KR101335454B1 - Novel Lactobacillus sp. strains and their use as probiotics - Google Patents

Abstract

The present invention relates to novel Lactobacillus genus lactic acid bacteria and their use as probiotics which inhibit harmful pathogens in the intestine, have excellent intestinal cell adhesion, have an immunostimulating effect, have no resistance transfer, and are excellent in acid resistance and bile resistance. The probiotic may be usefully used as feed additives and veterinary compositions of livestock.

Description

Novel Lactobacillus sp. Strain and its probiotic use {Novel Lactobacillus sp. strains and their use as probiotics}

The present invention relates to novel Lactobacillus genus lactic acid bacteria and their use as probiotics which inhibit harmful pathogens in the intestine, have excellent intestinal cell adhesion, have an immunostimulating effect, have no resistance transfer, and are excellent in acid resistance and bile resistance. The probiotic may be usefully used as feed additives and veterinary compositions of livestock.

The use of antibiotics in animal feed for livestock production has improved productivity, including preventing and treating livestock diseases such as diarrhea, promoting growth and improving feed efficiency, and contributing to increased income for livestock farmers.

Despite these effects of antibiotics, however, controversy has begun with the emergence of resistant strains, identification of residual antibiotics in livestock products, and human safety issues due to the transfer of resistance of bacteria from animals to humans, which has led to restrictions on the use of antibiotics. Antibiotic residue testing on livestock products is also being strengthened. Recently, the European Commission has banned the use of antibiotics added to feeds as animal growth promoters. In Korea, the total use of antibiotics in the livestock industry has declined to 1,595 tons in 2001, 1,211 tons in 2008 and 999 tons in 2009 (National Veterinary Research and Quarantine Service), especially in 2009, which are highly resistant to acquired antibiotics. (eg bacitracin, chlortetracycline, colistin, lincomycin, neomycin, oxytetracycline and penicillin) is prohibited from being used as animal feed. In addition, antibiotics, excluding insect repellent and anticoccidial, will be banned from animal feed in 2012.

As the use of antibiotics decreases due to the restriction and prohibition of the use of antibiotics, there is a growing interest in substances to replace antibiotics. Antibiotic alternatives currently in use include organic acids (acidifiers), probiotics, prebiotics, feed enzymes (enzymes), immune modulators (plant extracts) ) And minerals, among which probiotics are attracting the most attention.

Probiotics have generally been defined as healthful probiotic food additives (Lilly and Stillwell, Science 147: 747-748, 1965), but recently Salminen et al. (Int. J. Food Microbiol. 44: 93-106, 1998 ) Is defined as a living microbial agent in food and feed or dietary additives designed to promote human or animal health. Bacteria that are most commonly used as a probiotic is Lactobacillus and the like (Lactobacillus) as a lactic acid bacterium and Streptococcus (Streptococcus).

Preferred conditions for probiotics should be safe, tolerant to acids and bile, adherent to intestinal epithelium, antagonistic activity against pathogenic bacteria, It should have an immune boosting effect, and even if it has a resistance gene, it should not be a transfer of antibiotic resistance.

Although lactic acid bacteria (LAB) help intestinal microbial balance and have many useful benefits such as antimicrobial activity, prominent activity and anticancer activity, most of the lactic acid bacteria currently used as probiotics have not been evaluated as accurate probiotics. There are many, and the development of a system that can suppress the stealing of other lactic acid bacteria is an infinite reality. In addition, livestock probiotics are most ideally developed in the intestine of livestock, but many probiotics are not the same.

Therefore, the present inventors studied to develop a novel lactic acid bacterium satisfying the conditions as a probiotic, four kinds of lactic acid bacteria selected from the intestinal contents of pigs exhibit acid resistance and bile resistance, intestinal harmful microbial growth inhibitory activity and immune enhancing activity The present invention was completed by confirming that it can be used as an excellent probiotic having a healing effect on feed additives or pathogenic microbial infection of animals.

One object of the present invention is to provide a lactic acid bacteria strain isolated from pig intestinal contents, exhibiting acid resistance and bile resistance, exhibiting intestinal harmful microbial growth inhibitory activity and immune enhancing activity.

Still another object of the present invention is to provide a probiotic composition comprising the lactic acid bacteria strain or a culture thereof.

It is another object of the present invention to provide an additive for animal feed containing the probiotic composition.

It is another object of the present invention to provide a composition for treating or preventing intestinal pathogenic microbial infections, including the probiotic composition.

It is another object of the present invention to provide a method for inhibiting the growth of intestinal pathogenic microorganisms or preventing or treating diarrhea caused by the microorganisms by administering the probiotic composition to animals other than humans.

As one aspect for achieving the above object, the present invention relates to a lactic acid bacteria strain isolated from pig intestinal content, exhibits acid resistance and bile resistance, and exhibits intestinal harmful microbial growth inhibitory activity and immune enhancing activity.

Preferably, the lactic acid bacteria strain is Lactobacillus salivarius KACC91678P strain. The strain is also named PL9028 in the present invention.

Preferably, the lactic acid bacteria strain is Lactobacillus plantarum KACC91679P strain. The strain is also named PL9031 in the present invention.

Preferably, the lactic acid bacteria strain is Lactobacillus plantarum KACC91680P strain. The strain is also named PL9033 in the present invention.

Preferably, the lactic acid bacteria strain is Lactobacillus amylovorus KACC91681P strain. The strain is also named PL9034 in the present invention.

In another embodiment, the present invention is one or more strains selected from the group consisting of the Lactobacillus salivarius KACC91678P strain, Lactobacillus plantarum KACC91679P strain, Lactobacillus plantarum KACC91680P strain and Lactobacillus amyloborus KACC91681P strain. Or it relates to a probiotic composition comprising a culture thereof.

As another aspect, the present invention relates to an additive for animal feed comprising the composition.

As another aspect, the present invention relates to a composition for the treatment or prevention of intestinal pathogenic microbial infection disease comprising the composition.

Preferably, the pathogenic microorganism is Enterohemorrhagic Escherichia coli (EHEC), Escherichia coli O157: H7, Escherichia coli O78, Escherichia coli O26, Salmonella enteritidis , Salmonella typhimurium ( Salmonella typhimurium) typhimurium ), Salmonella cholerasuis ( Salmonella cholerasuis ) and Salmonella derby ( Salmonella derby ) may be selected from the group consisting of.

