KR20090105171A - Novel bacteriocin-producing lactic acid bacteria and mixed microbial composition using it - Google Patents

Abstract

PURPOSE: A lactobacillus producing anti-bacteiral ingredient and a probiotic composition containing the same are provided to use as an alternative material of antibiotics. CONSTITUTION: A probiotic composition contains Lactobacillus salivarium JWS 58, KACC91357P, Lactobacillus plantarum JWS 1354, KACC91358P, Enterococcus faecium JWS 833, KACC91359P, Pediococcus pentosaceus JWS 939, KACC91360P, and their anti-bacterial product.

Description

Lactobacillus producing antimicrobial substances and probiotic composition containing the same {Novel bacteriocin-producing lactic acid bacteria and mixed microbial composition using it}

The present invention relates to an antibacterial substance producing strain that inhibits pathogenic harmful bacteria and a probiotic composition using the same, and more particularly, to a lactic acid bacterium producing an antimicrobial substance and a probiotic composition containing the same.

Antibiotics have been widely used in the breeding and production of livestock for the treatment of disease, disease prevention, and improving the productivity of livestock. However, recently, the emergence of resistant strains due to misuse of antibiotics and the safety problems due to residual antibiotics in livestock products It is a trend that is gradually being regulated. In addition, as consumers' awareness of the safety of livestock products increases and the demand for non-antibiotic livestock products increases, the development of antibiotic substitutes is of great interest.

Antibiotic substitutes include probiotics, acidifers, immunomodulatory and enhancers, feed enzyme complexes, and functional natural extracts, but probiotics that benefit the host animal by improving the balance of intestinal microorganisms. Is the most sought after (Fuller, 1992). The main mechanism of probiotic for livestock is related to intestinal harmful bacteria and rot through the production and competitive exclusion of anti-pathogenic factors such as organic acids, hydrogen peroxide (H ₂ O ₂ ) and bacteriocin against enteropathogenic microorganisms. By reducing the strains, it promotes the growth and feed efficiency of livestock. It also improves the breeding environment of livestock by reducing manure excretion and suppressing odorous harmful gas.

Bacteriocin is a protein or peptide-based antibacterial substance secreted to the outside of the cell is known to kill or inhibit the growth of microorganisms in close relationship with the production strain. In addition, since it is decomposed by various proteolytic enzymes of the digestive system, there is an advantage that it is harmless to the human body and has no persistence. Recently, researches for utilizing it as a natural food preservative have been actively conducted. Currently, nisin, the only bacteriocin that is commercially available, has been used as a food preservative in processed cheeses, vegetables, canned fruits and fermented milk products in about 50 countries. In Korea, bacteriocins produced by lactic acid bacteria derived from traditional fermented foods (kimchi, salted fish) have been reported to inhibit the pathogenic strains related to food spoilage (Kwon et al., 2002).

Examples of bacteriocins used in the treatment of livestock diseases include the use of lacticin 3147 produced by Lactococcus lactis isolated from fermented dairy foods in the treatment of mastitis in cows (Ryan et al., 1998). A report that inhibited the growth of Salmonella and E. coli O157: H7 by administering bacteriocins, colicin and microcin, produced by E. coli to poultry or cattle (Gillor et al., 2004; Diez-Gonzalez, 2007). In addition, there have been reports of isolates and identification of bacteriocins from pigs (Kyung-Joon Park et al., 1995; Rodr et al., 2003) and poultry (Halami et al., 1999; Poeta et al., 2006). In addition, although it has been reported that lactic acid bacteria having antimicrobial properties against pathogenic microorganisms have been separated from feces and intestines of livestock for development as livestock probiotics, most of them are known to be organic acids, hydrogen peroxide, or low molecular weight substances which are not bacteriocins.

So far, there are few studies to separate the lactic acid bacteria producing bacteriocin from livestock to reveal their characteristics and mechanism of action, and to develop livestock probiotics using bacteriocin or bacteriocin producing strains.

