KR101101894B1 - Strains of lactobacillus fermentum lc272 having the production ability of vitamin k2 - Google Patents

Abstract

PURPOSE: A novel strain Lactobacillus fermentum LC272 with vitamin K2 productivity is provided to ensure antibiotic resistance, enzyme activity, and antibacterial activity. CONSTITUTION: A Lactobacillus fermentum LC272(KACC91565P) has vitamin K2 productivity. The strain is cultured in a modified MRS medium containing bromcresol purple and sodium azide. The strain is isolated from crude oil. The strain has antibacterial ability against Salmonella typhimurium and Staphylococcus aureu.

Description

Strains of Lactobacillus fermentum LC272 having the production ability of vitamin K2}

The present invention is in using the strain identified separating the strain, and characterized in that with the present invention relates to the novel strain Lactobacillus buffer momentum LC272 with vitamin K ₂ producing ability, and more particularly, vitamin K ₂ producing ability by fermentation useful starter It is about.

Vitamin K refers to the generic name for all groups of compounds with similar biological activity. This includes the 2-methyl-1,4-naphthoquinone nucleus with lipophilic side chains all at the 3 position. The three most known ingredients are phylloquinone (vitamin K ₁ ), the only form of vitamin K found in plants. Menaquinone, vitamin K _2, consists of a class of compounds with isoprenyl side chains of varying lengths. Menadione, vitamin K ₃ , is a provitamin that can be converted to vitamin K ₂ by animals.

Vitamin K compounds are predominantly distributed in foods, and among animal foods, eggs, butter, and liver are good sources and contain an amount of about 2-50 μg per 100 g of food. Green vegetables are also a good source and may include 30 to 800 μg per 100 g of food. Spinach, kale, Brussels sprouts and broccoli are good sources. Vegetable oils, and products made from vegetable oils, such as margarine and salad dressings, may also be good sources and include 10 to 300 μg of vitamin K per 100 g of oil. Olive oil and soybean oil are particularly rich in vitamin K. Although quantitative is difficult and can vary widely, some vitamin K is also made from intestinal bacteria.

The US daily recommended dose (RDA) for vitamin K starts at 10 μg per day for infants and increases to 65 μg per day for women and 80 μg per day for men. The best known role for vitamin K in humans is as a cofactor for the synthesis of six proteins involved in blood coagulation. These proteins are inactive proenzymes that are converted into active enzymes in the presence of calcium during the coagulation process. Such proteins include gamma-carboxy-glutamate, which is a specific amino acid. It is formed by the carboxylation of glutamic acid residues in proteins by the enzyme gamma-glutamyl carboxylase in a vitamin K-dependent reaction. Without vitamin K, normal forms of clotting factor cannot be synthesized. Proteins comprising gamma-carboxy-glutamate have been known under the generic name Gla protein. For some time the Gla protein was thought to be confined to the coagulation system and based on this, the RDA was calculated. However, enzymes with gamma-glutamyl-carboxylase activity are now known to be widely distributed in many other tissues and thus many functions of the Gla protein will be found. These proteins are particularly abundant in the kidneys and bones and are therefore thought to play a special role in these organs. Two Gla proteins are particularly abundant in bone. Bone Gla protein (BGP, commonly known as osteocalcin) contains three Gla residues and is present in large amounts in bone, dentin and cartilage.

Matrix Gla protein (MGP) is found in significant amounts in bone, dentin and cartilage. Many ongoing studies are trying to identify the role of these proteins, which appear to be involved in determining the strength and elasticity of structures. Renal Gla protein may be involved in the regulation of calcium secretion so that vitamin K may play a role in integrating the various mechanisms involved in maintaining bone strength (NC Binkley and JW Suttie, Vitamin K nutrition and osteoporosis, J). Nutr 1995; 125: 1812-21 and C Vermeer et al, Effects of vitamin K on bone mass and bone metabolism, J Nutr 1996; 126: 1187s-1191s).

