phosphoketolase (F-6-PPK) 6-PPK-positive strains were identified as Bifidobacterium. The fermentation patterns of the selected strains were determined by using API 50 CHL identification kit (bioMerieux Vitex, Inc, France) Bacteriology and Roy and Ward (Roy, D. and P. Ward, 1990. Evaluation of Rapid Methods for Differentiation of Bifidobacterium Species, J. Appl. Bacteriol. 69: 739-749).

Finally, the nucleotide sequence of the 16S rRNA gene was identified by analyzing the Microseq 16S rRNA gene kit (Perkin Elmer Applied Biosystem), and then the strain was stained with Gram stain and confirmed by optical microscope and electron microscope.

As a result, as shown in FIG. 1, the two strains selected were Gram-positive. As shown in FIG. 2, the type of bacteria observed by an electron microscope showed the form of bacterium as a form of Bifidobacterium.

In addition, the selected strain having the 16S rRNA BLAST search result (http://www.ncbi.nlm.nih.gov/blast) and the 16S rRNA sequence (SEQ ID NO: 1) (Fig. 4) Bifidobacterium longum subsp. infantis) ATCC 15697 (T) and showed a homology of 99.67%, and thus a bipyridinium the strain isolated by the present invention gambling Te ronggeom Solarium spp Infante tooth (Bifidobacterium longum ssp. infantis) were named BI9988 (hereinafter, BI9988), the strain KCTC 12747BP (Bifidobacterium longum ssp. infantis BI9988) with the international deposit was the Korea Research Institute of Bioscience and Biotechnology Microbial Resources Center (Korean Collection of Type Culture, KCTC ).

TP medium component ingredient amount Trypticase peptone (BBL) 10 g Protease peptone No.3 5 g Ammonium sulfate 3 g KH ₂ PO ₄ 2 g K ₂ HPO ₄ 1 g L-cysteine.HCl.H ₂ O 0.5 g MgSO ₄ 0.2 g Agar 15 g Sterilized 20% TOS ^{1 )} 50 mL Sterilized 30% sodium propionate 50 mL Distilled water 1 liter

¹⁾ Trans galacto oligo saccharide (pH 7.0)

< Example  2> Acid resistance  Confirm

In order to measure the acid resistance of the Bifidobacterium strain isolated in Example 1, the following experiment was conducted.

Specifically, the acid resistance test was carried out in an artificial gastric juice supplemented with 1000 U / ㎖ of pepsin in pH 3.0 medium for measurement in an environment similar to the digestive tract condition. The isolated strain BI9988 was cultured in a liquid medium for 24 hours, centrifuged, and the cells were recovered and washed three times with sterile physiological saline. The same amount as the supernatant was added to the artificial gastric juice and the reaction was carried out for 90 minutes under the same conditions as the culture conditions of Bifidobacterium. Then, the cells were diluted with Bifidobacterium agar (Modified) plate and counted viable cells.

As a result, as shown in Table 2, the acid resistance and resistance to infertility of BI9988 and BL9988 isolated in the present invention were higher than those of the reference strains ATCC15697 and ATCC15707 (Conway, PL, SL Gorback, and BR Goldin 1987. Survival of lactic acid bacteria in J. Dairy Sci. 70: 1-12, Ibrahim, SA and Bezkorovainy, A. 1993. Survival of bifidobacteria in the presence of bile salt. J. Sci. Food Agric. 62: 351 -354), indicating a high survival rate (Table 2).

Survival rate of Bifidobacterium strains in acidic conditions pH 7.0 pH 5.0 Survival rate (%) Bifidobacterium longum ssp. infantis BI9988 9.3 × 10 ⁸ 9.0 × 10 ⁸ 96.80% Bifidobacterium longum ssp. infantis ATCC15697 2.1 × 10 ⁹ 8.4 × 10 ⁸ 40.00%

< Example  3> My bile (Bile acid resistance)

The following experiments were conducted to determine the biliary properties of Bifidobacterium strains isolated in Example 1 above.

Specifically, in the biliary test, each Bifidobacterium culture treated for 90 minutes in an artificial gastric condition was used, and 0.3% bile (pork bile extract, Sigma) and 1000 U / ml trypsin Artificial bile solution. After centrifuging the artificial gastric juice, the artificial bile solution was added in the same amount as the supernatant, and further reacted for 90 minutes. The resultant was further diluted by comparing with the control, and the resultant was plated on Bifidobacterium agar (Modified) Were measured.