Preferably, the intestinal pathogenic microbial infection disease may be selected from the group consisting of diarrhea, enteritis, gastroenteritis, constipation, abdominal pain, bloating, cholera and food poisoning.

As another aspect, the present invention relates to a method for inhibiting the growth of intestinal pathogenic microorganisms or preventing or treating diarrhea caused by the microorganisms by administering the probiotic composition to animals other than humans.

Preferably, said animal comprises a pig.

Hereinafter, the present invention will be described in more detail.

We collected intestinal material from a total of 1,120 pig fecal samples over 2004 and 2006, from which Lactobacillus sp. Total 432 by morphology, gram staining, catalase reaction and m tiplex PCR analysis. Strains were isolated and identified, and the most effective four strains, PL9028, PL9031, PL9033 and PL9034 strains were selected through the inhibitory activity test on eight pig diarrhea-inducing bacteria. All four strains showed excellent inhibitory ability against 8 types of swine diarrhea-inducing bacteria, and all of them showed excellent inhibitory ability of 20% or more even in mildly acidic conditions (pH 5.5 to 6).

The four strains showed stability in use in hemolysis test, gelatin liquefaction test, harmful substance production test, and harmful enzyme test, and further showed acid resistance and bile resistance, thus containing low pH artificial environment and bile. It satisfies the condition of probiotic bacteria that must survive in an intestinal tract environment.

In addition, it is generally reported that tetracycline-based antibiotics are used the most among antibiotics for livestock and livestock products, and most of the lactobacillus lactic acid bacteria are inherently resistant to vancomycin. The strains also showed high resistance to both vancomycin and tetracycline over 128 ug / ml on antibiotic resistance MIC (minimum inhibitory concentration). However, penicillin of the penicillin series, which is the second most widely used after tetracycline antibiotics, was susceptible to two strains of PL9034 and PL9031, and all four strains were sensitive to ampicillin, imipenem, gentamicin, clindamycin and erythromycin. Indicated. In addition, the four strains had the antibiotic resistance gene tet gene and / or bla gene, it was confirmed that does not transfer the resistance gene.

In addition, the probiotic lactic acid bacteria can function when it is able to settle in the intestine, proliferate and inhabit. The four strains are all attached to the intestinal cells of pigs and humans, about 10 ⁴ CFU, inhabit the human intestine and enter the intestines The potential to grow under conditions has been identified. In addition, all four strains induced the secretion of TNF-α when treated alone, confirming the immunopotentiating effect. In particular, PL9031 and PL9034 strains exhibited a function of reducing excessive TNF-α secretion by LPS (lipopolysaccaride). Therefore, it is possible to minimize the septic shock induced by LPS during bacterial infection.

Based on all the above experimental results, the inventors of the present invention, the four strains are able to inhibit various intestinal pathogenic bacteria, exhibit acid resistance and bile resistance, good intestinal cell adhesion, immune enhancing effect, resistance Since there was no metastasis, the strains could be determined as probiotics to replace antibiotics. Thus, the present inventors have identified the strains through molecular phylogenetic analysis based on 16S rRNA sequences, and these strains were deposited in the National Institute of Agricultural Science, respectively, and assigned accession numbers KACC91678P, KACC91679P, KACC91680P, and KACC91681P. .

The strains can be used alone, of course, can be expected to complement and synergistic function even when mixed use. In addition, since the lactic acid bacteria are less viable at room temperature, and can be used as a feed additive, the lactic acid bacteria can be used as a probiotic to improve the growth and feed efficiency of livestock by replacing antibiotics.

The Lactobacillus salivarius KACC91678P strain of the present invention was isolated from pig intestinal material, is a rod-type Gram-positive strain, has a catalase negative reaction, and has a 16S rRNA sequence of SEQ ID NO: 1.

Lactobacillus plantarum KACC91679P strain, Lactobacillus plantarum KACC91680P strain of the present invention was isolated from porcine intestinal material, gram-positive strain of the rod type, shows a catalase negative reaction, SEQ ID NO: 16S rRNA Has a sequence.

The Lactobacillus plantarum KACC91680P strain of the present invention was isolated from porcine intestinal material, is a rod-type Gram-positive strain, has a catalase negative reaction, and has a 16S rRNA sequence of SEQ ID NO: 3.

The Lactobacillus amyloborus KACC91681P strain of the present invention was isolated from porcine intestinal material, enterococci and Gram-positive strains, catalase negative reaction, and has the 16S rRNA sequence of SEQ ID NO: 4.

The strains of the present invention can be improved or improved to have equivalent activity and excellent field stability or to exhibit superior antimicrobial activity by conventional physicochemical mutation methods and the like.

The present invention also includes at least one strain selected from the group consisting of Lactobacillus salivarius KACC91678P strain, Lactobacillus plantarum KACC91679P strain, Lactobacillus plantarum KACC91680P strain, and Lactobacillus amyloborus KACC91681P strain or a culture thereof. It provides a probiotic composition. At this time, the microbial culture medium contains various antimicrobial organic acids and nonprotein antimicrobial substances produced by the microorganisms, when included as an active ingredient of the probiotic composition can exhibit the same effect as the composition containing the strain.

In addition, the probiotic composition according to the present invention is suitable for ingestion by the livestock together with the lactic acid bacteria of the present invention, the other microorganisms known to have the activity of inhibiting the growth of harmful microorganisms and improving the balance of intestinal flora when ingested It may further comprise.

The probiotic composition of the present invention may further include conventional pharmaceutical carriers and excipients in addition to the active ingredient, and such compositions may be lyophilized or heat dried according to a conventional probiotic composition preparation method, and may be encapsulated or cultured. It may be in the form of a suspension or a dry powder.

The composition of the present invention includes probiotic lactic acid bacteria or its culture medium excellent in intestinal pathogenic bacteria suppression, acid resistance and bile resistance, and thus is excellent in inhibiting and treating various enteric pathogenic bacteria and normalizing the intestinal flora of the host. As well as preventing bacterial diseases, as a probiotic composition such as intestinal pathogenic bacterial inhibitors, immune enhancers, fungicides, digestive agents, suits and antibacterial agents can be usefully used in feed, food and pharmaceuticals. In addition, the microorganism of the present invention is particularly separated from the intestine of the pig and forms a normal intestinal microbial flora, which is particularly advantageous in raising livestock.