There is recent evidence that vitamin K has a clinically appropriate effect on bone. Controlled studies in women with osteoporosis have shown that 45 mg of vitamin K2 per day can reduce the risk of fracture and slow but not prevent the progressive loss of bone mineral density (M Shiraki et al, J Bone Mineral Res). 2000; 15: 515-521). In a prospective study of 72,000 nurses, women with the lowest five minutes of vitamin K intake (<109 μg per day) increased the risk of fracture (D Feskanich et al, Vitamin K intake and hip fractures in women: a prospective) study, Am J Clin Nutr 1999 69: 74-79). In men and women aged about 70 years or older, the risk of osteoporotic fractures gradually decreased as vitamin K intake increased. The risk was lowest at the highest quadrant of vitamin K intake above 262 μg per day for women and 234 μg per day for men (SL Booth et al, Dietary vitamin K intakes are associated with hip fracture but not with bone mineral density in elderly men and women, Am J Clin Nutr 2000; 71: 1201-8). The gradual effect of increasing daily intake can be important for bone health, especially in older people, with doses of vitamin K much higher than RDA.

It is estimated that there are about 2 million osteoporosis patients in Korea, and vitamin K is an essential cofactor for forming gamma carboxy glutamic acid (Gla) residues in proteins (Olson, Annu. Rev. Nutr. 4, 281, 1984). Since protein-containing proteins bind to calcium ions and affect blood coagulation and tissue calcification (Hauschka and Reid, J. Biol. Chem. 253, 9063, 1978), vitamin K deficiency is associated with intracranial hemorrhage in newborns (Ferland et al. clinical pain and bone fractures, such as J. Clin. Invest. 91, 1761, 1993, cause osteoporosis (Knapen et al. Ann. Int. Med. 111, 1001, 1989).

As osteoporosis emerges as an important medical problem in an aging society worldwide, many studies have been conducted to develop preventive and therapeutic drugs. However, calcium-based simple formulations or fluoro or sterol compounds are used as treatments, but their use is restricted due to side effects. to be.

If we look more specifically at vitamin K, vitamin K has vitamin K ₁ (phylloquinone) and vitamin K ₂ (menaquinone-n) in nature, and vitamin K ₁ is found in green vegetables and algae in plants. K ₂ is mainly produced by microorganisms and is classified as menaquinone 1-14 due to the presence of homologues along the length of the isophenyl group side chains (Shearer, Br. J. Haematol. 75, 156, 1990).

In a study of bacteria that produce vitamin K ₂ Tani et al. (1986) was the Flavobacterium meningosepticum mutant 34mg This MK content per culture solution L, it was was 5.5mg per g dry cell (Tani et al. J. Nutr. Sci. Vitaminol. 32 (2), 137, 1986). Sato et al. (2001) also found that 10% soybean extract, 5% glycerol, 0.5% yeast extract and 0.05% K ₂ HPO ₄ (pH 7.3) were isolated from natto-derived B. subtilis MH-1 and diphenylamine resistant mutant strains D200-41. The maximum MK content was 60 mg / l when cultured at 45 ° C. for 5 days after 1 day stirring culture at 37 ° C. (Sato et al. J. Biosci. Bioeng. 91 (1), 16. 2001). Tsukamoto et al. (2002) also found that vitamin K ₂ (menaquinone-7) plays an important role in the carboxylation of osteocalcin, a bone protein, and that Natto contains higher MK7 than other foods. They found that the MK7 content of Natto produced by B. subtilis mutant strain was 1719 mug / 100 g natto, which was 2 times higher than that of natto produced by commercial strains (Tsukamoto et. Al. Biosci. Biotechnol. Biochem. 65). (9), 2007).

Hojo et al. (2007) also found that propionibacteria produce tetrahydromenaquinone-9 (MK-9), a major menaquinone (vitamin K ₂ ) .Norwegian Jarlsberg and emmental cheeses from the fermented propionibacteria are known as appenzella cheese or groupware. The content of MK-9 was 200-650 ng / g higher than that of cheese (Hojo et al. J. Dairy Sci. 90, 4078, 2007).