As a result, as shown in Table 3, the biliary properties of BI9988, a strain of the present invention, were determined by the reference strain (ATCC15697 (Ibrahim, SA and Bezkorovainy, A. 1993. Survival of bifidobacteria in the presence of bile salt, J. Sci. . 62: 351-354)), indicating high biliary properties (Table 3).

Survival rate of Bifidobacterium strains in 0.25% bile salts Even 0.00% Even if 0.25% Survival rate (%) Bifidobacterium longum ssp. infantis BI9988 9.3 × 10 ⁸ 3.43 x 10 ⁸ 36.90% Bifidobacterium longum ssp. infantis ATCC15697 2.1 × 10 ⁹ 1.00 x 10 ⁸ 4.70%

< Example  4> Confirm antibiotic resistance

The antibiotic resistance of Bifidobacterium strains isolated in Example 1 was measured according to the ISO guidelines as follows.

Specifically, the antibiotics were tested in accordance with the ISO guidelines (ISO10932: 2010 (E), Milk and milk products-Determination of the minimal inhibitory concentration (MIC) of antibiotics applicable to bifidobacteria and non-enterococcal lactic acid bacteria Table 4 shows the results obtained using gentamicin, streptomycin, tetracycline, erythromycin, clindamycin, chloramphenicol, ampicillin, and vancomycin. As shown in FIG. The antibiotics to be tested were prepared in 2x and plated in a volume of 50 [mu] l each. Bifidobacterium strain BI9988 of the present invention cultured anaerobically for 48 hours at 37 DEG C was adjusted to 0.86% physiological saline to make McPhail turbidity 1, and then cultured in 2X 0.03% cystein-containing LSM medium (Cappel, Aurora, OH , USA) (LSM-cys). This was inoculated on the LSM-cys medium supplemented with the antibiotic and cultured anaerobically for 48 hours at 37 ° C. After 2 days, the growth of Bifidobacterium strain BI9988 of the present invention was confirmed and the minimal inhibitory concentration (MIC) was determined. Antibiotic resistance was determined according to the thresholds set by EFSA (European Food Safety Authority, 2012; 10 (6): 2740).

As a result, as shown in Table 5, the strain BI9988 of the present invention showed sensitivity to all antibiotics compared to other Bifidobacterium strains, and thus it was confirmed that there is no risk of antibiotic resistance metastasis because it has no antibiotic resistance.

Antibiotic concentration range used for MIC of Bifidobacterium strain of the present invention Antibiotic Concentration range (/ / mL) Ampicillin 0.032 to 16 Vancomycin 0.25 to 128 Gentamicin 0.5 to 256 Streptomycin 0.5 to 256 Erythromycin 0.016 to 8 Clindamycin 0.032 to 16 Tetracycline 0.125 to 64 Chloramphenicol 0.125 to 64

The minimum inhibitory concentration (MIC) of the Bifidobacterium strain BI9988 of the present invention Isolated Bifidobacterium strain sample No. 16SID results Minimum inhibitory concentrations (MICs) GEN STR TET ERY CLI CHL AMP VAN One The Bifidobacterium BI9988 (KCTC12747BP) 2 2 One 0.063 0.032 0.5 One 0.5 2 B. longum subsp. infantis 16 8 2 One 0.063 2 4 0.25 3 B. longum subsp. infantis 32 8 2 0.5 0.063 2 0.5 0.5 4 B. longum subsp. infantis 8 8 One 0.25 0.032 2 One 0.5 5 B. longum subsp. infantis 32 4 0.5 0.25 0.032 One 0.5 0.5 6 B. longum subsp . infantis 16 32 0.5 0.5 0.032 2 0.25 0.5 7 B. longum subsp . infantis 32 16 > 64 0.25 0.032 2 0.25 0.5 8 B. longum subsp . Infantis 32 One One 0.125 0.032 2 0.25 0.25 9 B. longum subsp . infantis 32 32 One 0.25 0.063 2 0.5 0.25 10 B. longum subsp . infantis 16 16 One 0.5 0.032 4 0.125 0.25 11 B. longum subsp . infantis 4 8 One 0.125 0.032 One 0.25 0.25 12 B. longum subsp . infantis 32 32 One 0.5 > 16 2 0.5 0.5 13 B. longum subsp . longum 8 4 4 0.125 0.032 One 0.5 0.25 14 B. longum subsp . longum 8 4 One One 0.032 4 0.25 0.25 15 B. longum subsp . longum One 8 One 0.25 0.032 4 0.125 0.25 16 B. longum subsp . longum 2 8 One 0.016 0.032 4 0.032 0.25 17 B. longum subsp . longum 32 16 64 0.125 0.032 4 0.125 0.25 18 B. longum subsp . longum 2 2 0.5 0.125 0.032 One 0.032 0.25 19 B. longum subsp . longum 16 32 0.5 0.5 0.032 2 2 0.25 20 B. longum subsp . longum 2 4 One 0.25 0.032 0.25 0.063 One 21 B. longum subsp . longum 4 16 One 0.5 0.063 2 0.5 0.25 22 B. faecale 16 64 0.5 0.25 0.032 One 0.063 0.25 23 B. 동화 2 2 0.5 0.125 0.032 0.25 0.063 0.5 24 B. 동화 4 8 0.5 4 One One 0.25 0.5 25 B. adolescentis 4 16 0.25 0.25 0.063 One 0.063 0.25 26 B. adolescentis 4 32 0.5 0.125 0.032 One 0.125 0.25 B. longum (ATCC reference strain) 16 16 0.5 0.25 0.063 One 0.25 0.5 Minimum inhibitory concentration cut-off values of Bifidobacterium 64 128 8 One One 4 2 2