Preferably, the probiotic composition of the present invention may be usefully used as an additive for animal feed or as a composition for treating or preventing intestinal pathogenic microbial infection diseases. In addition, the present invention may be usefully used to prevent the growth of intestinal pathogenic microorganisms or to prevent or treat diarrhea caused by the microorganisms by administering the probiotic composition to animals other than humans.

Livestock to which the probiotic of the present invention may be applied includes, but is not limited to, dogs, horses, cows, pigs, sheep, goats, chickens, ducks, turkeys, and geese, preferably pigs.

When the probiotic composition of the present invention is used as a feed additive, it is used as a substitute for the existing antibiotics to inhibit the growth of harmful intestinal bacteria and maintain the intestinal flora in a stable condition to improve the health of the livestock and improve the weight gain and meat quality of the livestock. And increase acid yield and immunity. At this time, it is preferable to include the novel Lactobacillus strain or its culture solution in an amount of 1 to 20 parts by weight, and more preferably 10 to 20 parts by weight based on 100 parts by weight of the feed.

In addition, the feed additive according to the present invention is not limited thereto, but may be prepared in the form of fermented feed, blended feed, pellet form, silage, and the like. The fermented feed may be prepared by fermenting an organic material by adding the strain of the present invention and various microorganisms or enzymes, and the blended feed may be prepared by mixing various kinds of general feed and the Lactobacillus strain of the present invention. . The feed in pellet form may be prepared by formulating the fermented feed or blended feed into a pellet machine, and silage may be prepared by fermenting the green feed with the Lactobacillus strain according to the present invention.

When the probiotic composition of the present invention is used as a composition for the treatment or prevention of enteric pathogenic microbial infection diseases, the enteric pathogenic microorganism is enterohemorrhagic Escherichia coli ; EHEC), E. coli O157: H7, E. coli O78, E. coli O26, Salmonella entera itty display (Salmonella enteritidis), S. typhimurium (Salmonella typhimurium ) Salmonella cholera suis cholerasui s) and Salmonella Derby (Salmonella derby ) may be selected from the group consisting of, but is not limited thereto. The intestinal pathogenic microbial infection disease may be selected from the group consisting of diarrhea, enteritis, gastroenteritis, constipation, abdominal pain, bloating, cholera and food poisoning, but is not limited thereto.

The composition for treating or preventing the intestinal pathogenic microbial infection disease can be administered orally or parenterally during clinical administration, and one or two or more pharmaceutically acceptable ingredients in an effective amount of the Lactobacillus strain or its culture as a main component. Conventional carriers or one or two or more additives may be selected to prepare compositions in conventional pharmaceutical formulations. The carrier may be used by selecting one or two or more from diluents, lubricants, binders, disintegrants, sweeteners, stabilizers, and preservatives, and one or two or more of the fragrances, vitamins, and antioxidants. Can be used.

The novel microorganisms of the genus Lactobacillus of the present invention are acid- and bile-resistant and probiotic to inhibit harmful enteric bacteria in the intestine, and replace the existing antibiotics to stabilize the intestinal microbial flora when administered to livestock to be caused by abnormal fermentation of harmful microorganisms in the intestine It can cure or prevent any of the possible symptoms, and improve the health and improve the livestock weight, improve the meat quality, increase the milk production and increase immunity.

1 is a result of confirming the adhesion pattern of PL9028, PL9031, PL9033 and PL9034 strain to IPI-21 cells, porcine intestinal cell line through gram staining.
Figure 2 shows the results obtained by gram staining the adhesion pattern of PL9028, PL9031, PL9033 and PL9034 strain to Caco-2 cell cells, a human intestinal cell line.
FIG. 3 shows TNF-a in cell control, LPS control group (LPS control), lactic acid bacteria alone group (gray bars) and LPS and lactic acid bacteria treatment group (white bars) for macrophage cell line RAW 264.7. It shows the degree of secretion.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  1. Collection of Intestinal Substances and Isolation of Lactic Acid Bacteria

1-1. Collection of Pig Intestinal Substances

Lactobacillus Lactic acid bacteria such as amylovorus , L. intestinalis , L. crispatus , L. plantarum , L. acidophilus , L. ruminis , L. saerimneri , L. reuteri , L. buchneri , L. murinus and L. mucosae It is present in the intestine, caecum and rectum and can be isolated from pig feces. Thus, 1,120 pig intestinal substances were collected eight times in 2004 and 2006 from healthy pigs slaughtered in slaughterhouses in Gyeonggi-do. Pig intestinal material was collected from a total of 1,120 pig fecal samples by collecting 572 pig fecal samples in four times in 2004 and 548 pig fecal samples in four times in 2006.

1-2. Isolation of Lactic Acid Bacteria

The primary separation of lactic acid bacteria was plated with 0.002% Bromophenol blue added De Man Rogosa (hereinafter MRS, Difco, Becton, Dickinson and Company sparks, MD, U.S.A) medium and incubated for 48 hours in a 37 ℃ incubator. Colonies in the form of lactic acid bacteria were harvested and subjected to primary passages. The colonies grown in culture were selected only for the bacteria that showed a negative reaction through a catalase reaction experiment, and then Gram stained to identify the Gram-positive and rod form strains with M μtiplex PCR (Yu-Li Song, et.al., FEMS Microbiology Letters, 187: 167-173, 2000).

Among the colonies grown in MRS medium, the strains catalase-negative and gram positive and rod-shaped by Gram staining were 519 strains in 2004 and 431 strains in 2006, totaling 950 strains. The strain identified as Lactobacillus sp. By M μltiplex PCR was 186 strains in 2004 and 246 strains in 2006, the last 432 strains.