The only study on vitamin K-producing lactic acid bacteria was published by Morishita et al. (1999). Lactococcus lactis ssp. When cremoris YIT 2001 and Leuconostoc lactis YIT 3001 were cultured in reduced skim milk or soymilk medium, the content of menaquinone (vitamin K ₂ ) was 29-123µg / L, suggesting that it can be used as a starter for dairy or other food fermentation. No cases have been developed (Morishita et. Al. J. Dairy Sci. 82, 1897, 1999). In Korea, Lee et al. (1998) produced Bacillus sp. LAM 97-44 strain is typical isoprenoid chain is a 7-menaquinone (MK-7) that has a bacterium belonging to the genus Bacillus, but the said (such as, thickening Korea Journal, 41 (3), 208, 1998) to produce vitamin K ₂ There is no research on lactic acid bacteria. Accordingly, if a product is made using lactic acid bacteria developed in Korea, it is considered that the import substitution effect of the product and lactic acid bacteria and the international competitiveness that can be exported abroad can be improved.

Lactobacillus fermentum, which has been found in the past, is isolated from plants and zoos, and Lactobacillus fermentum ME-3 strains have been reported as heterolactic acid strains with antibacterial and antioxidant effects (Kullisaar et al. Int. J. food Microbiol. 72, 215, 2002), n-3 fatty acid-containing functional spread cheeses can be prepared with Lactobacillus fermentum ME-3 strains and reported to reduce the amount of chemical antioxidants added (Jarvenpaa et al. J. Dairy Sci. 90 (7). ), 3171, 2007), and there are no studies on vitamin K ₂ production of Lactobacillus fermentum , and there are no cases of using it as a starter for fermented milk products.

Therefore, the present invention aims to contribute to people's lives health by excellent vitamin K ₂ producing ability than the conventional lactic acid bacteria main provide suitable novel strain in fermented milk produced, and provide fermented milk with the novel strain to a large number of.

The technical objects to be achieved by the present invention are not limited to the above-mentioned technical problems, and other technical subjects which are not mentioned can be clearly understood by those skilled in the art from the description of the present invention .

In order to solve the problems of the prior art, the present invention provides a Lactobacillus fermentum LC272 (KACC91565P) strain.

The strain may be characterized by having a vitamin Ｋ ₂ production ability.

The new strain Lactobacillus permanentum LC272 of the present invention is excellent in vitamin K ₂ production ability as compared to the conventional lactic acid bacteria, and serves as a functional strain having antibiotic resistance, enzyme activity, bile and pH resistance, and antibacterial activity. Therefore, it can be usefully used for the production of fermented milk using the new strain.

1 is a chromatogram of vitamin K ₂ (menaqinone-4) standard.
2 is a chromatogram of the eluate extracted from lactic acid bacteria.
Figure 3 is a graph showing the growth when inoculated with 10% reduced skim milk Lactobacillus Percentum LC272.
Figure 4 is a graph showing the pH change when inoculated with 10% reduced skim milk Lactobacillus Percentum LC272.
5 is a graph showing the growth of Lactobacillus permanent LC272 in MRS medium containing L-cysteine with or without bile addition.

The present invention relates to Lactobacillus fermentum LC272 (KACC91565P) strain.

The strain may be characterized by having a vitamin Ｋ ₂ production ability.

The strain may have functions such as antibiotic resistance, enzyme activity, bile resistance, pH resistance and antibacterial activity.

The strain may be cultured in a modified MRS medium added with bromcresol purple and sodium azide.

A novel strain of the present invention, Lactobacillus permanent LC272 (KACC91565P), has been isolated from crude oil and can be screened and identified according to the methods of the following examples and experimental examples. Identification of microorganisms is made on the basis of their morphological, physiological and biochemical properties. The novel lactic acid bacteria isolated from the crude oil was found to be suitable for the production of fermented milk because of excellent lactic acid and vitamin K ₂ production ability, and completed the present invention.

The present inventors incubated the strains contained in crude milk in modified MRS medium, followed by pure separation of each bacterium and selection of the bacterium that turned yellow as a temporary lactic acid bacterium, and the selected strains plated in an electroformed MRS medium, followed by aerobic culture. Pure water separation. As a result of identifying the purely isolated strains, the strains were Gram-positive bacillus, well grown with or without oxygen, and negative for catalase and motility. In addition, it did not grow at 15 ℃ while growing at 45 ℃, it was confirmed that belongs to Lactobacillus (Lactobacillus) did not generate ammonia from gas and alginic acid from glucose (glucose).