In addition, as consumer demand for health food increases, the consumption of duck meat per capita has more than tripled from 0.35 kg in 1994 to 1.75 kg in 2002, but little research has been done on ducks. have.

The present invention relates to a bacteriocin-producing lactic acid strain and a probiotic composition using the same.

Microorganisms used for livestock probiotics include Bacillus , Enterococcus , Lactobacillus , Pediococcus , Streptococcus , Yeast, etc. In consideration of intrinsic adhesion, fixability, and safety, it is preferable to prepare a probiotic using microorganisms isolated from animal species to be used. In order to develop probiotics for poultry, lactic acid bacteria producing antimicrobial substances such as organic acids and bacteriocins were isolated from domestic chickens, broilers or laying hens (Jin et al., 1997; Kim Sang-ho, 2002; Lee Jae-yeon, 2002; Stern et al., 2006). There are very few researches to develop probiotics by separating useful microorganisms from them. In the present invention, it is intended to provide a bacteriocin-producing lactic acid strain isolated from ducks.

Since more than 400 different microorganisms coexist in the intestine of the livestock, the use of complex probiotics consisting of different microorganisms of the same genus or microorganisms of different genus is advantageous to the intestinal survival of livestock than the probiotics consisting of single microorganisms. Improvements in growth and mortality have been reported (Timmerman et al., 2004). Therefore, in the present invention, a combination of bacteriocin-producing lactic acid strains isolated from ducks and various types of bacteriocins produced by them is intended to develop a complex probiotic that can replace antibiotics, in particular a complex probiotic suitable for breeding ducks.

To this end, in the present invention, microorganisms were isolated and identified from the small intestine and caecum of the four weeks mainly producing antimicrobial material, especially bacteriocin, and confirmed the characteristics of the antimicrobial material and the requirements of probiotics. Based on these findings, in the present invention, Lactobacillus salivarium JWS 58 (KACC91357P), four strains derived from the digestive organs of ducks and producing antibacterial substances; Lactobacillus plantarum JWS 1354, KACC91358P); Enterococcus JWS 833 ( Enterococcus faecium JWS 833, KACC91359P) and Pediococcus pentosassius JWS 939 ( Pediococcus pentosaceus JWS 939, KACC91360P).

Combination of several bacteriocins rather than a single bacteriocin shows a broader antimicrobial range and can be further extended by reducing the number of microorganisms resistant to bacteriocin. Therefore, in the present invention, in order to develop a more effective duck-only probiotics, a complex probiotic was composed of selected microorganisms and bacteriocin, and finally the effect of the complex probiotic was confirmed through a duck specification experiment. Based on the experimental results, the present invention provides a probiotic composition comprising the four strains and the antimicrobial substances produced by them. The probiotic composition of the present invention preferably comprises the strains at 1.0 × 10 ⁶ cfu / g to 1.0 × 10 ¹⁰ cfu / g, respectively.

In the present invention, new bacteriocin-producing strains were isolated and identified from the small intestine and caecum of the duck to prepare a complex probiotic, the complex probiotic according to the invention is excellent in the ability to inhibit pathogenic bacteria as confirmed through the duck specification test It is expected to be used as a substitute for antibiotics in livestock raising.

In the present invention, in order to develop a probiotic for livestock, in particular, duck as a substitute for antibiotics, microorganisms producing bacteriocins having strong antimicrobial activity against intestinal pathogenic bacteria from the small intestine and cecum of the duck were selected and identified. In the present invention, in order to develop a more effective probiotic, by selecting a microorganism and bacteriocin that can exert a synergistic effect among the selected microorganisms to improve the inhibitory ability against pathogenic bacteria when combined use. The newly isolated and identified bacteriocin-producing lactic acid bacteria in the present invention, Lactobacillus Salivarius JWS 58 ( Lactobacillus) inhibits intestinal pathogenic harmful bacteria and has probiotic properties. salivarium JWS 58, KACC91357P); Lactobacillus plantarum JWS 1354, KACC91358P); Enterococcus faecium JWS 833, KACC91359P) and Pediococcus pentosassius JWS 939 ( Pediococcus pentosaceus JWS 939, KACC91360P) and was deposited on March 13, 2008 by the Korea Institute of Agricultural Biotechnology (KACC).