(GEN: gentamicin, STR: streptomycin, TET: tetracycline, ERY: erthromycin, CLI: clindamycin, CHL: chloramphenicol, AMP: ampicillin,

< Example  5> Beta glucosidase (β- Glucosidase ) Active confirmation

Β-Glucosidase is an enzyme that degrades glycosides. When glycosides are degraded, they are divided into sugars and non-glycosides. Among the foods consumed, it is known that when the glycosides are decomposed, the reactivity is increased, and the decomposed glycosides are increased in reactivity and increased in the body. In particular, cleansing glycosides such as amygdalin and coumarin glycosides are not desirable when toxic groups are released after decomposition to produce too much.

The present inventors investigated the activity of the carcinogenic beta-glucosidase, which may be possessed by Bifidobacterium, in order to confirm the safety of the Bifidobacterium strain isolated in Example 1 above.

Specifically, the activity of β-glucosidase was confirmed by Ferhak and Pey (Ferhak JD and Pey EK, 1983. Effect of glucose and other sugars on the β-1,4-glucosidase of Thermonospora fusca Biotech. Bioeng. 25: 2855-2864). The cells were collected by centrifugation in a TP medium for about 12 hours, washed twice with 0.1 M phosphate buffer (pH 6.0), and then washed with acetone-toluene (9: 1) Was suspended in 0.4 ml of the same phosphate buffer containing 1/50 (v / v). Substrate PNPG (p-nitro-phenyl-β-D-glucopyranoside) was added at a final concentration of 1 mM and reacted at 45 ° C for a certain period of time. To stop the reaction, 0.6 ml of 0.5 M Na ₂ CO ₃ was added and the amount of p-nitrophenol liberated was measured at 400 nm by absorbance. Where 1 unit is defined as the number of μmoles of PNP liberated for one minute at 45 ° C.

As a result, it was confirmed that the activity of beta-glucosidase was not detected in the strain BI9988 of the present invention.

< Example  6> Food poisoning bacteria Inhibition  Confirm

The following experiment was conducted to measure the inhibitory effect of the Bifidobacterium strain isolated in Example 1 on the food poisoning bacteria.

Specifically, Escherichia coli ( Escherichia coli) was added to the TP culture filtrate of the strain BI9988 and the BHI (Brain Heart Infusion) coli O157: H7, CCARM 0012) , Salmonella typhimurium (Salmonella typhimurium, CCARM 8001), Salmonella Entebbe utility disk (Salmonella enteritidis, CCARM 8010), L. monocytogenes cytokines jeneseu (Listeria monocytogenes, CCARM 0019), Staphylococcus aureus (Staphylococcus aureus B, CCARM 0045), Vibrio parahaemolyticus , CCARM 7001) were inoculated at a final concentration of 1% and cultured at 37 ° C. After 24 hours, the number of each food poisoning bacteria in the culture was measured by the most probable number method (MPN). At this time, the number of viable cells was measured by inoculating Listeria monocytogenes into blood agar medium, Salmonella typhimurium, Salmonella enteritidis, Eromonas hydropila and E. coli O157: H7 on MacConkey medium. The colony forming unit (CFU) was measured and expressed as a log value for the culture filtrate of the strain BI9988 of the present invention.

As a result, as shown in Table 6, the survival rate of food poisoning bacteria cultured with the strain of the present invention was lowered, and it was confirmed that the strain BI9988 of the present invention produced substances inhibiting food poisoning bacteria and inhibited the growth of food poisoning bacteria (Table 6). Thus, it was confirmed that the strain of the present invention produces substances inhibiting the growth of a strain causing food poisoning.