On the other hand, 49 kinds of sugar fermentation tests were conducted to confirm the species name, and acid was generated from 13 species including lactose, and the results obtained by inputting the ATB identification system and the results obtained using the 16S rRNA Gene Sequence were in agreement with Lactobacillus. It turned out to be a momentum. Lactobacillus fermentum LC272 was one of the skim milk coagulation isolates from crude oil that had excellent skim milk coagulation rate and vitamin K ₂ production ability.

As a result of testing the growth of lactic acid bacteria by measuring the number of viable bacteria and pH of the new strain of the present invention, the optimum temperature is 40 ℃, the result of observing the growth through the antibiotic resistance test to determine the minimum inhibitory concentration (MIC) value, Compared to other antibiotics, it is more resistant to gentamicin and streptomycin, but more susceptible to penicillin-G, rifampicin and chloramphenicol. As a result of the enzyme activity test, the enzyme activity was high in leucine arylamidase and acid phosphatase.

In addition, β-glucuronidase, a carcinogenic enzyme that converts Benzopyrene into carcinogenic substances, was found to have no enzyme activity, indicating safety. Bile resistance was measured by testing the growth of lactic acid bacteria in MRS medium, and there was a difference of about 0.5 log between oxgall and no oxgall, but showed similar growth trend.

As a result of testing the pH resistance of the new strain of the present invention, comparing the pH 2, pH 3 and pH 4 with the bacterial count of the control pH 6.4 showed that there is no difference in the number of bacteria after 3 hours there is acid resistance. The antimicrobial activity test showed no inhibitory activity against Escherichia coli , but showed 83.57% inhibition against Salmonella typhimurium and 86.13% inhibition against Staphylococcus aureus . As described above, it was confirmed that the Lactobacillus pertumtum LC272 of the present invention was suitable for producing fermented milk.

The isolated Lactobacillus Percentum LC272 of the present invention was deposited on June 9, 2010 at the National Academy of Agricultural Sciences (KACC) and was assigned KACC91565P as an accession number.

Hereinafter, the content of the present invention will be described in more detail with reference to Examples and Experimental Examples. However, these examples and the like are presented to understand the contents of the present invention, but the scope of the present invention is not limited to these examples and the like.

Example 1 Isolation of Strains (Microorganisms)

Bromcresol purple and sodium azide were collected from the crude oil inspection centers in the Seoul Milk Central, Southern and Western Guidance Offices (Gyeonggi-do and Gangwon-do), Jeonbuk Livestock Testing Laboratory, and Gyeongbuk Livestock Sanitation Laboratory. ) Was added to the modified MRS medium (see Table 1 below) 0.3ml each was inoculated in a planar manner method and then cultured for 48 hours at 37 ℃.

Each colony after incubation was purely separated in electro-modified MRS medium, and the colony that turned yellow was selected as a potential lactic acid bacterium. The selected strains were plated with platinum three times in electro-modified MRS medium and then aerobicly cultured for pure separation. Pure isolates were inoculated in 10% reduced skim milk (Difco Laboratories, USA) and incubated at 37 ° C. for 24 hours to confirm clotting. Table 1 below relates to the composition of the modified MRS medium used to isolate the strains of the present invention.

ingredient Component ratio (g / L) Proteas Peptone # 3 10.0 Beef extract 10.0 Yeast extract 5.0 Lactose 20.0 Twin 80 1.0 Ammonium citrate 2.0 Sodium acetate 5.0 Magnesium sulfate 0.1 Manganese Sulfate 0.05 Dipotassium phosphate 2.0 Sodium azide 0.25 Bromcresol Purple 0.04

Example 2 Selection of Lactic Acid Strains with Excellent Vitamin K ₂ Production Capacity