In addition, the present invention examined the characteristics of the biochemical properties of the bacteriocin itself produced by the strains, strain resistance of the selected microorganisms acid resistance, bile acid resistance and digestive enzyme activity.

And based on these results, the composite probiotic composition was prepared, and the effect was confirmed through a duck specification test. The probiotic composition of the present invention is particularly suitable for breeding ducks, but its use is not limited thereto. The probiotic composition of the present invention may be used in various poultry including broilers, and may also be used in pigs, ruminants, pets, and fish farmed for fish. In addition, the probiotic composition of the present invention can also be used as an antibiotic substitute or a probiotic in foods and the like according to the antibacterial action.

The probiotic composition of the present invention is preferably prepared as follows. First, the four selected strains were cultured overnight in a 10 ml MRS liquid medium and used as mass production seed. The medium for mass production is 0.5-3% soypeptone or animal protein, 0.1-2.0% yeast extract, 0.5-5% glucose or lactose, 0.1-0.5% buffering reagent, and 0.001-0.01% minerals (magnesium, Calcium, manganese, etc.), and the culture conditions are incubated for 16-20 hours in an anaerobic state at 30 ~ 37 ℃. After the incubation was completed, the cells were recovered by centrifugation rapidly, and then mixed with a cryoprotectant (containing 0.5 to 5%, skim milk powder, maltodextrin, trehalose, lactose, mannitol, cyclodextrin, etc.) at a specific gravity of 1: 1. Lyophilize. The microbial freeze-dried raw materials completed by lyophilization were mixed at 1.0 × 10 ⁶ cfu / g to 1.0 × 10 ¹⁰ cfu / g level, more preferably at 1.0 × 10 ⁹ cfu / g level to prepare the probiotic composition of the present invention. do.

The probiotic composition for ducks of the present invention may be added to feed or fed directly to duck feed, and may replace the use of antibiotics.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the scope of the present invention is not limited by the following examples, and those of ordinary skill in the art to which the present invention pertains should be within the equivalent scope of the technical concept of the present invention and the claims to be described below. Of course, various modifications and variations are possible.

Example  One

Isolation and Selection of Microorganisms

After collecting the viscera of ducks from the slaughter production line of Juwonsan Duck and transferring them to the laboratory in refrigerated state, it was immediately used for the separation experiment of bacteriocin producing strains. After cutting the small intestine and caecum site, about 1 g of the contents were put in an anaerobic dilution, diluted appropriately, smeared in MRS or BHI agar medium and incubated at 37 ° C. for 48 hours. Colonies grown in each medium were randomly inoculated in an Eppendorf tube containing 1 ml of MRS or BHI liquid medium, incubated for 1-2 days, and then 10 μl of the culture was added to the MRS or BHI solid medium. Dried for 1 hour. Then, BHI or MRS soft agar (0.7%) inoculated with the indicator bacteria was added, and then cultured for about 1 day, the strains having the ability to inhibit pathogenic strains were first selected by checking the clear zone shown on the medium. Indicators used at this time include E. coli KCTC 1467, Salmonella typhymurium KCTC 2515, Staphylococcus aureus KCTC 1621, Listeria monocytogenes KCTC 3569, and Lactobacillus sake KCCM 40264 was used. Slightly modified spot-on-lawn method for the first selected strains to remove the antimicrobial effect of lactic acid among various antibacterial substances (organic acid, acetyl, H ₂ O _2, etc.) produced by lactic acid bacteria (Mayr et al., 1972) conducted a second round of selection. That is, the cultures of the first selected strains were centrifuged, sterilized using a 0.45 filter, and then neutralized to about 6.5 with a cell-free supernatant using 10N NaOH. The filtrate was dropped on a soft agar to which the indicator bacteria were added at 1.0 × 10 ⁷ cfu / ml, followed by culturing under the culture conditions of the indicator bacteria (approximately 37 ° C., daily culture), and then observing the formation of a clear zone by bacteriocin. The resulting strains were finally selected.