Inhibition of various food poisoning bacteria in the BI 9988 culture medium of Bifidobacterium strain of the present invention Strain Food poisoning bacteria (log CFU / ml) 0 hr 24 hr Escherichia coli ( E. coli O157: H7, CCARM 0012) 7.2 7.69 Escherichia coli ( E. coli O157: H7, CCARM 0012) + BI9988 culture medium 6.51 6.31 Salmonella typhimurium (Sal. Typhimurium, CCARM 8001) 7.3 9.34 Salmonella typhimurium (Sal. Typhimurium, CCARM 8001) + BI9988 culture 7.62 7.81 Sal . enteritidis ( CCARM 8010) 7.39 9.25 Sal . enteritidis ( CCARM 8010) + BI9988 culture medium 7.04 7.14 L. monocytogenes cytokines jeneseu (Lis. Monocytogenes, CCARM 0019) 6.9 9.04 L. monocytogenes cytokines jeneseu (Lis. Monocytogenes, CCARM 0019) + BI9988 culture 6.95 8.69 Staphylococcus aureus (Staph. Aureus B, CCARM 0045 ) 7 9.11 Staphylococcus aureus (Staph. Aureus B, CCARM 0045 ) + BI9988 culture 7.2 8.03 Vibrio parahaemolyticus (CCARM 7001) 6.78 8.56 Vibrio parahaemolyticus (CCARM 7001) + BI9988 culture medium 6.79 7.15

< Example  7> Anaerobic Bacterial Growth Inhibition  Confirm

If the number of lactic acid bacteria in the intestine decreases during the treatment of infectious disease with a large amount of antibiotics or long-term antibiotic treatment, the number of anaerobic bacteria, Clostridium difficile , which is a minority in the intestine, will increase overwhelmingly. Clostridium difficile causes enteritis such as diarrhea, making treatment of cancer patients more difficult. Thus, the ability of the strain BI9988 isolated in Example 1 to inhibit Clostridium difficile was measured.

Specifically, Clostridium dipicyl was inoculated into a Thio-glycolate liquid medium containing hemine (Hemin, 0.01 g / l) and L-cysteine (0.5 g / l) and cultured anaerobically at 37 ° C for 48 hours. The culture was inoculated into the BHI-Cys medium so that the final concentration of the cultured Clostridium difficile (CCARM 0007) was 1%. Then, the culture filtrate of BI9988 and the culture solution of BHI-Cys inoculated with Clostridium difficile Was mixed with paraffin oil and incubated anaerobically at 37 ° C. After 24 hours, the culture broth was diluted, and inoculated into blood agar medium, cultured in an anaerobic incubator at 37 ° C for 48 hours, and the viable cell count of Clostridium difficile was measured.

As a result, as shown in Table 7, when the strain of the present invention was cultured together with the strain of the present invention, the survival rate of Clostridium difficile was lowered, and it was confirmed that the strain BI9988 of the present invention inhibited the growth of Clostridium difficile Table 7).

Inhibition of Clostridium difficile in the culture medium of Bifidobacterium strain BI9988 of the present invention. Clostridium dipicylate (CFU / ml) Clostridium difficile CCARM 0007 (pH 7.0 BHI) 1.7 × 10 ⁵ Clostridium difficile CCARM 0007 (pH 7.0 BHI) + Bifidobacterium longum ssp. infantis BI9988 0.7 × 10 ⁶ Clostridium difficile CCARM 0007 (pH 5.8 BHI) 4.3 × 10 ⁵ Clostridium difficile CCARM 0007 + Bifidobacterium longum ssp. infantis BI9988 2.2 x 10 ⁵

< Example  8> Identification of the effect on macrophage immunological activity

It is the immune system that recognizes and responds to external harmful substances that infiltrate the human body. Human cells that respond to specific immunity include B cells, T cells, macrophages, and the like. Bifidobacterium has been reported to directly activate macrophages, which are immune cells, and thereby to increase the production of cytokines, etc. (Yasui, H. and M. Ohwaki, 1991. Enhancement of immune responses in Peyer's patch cells cultured with Bifidobacterium breve . J. Dairy Sci. 74: 1187-1195; Hatcher, GE and RS Lambrecht. 1993. Augmentation of macrophage phagocytic activity by cell-free extracts of selected lactic acid-producing bacteria. J. Dairy Sci. 76: 2485-2492). It is claimed that the immune stimulants of bifidobacteria include peptidoglycan, which is a component of the cell wall, and polysaccharides, both inside and outside the cell. These immune stimuli enhance the resistance to pathogenic bacterial infiltration and pathogenic bacterial infiltration from the outside, and can be induced by various types of TNF-α (tumor necrosis factor) or IL (interleukin) -6 (Laskin, DL and J. Pendino, 1995. Macrophages and inflammatory mediators in tissue injury, Annu Rev. Pharmacol., 35: 655-677). TNF-α has cytotoxic and cytotoxic effects on leukocyte adhesion and IL-6 is involved in the differentiation of activated B cells. .

