KR101335455B1 - 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. strains and their use as probiotics}

The present invention is directed to a novel lactobacillus lactic acid bacteria of the genus Lactobacillus excellent in inhibiting harmful pathogens in the intestine, has excellent intestinal cell adhesion, has an immunity enhancing effect, has no resistance transfer, has excellent acid resistance and bile resistance, and its use as a probiotic. The probiotic can be usefully used as feed additives and veterinary compositions for livestock.

By mixing antibiotics with livestock feed and using them in the livestock industry, productivity such as prevention and treatment of livestock diseases such as diarrhea, growth promotion and feed efficiency improvement, etc. was improved, and contributed to the increase of income of livestock farmers.

However, despite these effects of antibiotics, controversy began to arise due to the emergence of resistant strains, identification of residual antibiotics in livestock products, and human safety problems due to the transfer of resistance of bacteria from animals to humans. In addition, the residual test of antibiotics for livestock products is being strengthened. Recently, the European commission banned the use of antibiotics added to feed as a growth promoter in animals. In Korea, the total use of antibiotics in the livestock industry is decreasing to 1,595 tons in 2001, 1,211 tons in 2008, and 999 tons in 2009 (National Veterinary Science and Quarantine Service). The use of (eg bacitracycline, chlortetracycline, colistin, lincomycin, neomycin, oxytetracycline and penicillin) as animal feed was prohibited. In addition, antibiotics other than anthelmintic and anticoccidium are expected to be completely banned as animal feed from 2012.

As the use of antibiotics decreases due to such restrictions and bans on the use of antibiotics, interest in substitutes for antibiotics is increasing. Antibiotic substitutes currently in use include organic acids (acidifiers), probiotics, prebiotics, enzymes for feed addition, immunomodulators, and plant extracts. ) And minerals, but among them, probiotics are receiving the most attention.

Probiotics have generally been defined as probiotic food additives beneficial to health (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 live microbial preparation contained in foods and feeds or dietary additives designed to improve the health of humans or animals. 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 to be provided as a probiotic are safe, must have tolerance to acid and bile, must have adhesion to the intestinal epithelium, must have antagonistic activity against pathogenic bacteria, It should have an immunity enhancing effect, and even if it has a resistance gene, it should not transfer antibiotic resistance.

Although lactic acid bacteria (LAB) helps balance microbes in the intestine, and has many useful points such as antibacterial activity, bright-field activity, and anti-cancer activity, most of the lactic acid bacteria currently used as probiotics are not accurately evaluated as probiotics. There are many, and the development of a system that can suppress the theft of other lactic acid bacteria is also an infinite reality. In addition, it is most ideal to develop probiotics for livestock by separating them in the intestines of livestock, but strains used as many probiotics do not.

Accordingly, the present inventors studied to develop a new lactic acid bacteria that satisfies the conditions as a probiotic, and four types of lactic acid bacteria selected from pig intestine contain acid resistance and bile resistance, inhibiting the growth of harmful microorganisms in the intestine, and enhancing immunity. Therefore, the present invention was completed by confirming that it can be used as a feed additive or an excellent probiotic having a curative effect on infection of pathogenic microorganisms in animals.

One object of the present invention is to provide a lactic acid bacteria strain that is isolated from porcine intestine containing material, exhibits acid resistance and bile resistance, and exhibits activity to inhibit growth of harmful microorganisms in the intestine and enhance immunity.

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

Another object of the present invention is to provide an additive for animal feed comprising the probiotic composition.

Another object of the present invention is to provide a composition for the treatment or prevention of intestinal pathogenic microbial infectious diseases comprising the probiotic composition.

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

As an aspect for achieving the above object, the present invention relates to a lactic acid bacteria strain isolated from porcine intestine containing material, showing acid resistance and bile resistance, and exhibiting inhibitory activity to inhibit growth of harmful microorganisms in the intestine and immune enhancing activity.

Preferably, the lactic acid bacteria strain is Lactobacillus salivarius ( Lactobacillus salivarius) KACC91678P strain. The strain is also referred to as PL9028 in the present invention.

Preferably, the lactic acid bacteria strain is Lactobacillus plantarum ( Lactobacillus plantarum) KACC91679P strain. The strain is also referred to as PL9031 in the present invention.

Preferably, the lactic acid bacteria strain is Lactobacillus plantarum ( Lactobacillus plantarum) KACC91680P strain. The strain is also referred to as PL9033 in the present invention.

Preferably, the lactic acid bacteria strain is Lactobacillus amyloborus ( Lactobacillus amylovorus) KACC91681P strain. The strain is also referred to as PL9034 in the present invention.

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

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

In another aspect, the present invention relates to a composition for the treatment or prevention of intestinal pathogenic microbial infectious diseases comprising the composition.

Preferably, the pathogenic microorganisms are Enterohemorrhagic Escherichia coli (EHEC), Escherichia coli O157:H7, Escherichia coli O78, Escherichia coli O26, Salmonella enteratidis ( Salmonella enteritidis), S. typhimurium (Salmonella typhimurium), cholera, salmonella can be device (Salmonella It may be selected from the group consisting of cholerasui s) and Salmonella derby (Salmonella derby).

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

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

Preferably, the animal comprises a pig.

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

The present inventors collected intestinal material from fecal samples of a total of 1,120 pigs over 2004 and 2006, from which lactic acid bacteria ( Lactobacillus sp.) were 432 total through morphology, Gram staining, catalase reaction, and m μltiplex PCR analysis. The strains were isolated and identified, and four strains having the best effect, namely, PL9028, PL9031, PL9033, and PL9034 strains were selected through an inhibitory ability test for 8 kinds of porcine diarrhea-causing bacteria. All of the four strains exhibited excellent inhibitory ability against 8 species of porcine diarrhea-causing bacteria, and all showed an excellent inhibitory ability of 20% or more even in weakly acidic conditions (pH 5.5-6).