416 strains of microorganisms were isolated from the small intestine and caecum of the duck, and 76 strains with inhibitory ability against five indicator bacteria were selected first. As a result of performing spot-on-lawn method to remove microbial inhibition effect by organic acid, 4 strains showing antimicrobial activity were selected secondarily. The four strains producing the antimicrobial substances showed different antimicrobial ranges.

Example  2

Identification of microorganisms

To identify bacteriocin producing strains, morphological and biochemical properties were investigated according to Bergey's Manual of Determinative bacteriology (Holt et al., 1994). Gram staining, motility, catalase test, CO ₂ production and carbohydrate fermentation pattern using API 50 CHL kit (BioMerieux, France) were investigated. For the final strain identification, 16S rDNA nucleotide sequence of the isolate was determined to determine the known strain. Compared with their base sequence.

The microorganisms selected 2nd week had characteristics such as gram positive, catalase negative, and non-motility, and were classified as lactobacillus as a result of sugar fermentation experiment. In addition, 16S rDNA nucleotide sequence of the selected microorganisms was compared with other lactic acid bacteria registered in the GeneBank. As a result, each strain showed more than 97% homology, and finally, each strain was Lactobacillus sali. Barius JWS 58 ( Lactobacillus salivarius JWS 58), Lactobacillus plantarium JWS 1354 ( Lactobacillus plantarum JWS 1354), Pediococcus pentosassius JWS 939 pentosaceus JWS 939), and Enterococcus JWS 833 ( Enterococcus faecium JWS 833). Table 1 below shows the results of carbohydrate fermentation of the isolate strain.

Example 3

Preparation of Cell-free Supernatant

The selected bacteriocin producing strains were inoculated in MRS broth and incubated at 37 ° C. for 18 hours. Cells were removed by centrifuging the culture medium at 3,000 × g for 20 minutes to remove the cells, and the obtained supernatant was adjusted to a final pH of 6.5 using 1N NaOH, and then filtered through a membrane (0.2 μm pore size) to measure bacteriocin activity. -free supernatant was prepared.

Example 4

Antibacterial range investigation

Deferred antagonism method and spot-on-lawn method were used to confirm the antimicrobial range of selected bacteriocin-producing strains. In the former method, the culture medium was dropped in a soft agar containing indicator bacteria, incubated overnight at the optimum growth temperature of the indicator bacteria, and then the presence of inhibitory rings was examined. In the later method, the cell-free supernatant was dropped in a soft agar containing indicator bacteria (1.0 × 10 ⁷ cfu / mL), and then cultured overnight at the optimum growth temperature of the indicator bacteria.

In the deferred antagonism method, most of the indicator bacteria were grown due to the antimicrobial substance containing the organic acid produced in the selection strain.

In the spot-on-lawn method, most of the selected lactic acid bacteria showed strong inhibitory activity against the growth of L. monocytogenes . Lact. salivarius JWS 58 mainly inhibited the strains of the lactobacilli strains, but also inhibited the strains of enterococci and Leuconostoc mesenteroides . Ent . faecium JWS 833 also showed extensive inhibitory activity against various microorganisms. Ped. pentosaceus JWS 939 is more effective than L. monocytogenes, Staph. It has been shown to inhibit the growth of food decay and pathogenic microorganisms such as aureus .