Example 2-1 Subculture

The lactic acid strain purely separated in Example 1 was passaged twice or more in an MRS liquid medium to increase its activity, and then modified by the method of Takahashi et al. (Takashi et al., J. Dairy Sci., 82, 1897, 1999). Inoculated in the prepared Rogosa medium (see Table 2 below) and incubated for 48 hours at 30 ℃. The cells were recovered by centrifugation at 4,000 rpm for 15 minutes in modified Rogosa medium, washed twice with sterile physiological saline, and lyophilized. Lactic acid bacteria were inoculated in reduced skim milk culture (reconstituted skim milk culture, see Table 3 below) and used without recovery after incubation at 37 ° C for 18 hours. Table 2 below relates to the composition of the modified Rogosa medium, and Table 3 below relates to the composition of the reconstituted skim milk culture liquid.

ingredient Component ratio (g / L) Dextrose 20.0 Tryptic Case Peptone 20.0 Tryptopose peptone 3.0 Yeast extract 5.0 Triammonium citrate 2.0 K ₂ PO ₄ (Potassium Phosphate) 3.0 K ₂ HPO ₄ (Dipotassium Phosphate) 3.0 Magnesium sulfate 0.575 Iron sulfate 0.034 Manganese Sulfate 0.1 L-cysteine 0.2 Sodium acetate 1.0 Twin 80 1.0

ingredient Component ratio (g / L) Skim milk powder 100 Yeast extract 5 Calcium chloride 2 L-cysteine 0.3

Example 2-2 HPLC Analysis

The eluate and vitamin K ₂ (menaqinone-4) standards extracted from four lactic acid bacteria of M581, M301, M351 and LC272 in which vitamin K ₂ production was confirmed in the culture of 1. were analyzed by HPLC. Summarized in In addition, representative chromatograms were the same as those of FIGS. 1 and 2. Table 4 below using HPLC The vitamin K ₂ content produced from the selected lactic acid bacteria.

Selection strain Vitamin K ₂ Content (µg / L) Rogosa Badge Reduced skim milk M581 36.12 20.25 M301 38.54 27.65 M351 155.58 30.93 LC 272 184.94 63.93

* R ² = 0.9845

As can be seen in Table 4, the isolated lactic acid bacteria of lactic acid bacteria of LC 272, 184.94 ㎍ / L in Rogosa medium and 63.93 ㎍ / L in reduced skim milk medium, it was confirmed that excellent vitamin K ₂ production capacity The strain was selected.

Example 3 Identification of Strains

The taxonomy of microorganisms (strains) was performed according to the method of Hammes et al. (The prokaryotes, 1563-1578, 2nd Edition, Springer-Verlag Co. (1992)), the results are shown in Table 5 below.

These strains were both gram positive and bacillus, well grown with or without oxygen, and catalase and motility were negative. In addition, Lactobacillus did not grow at 15 ° C but at 45 ° C and did not produce gas from glucose and ammonia from arginine. It was found to be genus.

In order to determine the species of the strain, when the strain was subjected to 49 sugar fermentation tests using the 50CH kit (API bioMerieux, France), acid was generated from 13 species such as lactose and entered into the ATB identification system. The results obtained using the 16S rRNA Gene Sequence were in agreement with each other and proved to be Lactobacillus percentage. Lactobacillus fermentum LC272 was named as a strain that has excellent lactic acid and vitamin K ₂ production ability among skim milk coagulation strains isolated from crude oil.

The isolated Lactobacillus Percentum LC272 of the present invention was deposited on June 9, 2010 at the National Academy of Agricultural Sciences (KACC) and was assigned KACC91565P as an accession number. Table 5 below relates to the physiological and biochemical properties of the new strain Lactobacillus fermentum LC272 of the present invention.

Gram reaction
Cell morphology
Spore formation
motility
Aerobic growth
Anaerobic growth
Catalase reaction
Grow at 15 ℃
Grow at 45 ℃
Gas generation from glucose
Production of Ammonia from Arginine +
rod
-
-
+
+
-
_
+
-
- Party use ability Glycerol
Erythritol
D-Arabinose
L-Arabinose
Ribose
Glucose
Fructose
Mannose
Sorbos
Rhamnos
Dulcitol
Inositol
Mannitol
Sorbitol
α-methyl-D-mannoside
α-methyl-D-glucoside
N Acetyl Glucamine
Amigdalin
Arbutin
Esculin
Salinity
Cellobiose
Maltose
Lactose
Melibiose -
-
-
-
+
+
+
+
-
-
-
-
+
-
-
-
-
-
-
-
-
-
+
+
+ D-cyros
L-cyros
Adonitol
β-methyl-D-cyroside
Galactose
Sucrose
Trehalose
Inulin
Melezitos
Raffinos
Starch
Glycogen
Silitol
Genthiobiose
D-Turanos
D-Lithus
D-tagatose
D-after course
L-hukos
D-Arabitol
L-Arabitol
Gluconate
2-keto-gluconate
5-keto-gluconate
-
-
-
-
+
+
+
-
-
+
-
-
-
-
-
-
-
-
-
-
-
+
-
-