In order to measure the immunoreactivity of BI9988, the Bifidobacterium strain of the present invention isolated in Example 1, the present inventors used macrophage cell line RAW 264.7 cells (American Type Tissue IL-6, and TNF-a production ability of IL-1, IL-6, and IL-6.

Specifically, RAW 264.7 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco Lab., Chagrin falls, IL) for 2-3 days at 37 ° C, 5-8% CO ₂ , ⁵ cells / mL and cultured in 96-well flat-bottomed tissue culture plates (Corning, New York). Quantification of TNF-α and IL-6 was performed by Dong's method (Dong, W., Ji Azona-Olivera, KH Brooks, JE Linz, and JJ Pestka 1994. Elevated gene expression and production of interleukins- and during exposure to vomitoxin (deoxyhivalene) and cycloheximide in the EL-4 thymoma. Toxicol. Appl. Pharmacol. 127: 282-290). 50 μl of IL-coating buffer containing pure rat anti-TNF-α or IL-6 antibody (PharMingen, SanDiego, CA) was added to each well of a microtiter strep well (Immunolon Removal: Dynatech Laboratories Inc., Chantilly, VG) After being dispensed, it was left overnight at 4 ° C. The cells were washed with PBST (phosphate buffered saline containing Tween 80) and blocked with 30% BSA-PBST. After incubation at 37 ° C for 30 minutes, the cells were washed again, and 50 μL of the pre-cultured macrophages and the BI9988 cultured filtrate were added. After incubation at 37 ° C for one hour, 50 μl of biotinylated rat anti-TNF-α or IL-6 antibody was added, incubated at room temperature for 1 hour, and streptavidin peroxidase was added for another hour. When TMB (3,3 ', 5,5-tetramethylbenzidine) was added as a substrate and allowed to stand at room temperature, the reaction was stopped by adding 6 NH ₂ SO ₄ to change the color and the absorbance was measured at 450 nm.

As a result, as shown in Table 8, when the culture filtrate of the strain of the present invention was added, the amount of cytokines (TNF-a, IL-1 and IL-6) Secretion was activated, it was confirmed that the strain BI9988 of the present invention increased the immunological activity of macrophages (Table 8).

Influence (ng / mL) of the Bifidobacterium strain BI9988 of the present invention in the production of TNF-α, IL-1 and IL-6 by macrophage Raw 264.7. IL-1 IL-6 TNF-a Macrophage 1.89E-06 0.473 16.564 B. longum ssp . longum ATCC 15707 8.163 - 45.46 B. longum ssp . infantis ATCC 15697 0.39 - 38.266 B. longum ssp . infantis BI9988 503.16 2.629 47.239

< Example  9> Colorectal epithelial cells ( HT29 -MTX cell) Attachment  Confirm

In order to measure the adherence of the strain isolated in Example 1 to colon epithelial cells (HT29-MTX cell), the following experiment was conducted.

Specifically, HT29-MTX cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v / v) heat-inactivated fetal bovine serum and antibiotic mixture. At this time, antibiotics were finally administered to 50 mg / mL penicillin, 50 mg / mL streptomycin, 50 mg / mL gentamicin and 1.25 mg / mL amphotericin B, respectively. HT29-MTX cells were seeded in 30 mm culture dishes at 1 × 10 ⁵ cells / mL and cultured at 37 ° C. for 14 days in a 5% CO ₂ incubator (about 1 × 10 ⁷ cells / mL). All experiments were repeated three times. Bifidobacterium cultures were collected by centrifugation, washed twice with Dulbecco's PBS buffer (Sigma), and suspended in DMEM without antibiotics to a bacterial: intestinal cell concentration of 10: 1. To remove antibiotics, HT29-MTX monolayers were washed twice with Dulbecco's PBS and Bifidobacterium strain was added. After incubation at 37 ° C in a CO ₂ incubator for 1 hour, the supernatant was discarded and each well was gently washed three times with Dulbecco's PBS buffer to remove unattached bacteria. Cells were fixed with 4% fixative (100% of 35% formaldehyde, 16 g of Na ₂ HPO ₄ , ₄ g of distilled water to 1 L of NaH ₂ PO ₄ .H ₂ O) and Bifidobacteria were stained with Gram stain Respectively.