Based on the results, four selection strains, namely Lact. salivarius JWS 58, Ent. faecium 833, Ped. pentosaceus 939, Lact. Experiments were conducted on the properties of antimicrobial agents on plantarum 1354 and their application as probiotics. Table 2 shows the inhibitory effect on the pathogenic bacteria of the second selection strain.

Example  5

Investigation of Physicochemical Properties of Bacteriocin

In order to investigate the stability of bacteriocin to pH, the cell-free supernatant was adjusted to pH 2.0 to 10.0 with 10N NaOH and 10N HCl, and left at room temperature for 1 hour, and then the activity of bacteriocin was measured by spot-on-lawn method. It was. Thermal stability was measured for bacteriocin activity after the solution was treated for 30 minutes at 60 ° C., 30 minutes at 95 ° C., and 15 minutes at 121 ° C., respectively. In order to measure the activity change of bacteriocin according to the enzyme treatment, each enzyme was added to a final concentration of 1 mg / ml, and then reacted at 37 ° C. for 1 hour and heat treated at 80 ° C. for 10 minutes to stop enzymatic activity. Next bacteriocin activity was measured. Enzymes include proteinase K, protease, pepsin, trypsin, α-amylase, β-amylase, and catalase. Used. To investigate the effect of organic solvent treatment on bacteriocin activity, the same amount of cell-free supernatant and organic solvent were mixed and reacted at 37 ° C. for 1 hour, and then bacteriocin activity was measured. The organic solvent used at this time was ethanol, methanol, chloroform, acetone, acetonitrile, hexane, hexane, ethyl acetate, acetate and the like.

Table 3 shows the results of investigating the stability of bacteriocin against various enzyme treatment, heat treatment, pH and organic solvent treatment for the antimicrobial material produced from four selected strains. 1 is Lact . salivarius JWS 58 (A) and Ent. faecium As a result of inhibition of Listeria monocytogenes pathogens by JWS 833 (B), one of the photographs shows cell-free supernatant (CFS); 2 is catalase-treated CFS; 3 is proteinase K-treated CFS. Lact . salivarius JWS 58, Ent . faecium 833, and Ped . In case of pentosaceus 939, antimicrobial properties of cell-free supernatants were inactivated by proteinase K treatment, thereby losing its titer (FIG. 1), which proves that the antimicrobial material is a bacteriocin consisting of proteins or peptides. But Lact . The antimicrobial material of plantarum 1354 was not affected by proteases, suggesting that it is an organic acid other than bacteriocin or a small molecular weight material. Most of the bacteriocin activity produced in each strain was stable in the pH range of 2 to 10, but the organic solvent treatment showed a difference in activity by strain or by type of organic solvent. The following Table 3 compares the vitality of the antimicrobial agents against enzymes, organic acids, pH, and heat treatment.

Example 6

Acid and Bile Acid Resistance

The isolated strain was inoculated in MRS liquid medium and incubated overnight at 37 ° C., and then in pH 7.0, 3.0, 2.5, 2.3, and 2.0 buffer solutions prepared by calibrating 0.05M sodium phosphate buffer with HCl. Inoculation was performed at an initial inoculation concentration of 1.0 × 10 ⁷ cfu / ml. After the above solution was incubated at 37 ° C. for 2 hours, 1 ml dilution was applied to MRS or BHI agar medium for each concentration and incubated at 37 ° C. for 48 hours. And resistance to each acidic pH was measured by counting the number of colonies shown after the culture. Acid resistance test results are shown in FIG. 2.

The isolated strain was inoculated in MRS or BHI liquid medium in a certain amount and incubated overnight at 37 ° C., and then diluted with sterile saline solution, and then modified by the method of Gilliland et al. Plated in MRS agar medium containing 0.05%, 0.1%, 0.3%, 0.5% each and incubated for 48 hours at 37 ℃. And by counting the number of colonies appeared after the culture was measured bile acid resistance according to the bile acid concentration. The anti-bile acid test results are shown in FIG. 3.