Based on the above examples, Lactobacillus fermentum LC272 was used in the experiment while incubated in 10% reduced skim milk to determine whether it is suitable as a starter for fermented milk.

Experimental Example 1 Growth Experiment of Lactobacillus Percentum LC272

Growth of lactic acid bacteria was tested by measuring the number of viable cells, pH. The number of viable cells was inoculated with 150 ml of 10% skim milk in 1 drop of the strain with a pipette for 1 ml, and each sample incubated at 34, 37 and 40 ° C for 3 hours at 3 hours intervals was diluted in 0.1% peptone solution. Poured from the agar plate and hardened and counted by incubation for 48 hours at 35 ℃, the pH change was measured by temperature and time and the results are shown in Figures 3 and 4.

As shown in FIG. 3 and FIG. 4, it took 6 hours to logarithmically, and grew almost similarly at 37 ° C. and 40 ° C., and 40 ° C. was most suitable as pH showed the fastest acid production. Temperature was shown (see FIGS. 3 and 4).

Experimental Example 2 Antibiotic Resistance Test of Lactobacillus Percentum LC272

In the antibiotic resistance test, tryptic soy broth (Difco, USA) was used to determine the minimum inhibitory concentration (MIC) value by observing growth by a 2-fold dilution method, and the measured results are shown in Table 6 below. Indicated. Table 6 below relates to antibiotic susceptibility results of Lactobacillus permanent LC272.

Antibiotic Minimum inhibitory concentration (㎍ / ㎖) Aminoglycosides Amikacin 200.0 Gentamicin 1600.0 Kanamycin 10.0 Neomycin 120.0 Streptomycin * 2400.0 β-lactam meter Penicillin-G * 0.5 Methicillin 60 Oxacillin 400 Ampicillin 25 Gram-positive spectrum Bassitracin * 160 Rifampicin 0.1 Novobiocin 320 Lincomycin 40 Gram-Voice Spectrum Polymycin B * 160 Broad spectrum Chloramphenicol 2.5 Vancomycin * 7.8

*: units / ml

As can be seen in Table 6, it was found that Lactobacillus permanent LC272 is more resistant to gentamicin and streptomycin than other antibiotics, while it is highly susceptible to penicillin-G, rifampicin and chloramphenicol.

Experimental Example 3 Enzyme Activity Experiment of Lactobacillus Percentum LC272

Enzyme activity test was prepared by diluting the strain incubated for 18 hours at 37 ℃ in MRS liquid medium with physiological saline to prepare a sample of 10 ⁵ ~ 10 ⁶ cfu / ㎖, using API ZYM kit (API bioMerieux, France) After incubating at 37 ° C. for 5 hours, enzyme reaction was performed. Enzyme activity was expressed as a value of 0 to 5 by comparing the standard color table, summarized the results are shown in Table 7 for the enzyme activity comparison of Lactobacillus permanent LC272.

enzyme Enzyme activity Alkaline phosphatase
Esterase (C4)
Esterase Lipase (C8)
Lipase (C14)
Leucine Arylamidase
Valine arylamidase
Cystine arylamidase
Trypsin
Chymotrypsin
Acid phosphatase
Naphthol-AS-BI-phosphohydrolase
α-galactosidase
β-galactosidase
β-glucronidase
α-glucosidase
β-glucosidase
N-acetyl-β-glucosaminenidase
α-mannosidase
β-fucosidase One
4
4
0
5
2
2
One
One
5
4
0
2
0
0
0
0
0
0

*: Standard colors are specified in steps from 0 to 5, where 0 is negative and 5 is maximum intensity. 1 is 5 nanomoles (nanomoles), 2 is 10 nanomoles, 3 is 20 nanomoles, 4 is 30 nanomoles, 5 is 40 nanomoles or more enzyme activity.