Cells and Bifidobacterium were first removed from the culture dish with 0.1% TritonX-100, and this was serially diluted and inoculated into the MRSC solid medium by spreading method in 100 μl aliquots. CFU was then counted for 2 days in anaerobic conditions. All experiments were repeated three times and data were analyzed using SPSS 11.0 software for Windows (SPSS Inc., Chicago, IL, USA) (Gonzalez de los Reyes-Gavilan, Clara et al., 2011. Adhesion of even adapted Bifidobacterium strains to the HT29-MTX cell line modified to sequential gastrointestinal challenge simulated in vitro using human gastric and duodenal juices. Res. Microbiol. 162: 514-519; Kainulainena V., J. Reunanena, K. Hiippalaa, S. Guglielmettib , S. Vesterlundc, A. Palvaa and R. Satokaria, 2013. BopA does not have a major role in the adhesion of Bifidobacterium bifidum to intestinal epithelial cells, extracellular matrix proteins, and mucus, Appl. Environ Microbiol. 79: 6989- 6997).

As a result, as shown in FIG. 3, when Gram stain and observation under an optical microscope, BI9988 of the present invention was observed to bind well to HT29-MTX cells, which are intestinal cell lines (FIG. 3). In addition, as shown in Table 9, Bifidobacterium attached to HT29-MTX cells in one well longum ssp. exhibited a number of infantis BI9988, it was confirmed that the fungi are well attached to the cells (Table 9).

Bifidobacterium bound to HT29-MTX cells grown in one well longum ssp. The number of infantis BI9988 Number of bacteria attached to cells in one well Bifidobacterium longum ssp. longum ATCC 15707 1 x 10 ⁴ CFU / well Bifidobacterium longum ssp. infantis ATCC 15697 1 x 10 ⁴ CFU / well Bifidobacterium longum ssp. infantis BI9988 1x10 ⁶ CFU / welll

< Example  10> cholesterol Inhibition  Confirm

There are a number of reports that lactobacillus can lower blood cholesterol. In vitro, lactic acid bacteria have been reported to digest, decompose, or adsorb cholesterol as they grow on cholesterol-supplemented media (Gilliland, SE, CR Nelson and C. Maxwell 1985. Assimilation of Cholesterol by Lactobacillus acidophilus . : 377-381), and lactic acid bacteria secrete bile salt hydrolase to decongest the bile acid secreted into the small intestine to produce glycine or taurine separated bile bile acid (Klaver, FAM and R. van der Meer. 1993. The assumed assimilation of cholesterol by Lactobacilli and Bifidobacterium bifidum is due to their salt deconjugating activity. Appl. Environ. Microbio. 59: 1120-1124), the released bile acid is less resorbable than the conjugated bile acid, and consequently, the bile acid that absorbs the cholesterol is reduced, Therefore, the amount of sterol is reduced is the amount of blood cholesterol by increasing the down between cholesterol demand has been reported.

Therefore, the present inventors investigated the cholesterol-suppressing ability (resolution) of Bifidobacterium strains isolated in Example 1 using Rudel LL, MD Morris, Determination of cholesterol using o-phthalaldehyde. Lipid Res. 1973, 14: 364-366).

Specifically, the Bifidobacterium strain of the present invention was cultured at 37 占 폚 in MRS-Cys medium containing soluble cholesterol (7.2 mg). After incubation for 24 hours, the supernatant was centrifuged (4 ° C, 13,000 rpm) and 0.5 ml of the supernatant was separated. The supernatant was separated by adding 2 ml of 50% KOH and 3 ml of 95% ethanol and kept at 60 ° C for 10 min. After cooling, add 5 mL of hexane, mix well, add 3 mL of distilled water, and mix well. After separating the layers, the hexane layer was evaporated at 60 ° C with nitrogen gas, and 4 mL of o-phthaldehyde reagent (1 mL of 0.5 mg o-phthalaldehyde / glacial acetic acid) was added and left for 10 minutes . 2 ml of concentrated sulfuric acid was slowly added thereto and mixed well. After 10 minutes, the optical density was measured at 550 nm to show the content of residual cholesterol.

As a result, as shown in Table 10, when the strain of the present invention was added, the content of cholesterol remaining in the culture medium was lower than that of the reference strain. Therefore, the BI9988 strain of the present invention has a cholesterol- (Table 10).

The ability of the Bifidobacterium strain BI9988 of the present invention to inhibit cholesterol. MRS MRS + Bifidobacterium longum ssp. longum ATCC 15707 5.75 mg 6.00 mg Bifidobacterium longum ssp. infantis ATCC 15697 5.49 mg 5.49 mg Bifidobacterium longum ssp. infantis BI9988 4.47 mg 5.49 mg

* The amount of cholesterol remaining after the addition of 7.2 mg of cholesterol.