Example  2. Pathogens Inhibition  Test ( antimicrobial activity test Selection of lactic acid bacteria through)

2-1. Pathogen Inhibition  Test

The well test was performed to select strains with inhibitory ability against pig diarrhea-inducing bacteria among the 432 lactic acid bacteria selected primarily in Example 1. First, swine diarrhea-causing bacteria include E. coli (EHEC, CCARM1250, swine), E. coli O157: H7 (CCARM1239, swine), E. coli 078 (ETEC, CCARM230, swine), E. coli 026 (EPEC, CCARM228, swine), S. enteritidis ( CCARM134, swine), S. typhimurium DT104 (CCARM125, human), S. cholerasuis ( CCARM43, swine) and S. derby ( CCARM42, swine). The lactic acid bacteria selected in Example 1 were each incubated for 24 hours at 37 ° C. in 100 ml MRS medium, followed by centrifugation (10,000 × g, 15 min). The supernatant was mixed with the same amount of ethyl acetate. After shaking vigorously for 1 hour, the ethyl acetate layer was concentrated to 1/100 with an evaporator. Porcine diarrhea-inducing bacteria were inoculated into M μler-Hington (hereinafter referred to as MH) solid medium according to MacFarland 0.5, and 100 μl of lactic acid bacterium concentrate was injected with a sterile Pasteur pipette. MRS broth was extracted and used as a negative control. After incubation overnight at 35 ℃ to check the presence and size of the inhibitory ring, a total of four kinds of lactic acid bacteria were finally selected.

Table 1 shows the results of measuring the size of the inhibitory ring of the negative control and the final selection strains PL9028, PL9031, PL9033 and PL9034. No diarrhea-causing bacteria showed inhibitory activity in the control MRS broth extract, but Lactobacillus PL9028, PL9031, PL9033 and PL9034 showed excellent inhibition against all 8 pathogens. Thus, Lactobacillus PL9028, PL9031, PL9033 and PL9034 were assigned accession numbers KACC91678P, KACC91679P, KACC91680P and KACC91681P to the National Institute of Agricultural Science, respectively.

Pathogen Size of restraint ring (mm) Control group PL9028 PL9031 PL9033 PL9034 E. coli ( EHEC) 6 21 17 21 18 E. coli O157: H7 6 22 20 24 18 E. coli 078 ( ETEC) 6 23 21 25 19 E. coli 026 (EPEC) 6 22 18 22 18 S. enteritidis 6 23 20 25 19 S. typhimurium DT104 6 24 20 25 20 S. cholerasuis 6 24 20 25 19 S. derby 6 23 20 24 19

2-2. pH In accordance Inhibition

In order to exclude the inhibitory ability according to the acidity of lactic acid bacteria, the pH of the lactic acid bacteria culture medium was examined to increase the pH to weak acidity (pH 5.5 ~ 6.0) and then check whether there is an inhibitory ability. The lactic acid bacteria were inoculated in MRS liquid medium and incubated at 35 ° C. for 24 hours. The cells were removed by centrifugation (10,000 xg, 15 minutes at 4 ° C.) and the same amount of Mueller-Hinton as the lactic acid culture supernatant (hereinafter MH (2X)). After mixing with the medium was adjusted to three pH 6, 5.75, 5.5 with saturated TRIS and filtered through a 0.45 ㎛ filter. The diarrhea-inducing bacteria used in Example 2-1 were inoculated to 10 ⁶ CFU / mL and incubated at 35 ° C. for 24 hours, and the optical density was measured at an absorbance of 450 nm. As a control, MRS liquid medium was used instead of the supernatant of lactic acid bacteria.

Table 2 shows the growth inhibition rate (%) of diarrhea-inducing bacteria of each lactic acid bacteria culture medium according to pH. No diarrhea-causing bacteria showed inhibitory effect in the control MRS broth extract, but Lactobacillus PL9028, PL9031, PL9033 and PL9034 showed more than 20% inhibition of all 8 pathogens regardless of pH. At pH 5.5, each lactic acid bacterium inhibited many diarrhea-causing bacteria.

Control group ³ PL9028 PL9031 PL9033 PL9034 E. coli ( EHEC) pH 6 ¹ 0.00 ² 24.1 46.1 45.9 48.1 pH 5.75 0.00 46.8 60.7 55.7 61.9 pH 5.5 0.00 50.7 80.7 80.3 75.0 E. coli O157: H7 pH 6 0.00 29.6 45.5 47.0 40.8 pH 5.75 0.00 40.9 54.1 53.3 53.7 pH 5.5 0.00 43.2 75.6 81.6 68.1 E. coli 078 (ETEC) pH 6 0.00 27.5 37.8 46.2 41.2 pH 5.75 0.00 0.0 39.8 22.5 25.9 pH 5.5 0.00 0.0 82.5 36.4 75.7 E. coli 026 (EPEC) pH 6 0.00 26.4 52.6 44.8 56.0 pH 5.75 0.00 13.1 71.4 60.0 71.5 pH 5.5 0.00 39.5 92.1 71.3 89.0 S. enteritidis pH 6 0.00 29.4 43.0 46.4 48.4 pH 5.75 0.00 43.7 60.6 61.9 61.0 pH 5.5 0.00 23.4 89.1 59.2 79.5 S. typhimurium DT104 pH 6 0.00 39.6 47.5 73.6 66.4 pH 5.75 0.00 50.4 63.0 75.1 81.2 pH 5.5 0.00 58.2 96.5 85.3 95.8 S. cholerasuis pH 6 0.00 34.7 45.9 47.6 53.5 pH 5.75 0.00 47.4 59.6 61.4 65.5 pH 5.5 0.00 55.4 86.8 84.8 78.6 S. derby pH 6 0.00 35.2 51.7 42.1 54.6 pH 5.75 0.00 49.1 65.4 59.3 66.2 pH 5.5 0.00 52.9 83.0 81.7 77.3 ¹ : pH of culture supernatant
² : growth inhibition rate (%) = MRS absorbance
³ : growth inhibition rate in the absence of lactic acid bacteria = (absorbance in MRS medium-absorbance in each pH medium) / absorbance in MRS medium X 100

Example  3. Safety Test of Selected Lactic Acid Bacteria

3-1. Stability experiment

For the hemolysis test, test bacteria were inoculated into the blood agar medium and incubated at 37 ° C. for 24 hours, and the hemolysis was observed and recorded.