Lact. salivarius JWS 58 maintained a high survival rate of more than 50% of the initial inoculation concentration after 2 hours at pH 2.5, but the remaining starter strains began to die rapidly below pH 2.5 and rarely survived at pH 2.0 (Fig. 2). In addition, bile acid resistance experiments were performed on MRS or BHI agar medium containing 0.05%, 0.1%, 0.3%, and 0.5% (w / v) of bile acid (oxgall), respectively. It was shown that colonies normally formed without inhibition (FIG. 3).

To act as a probiotic in the intestines of livestock, it must be resistant to bile acids that pass through the stomach with a low pH and are secreted into the duodenum. Results of acid resistance tests on selected strains, Lact. Only salivarius JWS 58 had relatively high acid resistance, while other strains showed acid resistance of common microorganisms, but bile acid resistance was very high. This suggests that non-intestinal strains do not grow in medium containing more than 0.3% bile (Khattab and Abou-Donia, 1987). It seems to be.

Example 7

Enzyme activity investigation

The inoculated strains were inoculated in MRS or BHI broth and incubated overnight at 37 ° C., and the culture solution was centrifuged at 4 ° C. for 10 minutes to filter the supernatant with a 0.22 μm syringe filter.

Amylase and cellulase activity was measured using a modification of Miller (1959) DNS (dinitrosalicyclic acid) method (Khasin et al., 1993). 50 μl of the culture solution was mixed with 0.1 M Tris-HCl enzyme reaction solution (pH 7.0) containing 950 μl of 0.5% substrate (starch, carboxymethylcellulose), and reacted at 37 ° C. for 10 minutes, and then 1 ml of DNS was added. The reaction was stopped. The mixture was left to stand in boiling water for 5 to 7 minutes to develop color and immediately cooled, and then absorbance was measured at 540 nm using UV spectrophotometer (UV spectrophotometer, Unico, USA) by adding 8 ml of distilled water. One unit of amylase and cellulase activity was defined as the amount of enzyme that produced 1 equivalent reducing sugar (glucose) for 1 minute at 37 ° C. The activity of lipase was used by modifying the method of Lesuisse et al. (1993). After mixing 0.5 ml of the culture solution and 0.5 ml of the substrate solution (1% 4-nitrophenyl butyrate in 50 mM Tris-HCl, pH 7.0) and reacting at 37 ° C. for 10 minutes, the absorbance was measured at 405 nm using a UV spectrophotometer. It was. One unit of lipase was defined as the amount of enzyme that produced 1 product (4-nitrophenol) at 37 ° C. for 1 minute. The activity of protease was investigated by modifying the method of Yanagida et al. (1986). After mixing 2.5 ml of a 0.7% casein (from bovine milk, Sigma) solution and 0.5 ml of the culture solution for 30 minutes at 37 ° C, 2.5 ml of 10% TCA (trichloroacetic acid) solution was added to stop the reaction. This was centrifuged at 3,500 rpm for 10 minutes at 4 ° C, the supernatant was taken, and the absorbance was measured at 275 nm with a UV spectrophotometer. Protease activity was defined as 1 unit liberating 1 mole of tyrosine for 1 minute at 37 ° C. The enzyme activity investigation results are shown in Table 4 below.

As a result of investigating the secretory enzyme activity of 4 different types of duck small intestine and cecum-derived lactobacillus, protease enzyme activity was relatively low while amylase activity was higher than that of other enzymes. Especially Ped . pentosaceus JWS 939 and Lact . plantarum JWS 1354 had relatively high amylase and cellulase enzyme activities.