< Formulation example  1> Manufacturing of food

Foods comprising the Bifidobacterium BI9988 strain of the present invention and a culture thereof were prepared as follows.

<1-1> Preparation of cooking seasoning

100 parts by weight of cooking sauce was mixed with 1 to 12 parts by weight of the Bifidobacterium strain BI9988 of the present invention and the culture thereof to prepare cooking seasonings for improving the bowel function.

<1-2> soup  And gravies

1 to 12 parts by weight of the Bifidobacterium strain BI9988 strain of the present invention and its culture were added to 100 parts by weight of soup and juice to prepare a meat processing product for improving intestinal function, a soup noodle and a juice.

<1-3> Manufacture of dairy products

Bifidobacterium BI9988 strain of the present invention and 1 to 12 parts by weight of the culture thereof were added to 100 parts by weight of milk and various dairy products such as butter and ice cream were prepared using the milk.

<1-4> Solar  Produce

The Bifidobacterium BI9988 strain of the present invention and its culture were depressurized and concentrated in a vacuum concentrator, sprayed and dried with a hot air drier, and the resulting dried product was pulverized to a size of 60 mesh with a pulverizer to obtain a dry powder.

The grains, the seeds, and the Bifidobacterium BI9988 strain of the present invention and the culture thereof were mixed to prepare an electric wire.

< Formulation example  2> Manufacturing of beverages

<2-1> Production of carbonated beverage

5 to 10 parts by weight of sugar, 0.05 to 0.3 parts by weight of citric acid, 0.005 to 0.02 parts by weight of caramel, 0.1 to 1 part by weight of vitamin C, the Bifidobacterium BI9988 strain of the present invention and 10 parts by weight of the culture solution thereof were mixed, PL 9039 to 94 parts by weight of purified water to prepare a syrup. The syrup was sterilized at 95 to 98 ° C for 20 to 180 seconds and mixed with cooling water at a ratio of 1: 4. Then, 0.5 to 0.82 parts by weight of carbon dioxide was injected, .

<2-2> Production of functional beverage

Bifidobacterium BI9988 strain of the present invention and Bifidobacterium BI9988 strain of the present invention were added to 0.1 part by weight of vitamin C, 5.8 parts by weight of fructose, 3.8 parts by weight of white sugar, 0.12 part by weight of citric acid, 0.03 part by weight of malic acid, 0.04 part by weight of sodium citrate, And 10 parts by weight of the culture solution thereof were mixed and dissolved completely in the metered water. The dissolved beverage was adjusted to the purified water to be weighed so that the total amount was 100 parts by weight.

<2-3> Manufacture of health drinks

0.5 parts by weight of liquid fructose, 2 parts by weight of oligosaccharide, 2 parts by weight of sugar, 0.5 part by weight of salt, and 75 parts by weight of water and 10 parts by weight of the Bifidobacterium BI9988 strain of the present invention and the culture solution thereof were homogenized, And then packaged in small containers such as glass bottles and plastic bottles to produce health drinks.

<2-4> Preparation of vegetable juice

A vegetable juice for health promotion was prepared by adding 10 g of the dried Bifidobacterium strain BI9988 and the culture solution thereof to 1 liter of tomato or carrot juice.

<2-5> Preparation of fruit juice

10 g of the dried product of Bifidobacterium BI9988 strain of the present invention and its culture solution was added to 1 L of apple or grape juice to prepare fruit juice for health promotion.

< Formulation example  3> Preparation of fermented milk

Using the skim milk powder, the raw material oil having the non-skimmed milk solids content adjusted to 8 to 20 parts by weight was sterilized at 72 to 75 캜 for 15 seconds. The sterilized raw material oil was cooled to a predetermined temperature, and then Bifidobacterium BI9988 strain was inoculated at a concentration of 10 ⁶ CFU / ml until a pH of 4 to 5 was reached. After completion of the culture, the culture was cooled. 0.1 to 20 parts by weight of dietary fiber, 0.5 to 30 parts by weight of glucose, 0.1 to 15 parts by weight of oligosaccharide, 0.001 to 10 parts by weight of calcium and 0.0001 to 5 parts by weight of vitamins were dissolved to prepare syrup . The syrup thus produced was sterilized, cooled, mixed with the culture solution at a predetermined ratio, and stirred to prepare a fermented milk by packing in a homogenized container.