As can be seen in Table 7, Lactobacillus permanent LC272 showed high enzyme activity in leucine arylamidase and acid phosphatase. Β-glucuronidase, a carcinogenic enzyme that converts benzopyrene to carcinogenic substances, was found to have no enzymatic activity, indicating safety.

Experimental Example 4 Bile Tolerance Test of Lactobacillus Percentum LC272

Bile resistance test was measured according to the method of Gilliland and Walker (Gilliland & Walker, J. Dairy Sci., 73, 905, 1990), the results are shown in FIG. As can be seen in Figure 5, there was a difference of about 0.5 log value between the addition and the addition of oxgall, but showed a similar growth trend, it can be concluded that the overall resistance to bile.

Experimental Example 5 pH Tolerance Test of Lactobacillus Percentum LC272

pH resistance test was measured according to Clark et al. (Clark et al., Cultured Dairy Products J., 28 (4), 11, 1993), the results are shown in Table 8 below. Table 8 below relates to the results of the measurement of resistance of Lactobacillus permanent LC272 by pH and incubation time, and the unit is CFU per ml (Log number).

Incubation time (hr) pH 2 pH 3 pH 4 pH 6.4 0 7.8 7.8 7.9 7.9 One 7.6 7.6 7.8 7.9 2 7.7 7.8 7.9 7.9 3 7.8 7.9 7.9 7.9

As can be seen in Table 8, as compared to the control pH 6.4, even the strong acid pH 2 was not affected by the strain of the present invention was found to be acid resistant.

Experimental Example 6 Antimicrobial Activity of Lactobacillus Percentum LC272

Antimicrobial activity was measured according to Gilliland & Speck, J. Food Prot., 40 (12), 820, 1977, and the results are shown in Table 9 below. Table 9 summarizes the results of the degree of inhibition of food poisoning bacteria of Lactobacillus permanent LC272 in MRS medium.

Food poisoning bacteria Growth % Inhibition Food poisoning bacteria ^a L. fermentum 272 ^a CFU / mL pH CFU / mL pH Escherichia coli 2.80 × 10 ⁷ 6.65 5.25 × 10 ⁸ 4.81 0 Salmonella typhimurium 7.00 × 10 ⁶ 6.48 1.15 × 10 ⁶ 4.75 83.57 Staphyloccous aureus 1.55 × 10 ⁸ 6.53 2.15 × 10 ⁷ 4.83 86.13

*: Initial number of Lactobacillus permanent LC272: 1.54 × 10 ⁶ CFU / mL

^a : measured after 6 hours incubation at 37 ℃

As can be seen in Table 9, Lactobacillus permanent LC272 was not inhibitory against Escherichia coli , but 83.64% against Salmonella typhimurium , Staphylococcus aureus ) showed 86.12% inhibition.

On the basis of the embodiments with reference to the accompanying drawings it was described in detail. However, this is not intended to limit the scope of the present invention to this intended to illustrate the present invention, there are various equivalents that can replace them. In addition, the terms or words used in the specification and claims should not be construed as being limited to the common or dictionary meanings, and the inventors should properly define the concept of terms in order to best explain their invention in the best way. It should be interpreted as meaning and concept corresponding to the technical idea of the present invention based on the principle that it can.

The present invention strain Lactobacillus Percentum LC272 is excellent in the ability to produce vitamin K ₂ and further has the ability of antibiotic resistance, enzyme activity, bile and pH resistance and antimicrobial activity can be used as a useful starter for fermentation of many consumers It is expected to contribute to the health of the living, to generate profits for the producers, and ultimately to the national industrial development.

Institute of Agricultural Biotechnology KACC91565 20100609

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Claims (2)

Lactobacillus fermentum LC272 strain (KACC91565P).
The method according to claim 1, wherein the strain is Lactobacillus Permantum LC272 strain (KACC91565P), characterized in that having the ability to produce vitamin Ｋ ₂ .

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