In contrast to human probiotics, livestock probiotics are more important for livestock productivity and economics, so increasing feed efficiency and gain rate is very important. Alam et al. (2003) found that when artificial digestion enzymes were added to broiler feed, the growth rate, feed efficiency, and yield of broilers were increased, and Jin et al. (1999) fed feed supplemented with Lactobacillus . Amylase activity in the small intestine of a broiler chicken was increased very high, while protein and fat-related enzyme activities were not affected. As a result of measuring a digestive enzyme activity for the 5 strains selected as the antimicrobial substance-producing strain from the duck, Ped. pentosaceus JWS 939 and Lact . The plantarum 1354 strain showed very high amylase activity, similar to the results of Jin et al. (1999). Given that feeds generally fed to poultry consist primarily of vegetable and small amounts of animal protein in grain feed (starch), the selection and use of microorganisms with high amylase activity may be a major selection requirement for livestock probiotics. have.

Example  8

Probiotic Preparation

The four selected strains were incubated overnight in 10 ml of MRS liquid medium to be used as mass production seed. The composition of the medium for mass production is shown in Table 5 below, and incubated at 37 ° C. for 16 hours under anaerobic conditions. After the incubation was completed, the cells were harvested by centrifugation quickly, and then mixed with a cryoprotectant at a specific gravity of 1: 1, and then lyophilized. The composition of the cryoprotectant used is shown in Table 6 below. The microbial freeze-dried raw materials completed by lyophilization were mixed at a level of 1.0 × 10 ⁹ cfu / g to prepare a probiotic for duck specification test.

Example  9

Specification test

In order to verify the efficacy of the bacteriocin-containing complex probiotic of the present invention, a specification test was conducted on a 1-day-old broiler duck. Specimen experiments were conducted up to 45 days of age, divided into 10 animals and 3 repetitions. The control group was fed only a general formula feed, the treatment was performed by adding the bacteriocin-containing complex probiotics prepared in Example 8 at a level of 0.1% by weight. The test results are shown in Table 7 below in comparison with the control.

In the control group, one animal died naturally, but the treatment group administered the bacteriocin-containing complex probiotic of the present invention showed a 100% growth rate. In the productivity comparison, the weight gain and feed efficiency were significantly higher in the treatment group than in the treatment group.

Bacteriocin-containing complex probiotics according to the present invention are excellent in inhibiting ability against pathogenic bacteria, and are expected to be excellent in intestinal adhesion, fixability, and safety when they are isolated from the digestive organs of ducks. Therefore, the composite probiotic composition of the present invention is expected to be widely used industrially as an antibiotic replacement in duck breeding, and further, it is expected to be utilized in other poultry or livestock due to its excellent antibacterial action.

1 is Listeria by Lactobacillus salivarius JWS 58 (A) and Enterococcus fascium JWS 833 (B). monocytogenes is the result of inhibition of pathogens.

2 is an acid resistance test result.

Figure 3 is a test result of bile acid resistance.

Claims (6)

Lactobacillus salivarium JWS 58, KACC91357P); Lactobacillus plantarum JWS 1354, KACC91358P); Enterococcus JWS 833 ( Enterococcus faecium JWS 833, KACC91359P); Pediococcus pentosassius JWS 939 pentosaceus JWS 939, KACC91360P) and a probiotic composition comprising the antimicrobial substances they produce. The probiotic composition of claim 1, wherein the strain comprises 1.0 × 10 ⁶ cfu / g to 1.0 × 10 ¹⁰ cfu / g, respectively. Lactobacillus JWS 58 ( Lactobacillus, Lactobacillus) salivarium JWS 58, KACC91357P) Lactobacillus JWS 1354 ( Lactobacillus, Lactobacillus) plantarum JWS 1354, KACC91358P). Enterococcus, which is derived from the digestive tract of ducks and produces antimicrobial substances JWS 833 ( Enterococcus faecium JWS 833, KACC91359P). Derived from the digestive tract of ducks and produces antimicrobial substances and Pediococcus pentosassius JWS 939 ( Pediococcus pentosaceus JWS 939, KACC91360P).

Images