< Formulation example  4> lactic acid bacteria Spindle  Produce

Bifidobacterium strain BI9988 was inoculated to MRS medium at a concentration of 10 ⁶ CFU / ml and subjected to pH-control fermentation at 37 ° C for 18 to 24 hours. After completion of the culture, the cells were recovered by centrifugation at 10,000 xg at 4 ° C. The recovered cells were mixed with the same amount of 5% skim milk, 2.5% whey and 5% sucrose, and then powdered through a freeze dryer. The dried powder of Bifidobacterium BI9988 thus prepared was diluted with trehalose and prepared to have a viable cell count of 1 x 10 &lt; ¹¹ &gt; CFU / g or more.

< Formulation example  5> Preparation of lactic acid bacteria preparation

A lactic acid bacterial preparation such as a lactic acid bacterium food, a dressing agent and the like was prepared using the lactic acid bacterium produced in <Formulation Example 4>. 10 parts by weight of oligosaccharide, 20 parts by weight of anhydrous glucose, 5 parts by weight of fructose fructose, 2 parts by weight of vitamin C, 2 parts by weight of fruit powder, 20 parts by weight of a dry powder of Bifidobacterium BI9988 (viable cell count 1 x 10 ¹⁰ CFU / 5 parts by weight of aloe, 15 parts by weight of dietary fiber, and 18 parts by weight of diaphoresis were mixed and sprayed on a stick or bottle by a predetermined amount. The lactic acid bacterial preparation thus prepared maintained a viable cell count of 5 × 10 ⁸ CFU / g or more.

Korea Biotechnology Research Institute KCTC12747BP 20150122

<110> LEE, Yeonhee <120> Bifidobacterium longum ssp. infantis BI9988 isolated from Korean          longevity village and having high nutraceutical activities <130> 2015P-01-027 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 613 <212> DNA <213> Bifidobacterium longum subsp. infantis <400> 1 gt; acaccggaat agctcctgga aacgggtggt aatgccggat gctccagttg atcgcatggt 120 cttctgggaa agctttcgcg gtatgggatg gggtcgcgtc ctatcagctt gacggcgggg 180 taacggccca ccgtggcttc gacgggtagc cggcctgaga gggcgaccgg ccacattggg 240 actgagatac ggcccagact cctacgggag gcagcagtgg ggaatattgc acaatgggcg 300 caagcctgat gcagcgacgc cgcgtgaggg atggaggcct tcgggttttta aacctctttt 360 atcggggagc aagcgtgagt gagtttaccc gttgaataag caccggctaa ctacgtgcca 420 gcagccgcgg taatacgtag ggtgcaagcg ttatccggaa ttattgggcg taaagggctc 480 gtaggcggtt cgtcgcgtcc ggggtgaaag tccatcgctt aacggtggat ccgcgccggg 540 tacgggcggg cttgagtgcg gtaggggaga ctggaattcc cggtgtaacg gtggaatgtg 600 tagatatcgg gaa 613

Claims (13)

Accession No. KCTC 12747BP novel Bifidobacterium subspecies ronggeom deposited by Infante tooth (Bifidobacterium longum ssp. Infantis) strain.
The strain according to claim 1, wherein the strain has the 16S rRNA sequence represented by SEQ ID NO: 1.
A prophylactic or antimicrobial composition comprising the strain of claim 1 or a culture thereof as an active ingredient.
delete The probiabant composition according to claim 3, wherein the strain or a culture thereof has an antibacterial activity.
[4] The composition according to claim 3, wherein the strain or the culture solution thereof is excellent in acid resistance and biliary cholesterol.
The probiabant composition according to claim 3, wherein the strain or a culture solution thereof has excellent adherent cell-cell ability.
The probiabant composition according to claim 3, wherein the strain or a culture thereof has an immunostimulating activity.
4. The method according to claim 3, wherein the strain or a culture thereof has an immunomodulating activity by increasing secretion of TNF-a, IL-6, or IL-l [beta] in an immune cell.
4. The prophylactic or therapeutic composition according to claim 3, wherein the strain or a culture thereof inhibits cholesterol.
A health food containing the strain of claim 1 or a culture thereof as an active ingredient.
12. The health food according to claim 11, wherein the strain or the culture liquid thereof promotes intestinal health activity by inhibiting intestinal pathogenic microorganism growth.
12. The method of claim 11, wherein the food product is selected from the group consisting of ice cream, milk, soy milk, yoghurt, milk products including cheese, and soy milk products, meat, sausages, bread, chocolate candies, snacks, confections, pizza, Wherein the composition is any one selected from the group consisting of various soups, beverages, tea, drinks, alcoholic beverages, capsules or sticks.

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