For gelatin liquefaction test, MRS gelatin medium (0.3 g beef extract, 0.5 g peptone, 12 g gelatin, 100 ml MRS broth) was inoculated with the test bacteria and experimented for 6 weeks at 35 ℃. After incubation with the control group was not inoculated and cooled for 4 hours at 4 ℃ was confirmed. When the medium was not solidified even after refrigerating, it was regarded as a positive reaction.

For the generation of harmful substances, the presence of harmful substances such as ammonia, indole and phenylpyruvic acid was examined.

For harmful enzyme identification test, it was confirmed whether or not harmful enzymes such as beta-glucuronidase and beta-glucosidase.

Table 3 shows the results of the stability experiment. The strains showing α-hemolysis were PL9028 and PL9031. The pathogenicity of bacteria is often dependent on cell invasion ability, but cell invasion requires proteolytic ability. In the gelatin liquefaction experiment to investigate the protein resolution, all four strains were negative. In addition, no ammonia, a harmful metabolite, was produced in all four strains, nor indole and phenylpyruvic acid. In addition, it was confirmed that the production of beta-glucuronidase and beta-glucosidase among enzymes known to be harmful enzymes produced by some intestinal microorganisms did not produce all.

Inspection items PL9028 PL9031 PL9033 PL9034 Hemolysis alpha alpha - - Gelatin liquefaction - - - - Urease production - - - - Indole generation - - - - Phenylpyruvic Acid Production - - - - Beta-glucuronidase production - - - - Beta-glucosidase production - - - -

 3-2. Acid resistance  And My bile  inspection

Acid resistance experiments were carried out in artificial gastric juice with 1000 U / ml pepsin added to pH 3.0 medium for measurement in an environment similar to the digestive tract conditions of pigs. The isolated lactic acid bacteria strains were incubated in liquid medium for 24 hours, and then centrifuged to recover the cells and washed three times with sterile saline. Artificial gastric juice was added in the same amount as the supernatant, and then reacted for 90 minutes under the same conditions as in the culture conditions of lactic acid bacteria, diluted with the control group, and plated on an MRS plate to count the number of viable cells to check the acid resistance.

Bile resistance experiments were carried out in each of the lactic acid bacteria cultures treated for 90 minutes in artificial gastric juice conditions, and was performed in artificial bile solution added 0.3% bile (pork bile extract, Sigma) and 1000 U / ml trypsin to pH 7.0 medium . The culture solution treated with artificial gastric juice was centrifuged, and then reacted for 90 minutes with the addition of the same amount of artificial bile as the supernatant. After dilution with the control group, the culture solution was counted on the MRS plate to check the degree of bile resistance.

Table 4 shows the acid and bile resistance test results. The PL9028, PL9031 and PL9033 strains survived almost 100% even after the treatment of artificial gastric juice and artificial bile, and PL9034 showed acid resistance and bile resistance even though 1/100 CFU was decreased.

 (Unit: CFU) 0 min In ^a simulated gastric fluid
90 mins In artificial bile fluid ^b
90 mins PL9028 2.86 x 10 ⁷ 2.23 x 10 ⁷ 5.40 x 10 ⁶ PL9031 1.80 x 10 ⁷ 1.54 x 10 ⁷ 2.27 x 10 ⁷ PL9033 7.10 x 10 ⁷ 5.80 x 10 ⁷ 6.30 x 10 ⁷ PL9034 6.05 x 10 ⁵ 6.00 x 10 ³ 4.00 x 10 ^{3 a} : Treats gastric juice
^b : After treatment with artificial gastric juice, artificial bile Processed

Example  4. Antibiotic Sensitivity Test of Selected Lactic Acid Bacteria

Antibiotic susceptibility testing was performed using Cation-adjusted Mueller-Hinton broth with lysed horse blood (hereinafter referred to as the method of the Clinical and Laboratory Standards Institute, CLSI, M45-A, Vol. 26, No. 19, 2006). CAMHB 2.5-5%) medium was used for broth microdilution method. Briefly, the selected lactic acid bacteria were inoculated in MRS agar and incubated overnight in a 5% CO ₂ incubator, and the cells were turbidized in sterile saline (0.85% NaCl) to a MacFarland value of 0.5 so that the final 5 x 10 ⁵ CFU / μl. The medium was inoculated with antibiotics. Imipenem was used by the Korea Research Institute of Chemical Technology (KRICT) and all other antibiotics were purchased from Sigma (St. Louis, Mo., USA). Antibiotic concentrations were used at 0.25 to 128 mg / ml. ATCC25922 and ATCC49619 were used as QCs for antibiotics and the results were analyzed according to the National Committee for Clinical Laboratory Standard (NCCLS) guidelines.

Table 5 shows the antibiotic resistance test results of each lactic acid bacteria. All of the selected lactic acid bacteria showed at least 128 highly resistant to vancomycin and tetracycline. PL9028 and PL9033 showed resistance to penicillin. All four strains were sensitive to ampicillin, imipenem, gentamicin, clindamycin and erythromycin.

 (Unit: ug / ml) penicillin Ampicillin Imipenem Gentamicin Vancomycin Clindamycin Erythromycin Tetracycline PL9028 64 4 ≤ 0.25 One > 128 ≤ 0.25 ≤ 0.25 > 128 PL9031 4 2 ≤ 0.25 ≤ 0.25 > 128 ≤ 0.25 ≤ 0.25 > 128 PL9033 64 4 ≤ 0.25 0.5 > 128 ≤ 0.25 ≤ 0.25 > 128 PL9034 One One ≤ 0.25 ≤ 0.25 > 128 ≤ 0.25 ≤ 0.25 > 128 KCTC3018 8 2 ≤ 0.25 0.5 > 128 ≤ 0.25 ≤ 0.25 64 KCCM40210 0.5 0.5 ≤ 0.25 0.5 > 128 ≤ 0.25 ≤ 0.25 2 ATCC49619 0.25-1 0.06-0.25 0.03-0.12 - 0.12-0.5 0.03-0.12 0.03-0.12 0.12-0.5 ATCCC25922 - - - 0.25-1 - - - - standard* S ≤8 ≤8 ≤0.5 ≤4 ≤4 ≤0.5 ≤0.5 - I - - - 8 8-16 1-2 1-4 - R - - - ≥ 16 ≥32 ≥4 ≥8 - ATCC49619: Streptococcus pneumoniae ;
ATCC25922: E. coli ;
*: MIC criteria according to the guidelines set by the CLSI, where S is susceptible, I is moderately sensitive, and R is resistant.

Example  5. for intestinal cells Attachment  Experiment

5-1. For porcine intestinal cells Attachment

Pig intestinal cell line IPI-21 cells (Boar miniature male ileum, ECACC) were heat-inactivated 10% Fetal bovine serum (hereinafter referred to as FBS) and 0.024 IU / mL of insulin (from pancreas of bovine, Sigma, St. Louis, Mo) , USA) incubated in DEME medium (D μlbecco's modified minimal essential medium, Gibco, BRL. USA), and then inoculated into 6 well plates by 1.0 X 10 ⁵ , followed by post-confluence. The experiment was conducted. Three washes were performed with 10 mM phosphate buffer saline (PBS), and fresh DMEM medium was dispensed. Lactobacillus passaged in MRS (about 1 X 10 ⁸ CFU / ㎖) was washed three times with PBS and then inoculated in a 6-well plate and incubated in a 37 ℃ CO ₂ incubator for 1 hour. After culturing, the cells were washed three times with 10 mM PBS to remove each unattached lactic acid bacteria, and then fixed with 4% fixative solution. Gram staining was used to observe attachment using an optical microscope (Nikon, eclipse80i, JAPAN). It was. In addition, for accurate counting, the plate washed with PBS was treated with 0.1% TritonX-100 to recover cells and lactic acid, followed by serial dilution, and then plated into 100 μl of agar plate. The cultures were counted after incubation in a 37 ° C. incubator for 2 days. The control group was an untreated group.

The number of lactic acid bacteria attached to intestinal cells is shown in Table 6, and the adhesion pattern through Gram staining is shown in FIG. 1. PL9034 was most attached at about 3.37 × 10 ⁴ , and PL9028, PL9031 and PL9033 were attached as much as 10 ⁴ , respectively. In addition, PL9034 was confirmed to be enterobacteria with different properties from other selected lactic acid bacteria as shown in the Gram staining photograph.

Number of lactic acid bacteria attached to IPI-21 cells (unit: CFU) PL9028 1.02 x 10 ⁴ ± 1.74 x 10 ³ PL9031 2.13 x 10 ⁴ ± 6.18 x 10 ³ PL9033 9.73 x 10 ³ ± 7.19 x 10 ² PL9034 3.37 x 10 ⁴ ± 8.99 x 10 ³

5-2. For human intestinal cells Attachment

Human intestinal cell line, Caco-2 cells (Human intestinal cell line, KCLB) was incubated in 6 well plates at 1.0 X 10 ⁵ cells / well. After confirming the post-confluence state, the experiment was conducted. The medium used for the culture was used by adding 20% inactivated FBS (Gibco, BRL. USA) to DMEM (Gibco, BRL. USA), and experimented by culturing in a 10% CO ₂ incubator at 37 ° C. Lactobacillus adhesion experiment was performed in the same manner as in the case of IPI-21 cells. The control group was an untreated group.

The number of each lactic acid bacteria attached to the intestinal cells is shown in Table 7, and the adhesion pattern through Gram staining is shown in FIG. PL9034 was most attached at about 1.79 × 10 ⁵ , and PL9028, PL9031 and PL9033 were attached as much as 10 ⁴ , respectively. In addition, PL9034 was confirmed to be enterobacteria with different properties from other selected lactic acid bacteria as shown in the Gram staining photograph.

Number of lactic acid bacteria attached to Caco-2 cells (unit: CFU) PL9028 1.69 x 10 ⁴ ± 8.06 x 10 ³ PL9031 1.22 x 10 ⁴ ± 8.29 x 10 ³ PL9033 9.77 x 10 ³ ± 5.78 x 10 ² PL9034 1.79 x 10 ⁵ ± 4.53 x 10 ⁴

Example  6. Immune-related experiment

RPMI1640 supplemented with macrophage RAW 264.7 (mouse monocyte; macrophage from KCLB (Korean Cell Line Bank)) with 10% FBS (Gibco BRL, Grand island, NYUSA) and 1% antibiotic-antimycotics (Gibco BRL, Grand island, NYUSA) (Roswell Park Memorial Institute-1640, Gibco BRL, Grand island, NYUSA) medium was inoculated at 1.0 x 10 ⁵ / well in 96 wells and stimulated cells with 1 ug / ㎖ of LPS (lipopolysaccaride). Each lactic acid bacteria subcultured in MRS broth was washed three times with PBS, suspended in RPMI1640 so that the final bacterial count was 1.0 X 10 ⁸ CFU / well, aliquoted into plates and incubated in a CO ₂ incubator at 37 ° C for 24 hours. It was. The supernatant of only reacting LPS, reacting only lactic acid, and reacting LPS and lactic acid were collected, made cell-free, and stored at -70 ° C for use in immuno-enhanced cytokine experiments. TNF-α Cytokine kit (Biosource, 542 Flynn Road Camarillo, CA 93012, USA) was used. 50 μl of cell culture supernatant was analyzed in triples according to the manufacturer's protocol. Optical density (OD) was read at absorbance 450 nm with a SPECTRAmax 250 Microplate Spectrophotometer (Molec μlar Devices, Sunnyvale, California, USA). The standard of each cytokine to determine the concentration of cytokine in the sample was generalized to the manufacturer's standard, and the OD value by absorbance was measured using the SOFTmaxPRO ELISA (Molec μlar Devices, USA). The change was measured.

The cytokine values in the immunopotentiation experiments were tested in triple, and the mean, standard deviation, and significance were analyzed by PASW Statistics 17 version (spss Inc. USA), using one-way ANOVA test. The control group was compared with the control group to find significance.

TNF-α, a proinflammatory cytokine, is secreted when non-pathogenic bacteria (lactic acid bacteria) stimulate macrophage cells, resulting in high levels of cytokine mRNA and secretion of IL-10. In the present invention, as a result of measuring the concentration of TNF-α secreted from the macrophage (Raw 264.7) by the above method, compared to the control group of the non-treated group, all the TNF-α involved in cellular immunity increased when only lactic acid bacteria treatment It was. In addition, TNF-α secreted more than 2000 pg / ml when stimulating macrophages with LPS, and PL9031 and PL9034 secreted TNF-α more significantly than LPS alone when LPS was stimulated and treated with Lactobacillus. Decreases (FIG. 3). As a result, all four strains had an immune enhancing effect because they induced TNF-α when only lactic acid bacteria were treated. Especially, PL9031 and PL9034 also showed a function of lowering excessive TNF-α secretion by LPS. It is thought to have a function to minimize the septic shock induced by LPS.

Example  7. Experiment of Resistance Transfer Gene of Lactic Acid Bacteria

7-1. Identification of resistance genes of lactic acid bacteria

Genomic DNA of lactic acid bacteria was performed by modifying Cetyltrimethylammonium bromide extraction method (hereinafter CTAB, Murray and Thompson, 1980). The lactic acid bacteria were inoculated in 5 ml of MRS medium and cultured overnight. The suspension was washed with Tris-EDTA (TE) buffer and pellets mixed with 567 μL TE buffer, 5 μl RNaseA, 15 μl 10% SDS, and 20 unit mutanolysin to repeat pipetting. The suspension was incubated at 37 ° C. for 1 hour with good mixing, then frozen at −70 ° C. for 30 minutes and dissolved at 37 ° C. for 10 minutes. 4 μl proteinase K (20 mg / ml) was added and incubated for 1 hour at 37 ° C. with good mixing until dissolution. 100 μl of 5 M NaCl solution was added and mixed well. 80 μl of a CTAB / NaCl (10% w / v; 0.7 M) solution was added to the suspension and incubated at 65 ° C. for 10 minutes. The lysate was then extracted with approximately equal amounts of chloroform / isoamyl alcohol (24; 1) and phenol / chloroform / isoamyl alcohol (25: 24: 1). Finally, genomic DNA was precipitated at -20 ° C. for 30 minutes by adding one-fold of isopropanol, and centrifuged to resuspend in 50 μl TE buffer.

In order to examine the antibiotic resistance gene of lactic acid bacteria, well-known determinants were confirmed by PCR. Genes related to tetracycline (tet genes) and erythromycin (erm genes), clindamycin (lnu (A) gene) and beta-lactam antibiotic resistance were identified by PCR amplification method and PCR conditions were performed as described in Table 8. It was. As a result, the antibiotic resistance gene of each lactic acid bacteria is shown in Table 9.

Resistance gene primer SEQ ID NO: PCR conditions Size (bp) aad (E) 5'-GCAGAACAGGATGAACGTATTCG-3 '
5'-ATCAGTCGGAACTATGTCCC-3 ' 5
6 30 seconds at 94 ℃
30 seconds at 55 ℃
30 seconds at 72 ° C .;
30 cycles 369 bla 5'-CAT ART TCC GAT AAT ASM GCC-3 '
5'-CGT STT TAA CTA AGT ATS GY-3 ' 7
8 30 seconds at 95 ℃
1 minute at 51 ° C,
45 sec at 72 ° C .;
35 cycles 297 cat 5'-TTA GGT TAT TGG GAT AAG TTA-3 '
5'-GCA TGR TAA CCA TCA CAW AC-3 ' 9
10 30 seconds at 95 ℃
1 minute at 48 ° C
45 sec at 72 ° C .;
35 cycles 300 erm (A) 5'-AAGCGGTAAACCCCTCTGA-3 '
5'-TTCGCAAATCCCTTCTCAAC-3 ' 11
12 30 seconds at 94 ℃
30 seconds at 55 ℃
30 seconds at 72 ° C .;
30 cycles 190 erm (B) 5'-TTTTGAAAGCCGTGCGTCTG-3 '
5'-CTGTGGTATGGCGGGTAAGTT-3 ' 13
14 30 seconds at 94 ℃
30 seconds at 55 ℃
30 seconds at 72 ° C .;
30 cycles 202 erm (C) 5'-AATCGTCAATTCCTGCATGT-3 '
5'-TAATCGTGGAATACGGGTTTG-3 ' 15
16 30 seconds at 94 ℃
30 seconds at 55 ℃
30 seconds at 72 ° C .;
30 cycles 299 lnu (A) 5'-GGT GGC TGG GGG GTA GAT GTA TTA ACT GG-3 '
5'-GCT TCT TTT GAA ATA CAT GGT ATT TTT CGA TC-3 ' 17
18 30 seconds at 95 ℃
30 seconds at 55 ℃
4 minutes at 60 ° C;
25 cycles 323 tet (K) 5'-CAATACCTACGATATCTA-3 '
5'-TTGAGCTGTCTTGGTTCA-3 ' 19
20 30 seconds at 94 ℃
30 seconds at 50 ° C
30 seconds at 72 ° C .;
30 cycles 352 tet (L) 5'-TGGTCCTATCTTCTACTCATTC-3 '
5'-TTCCGATTTCGGCAGTAC-3 ' 21
22 30 seconds at 94 ℃
30 seconds at 53 ℃
30 seconds at 72 ° C .;
30 cycles 385 tet (M) 5'-GGTGAACATCATAGACACGC-3 '
5'-CTTGTTCGAGTTCCAATGC-3 ' 23
24 30 seconds at 94 ℃
30 seconds at 55 ℃
30 seconds at 72 ° C .;
30 cycles 401 tet (O) 5'-AGCGTCAAAGGGGAATCACTATCC-3 '
5'-CGGCGGGGTTGGCAAATA-3 ' 25
26 30 seconds at 94 ℃
30 seconds at 55 ℃
30 seconds at 72 ° C .;
30 cycles 1723 tet (Q) 5'-AGAATCTGCTGTTTGCCAGTG-3 '
5'-CGGAGTGTCAATGATATTGCA-3 ' 27
28 30 seconds at 94 ℃
30 seconds at 63 ℃
30 seconds at 72 ° C .;
30 cycles 169 tet (S) 5'-ATCAAGATATTAAGGAC-3 '
5'-TTCTCTATGTGGTAATC-3 ' 29
30 1 minute at 94 ° C,
1 minute at 55 ℃
2 minutes at 72 ° C .;
30 cycles 573 tet (T) 5'-AAGGTTTATTATATAAAAGTG-3 '
5'-AGGTGTATCTATGATATTTAC-3 ' 31
32 30 seconds at 94 ℃
30 seconds at 46 ℃
30 seconds at 72 ° C .;
30 cycles 169 tet (W) 5'-GGMCAYRTGGATTTYWTIGC-3 '
5'-TCIGMIGGIGTRCTIRCIGGRC-3 ' 33
34 Touchdown 1187 tet B (P) 5'-AAAACTTATTATATTATAGTG-3 '
5'-TGGAGTATCAATAATATTCAC-3 ' 35
36 30 seconds at 94 ℃
30 seconds at 46 ℃
30 seconds at 72 ° C .;
30 cycles 169

Antibiotic resistance genes bla cat linA aad (E) erm (A) erm (B) erm (C) tet (K) tet (M) tet (L) tet (O) tetB (P) tet (Q) tet (S) tet (T) tet (W) PL9028 - - - - - - - - + + - - - - - + PL9031 - - - - - - - - - - - - - - + + PL9033 + - - - - - - - - - - - - - - + PL9034 - - - - - - - - - - - - - - - +

7-2. Check for resistance

Resistant strain whether the strain is Enterococcus faecalis , JH 2-2; Lactococcus lactis , LMG19460). Neve et al. ( J. Bacteriol . vol. 157, 833-838, 1984) was modified by the modified filter mating method. Donor strain (lactic acid bacteria resistant to tetracycline) and receptor strain ( Enterococcus faecalis , JH2-2 (resistant only to rifampicin), Lactococcus lactis , LMG19460 (resistant to rifampicin), was incubated overnight on MRS broth. After incubation the donor: recipient ratio was mixed at 10: 1. Filtered with 0.45 μm nitrocellulose filter, the filter was placed on an MRS agar plate and mated for 24 hours in a 35 ° C. incubator. After mating, the filter was suspended in MRS broth, and the medium was diluted in steps to separate and count donor, receptor and transconjugant cells and plated on MRS agar plates containing appropriate antibiotics. Lactococcus lactis was tested under anaerobic conditions. The final concentration of tetracycline is 64 ug ㎖ ^-1, rampa refuge is 50 ug ㎖ ^- was the ^first. As a result, all four strains with the tet gene did not transfer the resistance gene.

Example  8. Identification of Lactobacillus

Strains were identified by comparing 16S rRNA nucleotide sequences of genes with the nucleotide sequences of known strains to determine the correct species for 4 strains of Lactobacillus genus that have an inhibitory effect on pathogenic bacteria. Identification results are shown in Table 10. In molecular phylogenetic analysis based on 16S rRNA sequences, PL9028 is a standard strain of Lactobacillus salivarius with 99% 16S rRNA homology. salivarius strain CH-9) was identified as the strain showing the highest molecular systemic relationship, PL9031 was 100% 16S rRNA homology, the highest molecular system in the Lactobacillus plantarum strain MiLAB14 ( Lactobacillus plantarum strain MiLAB14) It was identified as a strain showing a flexible relationship. In addition, PL9033 is a standard strain of Lactobacillus plantarum with 99% 16S rRNA homology. plantarum strain BDLP0001) was identified as the strain showing the highest molecular systemic relationship. PL9034 was the standard strain of Lactobacillus amyloborus with 100% 16S rRNA homology. amylovorus strain LAB16) was identified as the strain showing the highest molecular relationship.

Accession number 16S rRNA sequence Species PL9028 KACC91678P SEQ ID NO: 1 L. salivarius PL9031 KACC91679P SEQ ID NO: 2 L. plantarum PL9033 KACC91680P SEQ ID NO: 3 L. plantarum PL9034 KACC91681P SEQ ID NO: 4 L. amylovorus

Agricultural Biotechnology Research Institute KACC91678P 20111026 Agricultural Biotechnology Research Institute KACC91679P 20111026 Agricultural Biotechnology Research Institute KACC91680P 20111026 Agricultural Biotechnology Research Institute KACC91681P 20111026

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Claims (12)

Indicating the characteristics of the following, Lactobacillus Planta room (Lactobacillus plantarum) KACC91679P strain and Lactobacillus Planta room (Lactobacillus plantarum) probiotic composition comprising a strain or culture fluid thereof one or more kinds selected from the group consisting of KACC91680P strains:
(a) exhibits acid and bile resistance;
(b) Enterohemorrhagic Escherichia coli (EHEC), Escherichia coli O157: H7, Escherichia coli O78, Escherichia coli O26, Salmonella enteritidis , Salmonella typhimurium , Salmonella cholera suis s) and Salmonella derby (Salmonella derby) represents the activity of inhibiting the growth;
(c) exhibits adhesion to enterocytes;
(d) does not transfer antibiotic resistance genes; And
(e) inducing secretion of TNF-α from macrophages upon treatment with the Lactobacillus plantarum strain only.
An additive for animal feed comprising the composition of claim 1.
A composition for the treatment or prophylaxis of intestinal pathogenic microbial infection diseases, comprising the composition of claim 1, selected from the group consisting of diarrhea, enteritis, gastroenteritis, abdominal pain and food poisoning.
Enterohemorrhagic Escherichia coli (EHEC), E. coli O157: H7, E. coli O78, E. coli O26, Salmonella enteritidis , Salmonella typhimurium ( Salmonella typhimurium ), comprising the composition of claim 1 number of devices (Salmonella cholerasui s) and Salmonella derby (Salmonella derby) antimicrobial composition for intestinal pathogenic microorganisms is selected from the group consisting of.
delete The composition of claim 1 is administered to an animal other than a human, and selected from the group consisting of enterococci Escherichia coli, Escherichia coli O157: H7, Escherichia coli O78, Escherichia coli O26, Salmonella enteritidis, Salmonella typhimurium, Salmonella cholerasus and Salmonella derby. A method for inhibiting the growth of intestinal pathogenic microorganisms or preventing or treating diarrhea caused by the microorganisms.
delete The method of claim 6, wherein the animal is a pig.
delete Lactobacillus plantarum KACC91679P strain.
Lactobacillus plantarum KACC91680P strain.
delete

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