The above four strains exhibited stability in use in hemolysis, gelatin liquefaction test, hazardous substance generation test, and harmful enzyme test, and further showed acid resistance and bile resistance. It satisfies the conditions as probiotic bacteria that must survive in the intestinal tract environment.

In addition, it is reported that the use of tetracycline antibiotics is the most common among antibiotics used for livestock and livestock products, and most of the lactobacilli genus are known to have intrinsic resistance to vancomycin. The strain also showed high resistance to more than 128 ug/ml to both vancomycin and tetracycline in the antibiotic resistance MIC (minimum inhibitory concentration) test. However, penicillin-based penicillin, which is widely used after tetracycline-based antibiotics, was within the susceptibility range of two strains PL9034 and PL9031, and all four strains were susceptible to ampicillin, imipenem, gentamicin, clindamycin and erythromycin. Indicated. In addition, the four strains had the antibiotic resistance gene tet gene and/or bla gene, but it was confirmed that the resistance gene was not transferred.

In addition, the probiotic lactic acid bacteria can settle in the intestine, proliferate and inhabit in the intestine, and all of the four strains have ^{about 10 4} CFU attached to the intestinal cells of pigs and humans, so that they live in the human intestine and The potential to grow under conditions has been identified. In addition, since all four strains induced the secretion of TNF-α when treated alone, the immunity enhancing effect could be confirmed.In particular, the PL9031 and PL9034 strains showed a function of lowering the secretion of excessive TNF-α by LPS (lipopolysaccaride). Therefore, septic shock induced by LPS during bacterial infection can be minimized.

Based on all the above experimental results, the present inventors found that the above four strains can suppress several intestinal pathogenic bacteria, exhibit acid resistance and bile resistance, have excellent intestinal cell adhesion, have an immunity enhancing effect, and have resistance. Since there is no metastasis, the strains could be determined as probiotics to replace antibiotics. Accordingly, the present inventors identified the strain through molecular phylogenetic analysis based on the 16S rRNA nucleotide sequence, and each of these strains was deposited with the National Institute of Agricultural Sciences Agricultural Genetic Resources Center, and were given accession numbers KACC91678P, KACC91679P, KACC91680P and KACC91681P. .

The strains can be used alone, as well as complementary and synergistic functions can be expected even when used in combination. In addition, since this lactic acid bacteria is a type of bacteria that can be used as feed additives and less proliferating at room temperature, it can be used as a probiotic that can promote the growth of livestock and improve feed efficiency by replacing antibiotics.

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

Lactobacillus plantarum KACC91679P strain of the present invention, Lactobacillus plantarum (Lactobacillus plantarum) KACC91680P strain was isolated from pig intestinal material, rod-shaped Gram-positive strain, showing a catalase negative reaction, 16S rRNA of SEQ ID NO: 2 Have a sequence.

The Lactobacillus plantarum KACC91680P strain of the present invention was isolated from porcine intestinal material, is a rod-shaped Gram-positive strain, exhibits 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, is an Enterococci and Gram-positive strain, exhibits a catalase negative reaction, and has a 16S rRNA sequence of SEQ ID NO: 4.

The strain of the present invention can be improved or improved to have an activity equivalent to that of a conventional physicochemical mutation method, and to exhibit excellent field stability or superior antimicrobial activity.

The present invention also includes one or more strains selected from the group consisting of Lactobacillus salivarius KACC91678P strain, Lactobacillus plantarum KACC91679P strain, Lactobacillus plantarum KACC91680P strain, and Lactobacillus amyloboros KACC91681P strain or a culture solution thereof. It provides a probiotic composition. At this time, the microbial culture medium contains various antimicrobial organic acids and non-protein antimicrobial substances produced by microorganisms, so that when included as an active ingredient of a probiotic composition, the same effect as the composition including the strain may be exhibited.

In addition, the probiotic composition according to the present invention is suitable for ingestion by livestock along with the lactic acid bacteria of the present invention, inhibiting the growth of harmful microorganisms when ingested, and improving the balance of intestinal flora. It may contain additionally.

The probiotic composition of the present invention may additionally contain conventional pharmaceutical carriers and excipients in addition to the above active ingredients, and these compositions may be freeze-dried or heat-dried according to a conventional probiotic composition manufacturing method, and encapsulated form or culture It can be in the form of a suspension or dry powder.

The composition of the present invention contains a probiotic lactic acid bacteria having excellent inhibition of intestinal pathogenic bacteria, acid resistance, and bile resistance, or a culture solution thereof, so that infection inhibition and treatment effects of various intestinal pathogenic bacteria are excellent, and by normalizing the intestinal flora of the host. In addition to being able to prevent bacterial diseases, as a probiotic composition such as an intestinal pathogenic bacteria inhibitor, an immune enhancing agent, a fungicide, a digestive agent, an intestinal agent and an antidiarrheal agent, it can be usefully used in feed, food, and medicine. In addition, the microorganism of the present invention is isolated from the intestinal tract of a pig and forms a normal intestinal microbial colony, which is particularly advantageous for livestock breeding.

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 infectious diseases. In addition, the present invention can be usefully used to inhibit the growth of pathogenic microorganisms in the intestine or prevent or treat diarrhea caused by the microorganisms by administering the probiotic composition to animals other than humans.

Livestock to which the probiotics of the present invention can be applied include, but are not limited to, dogs, horses, cows, pigs, sheep, goats, chickens, ducks, turkeys and geese, and are preferably pigs.

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

In addition, the feed additive according to the present invention may be prepared in a form such as fermented feed, blended feed, pellet form, and silage, but is not limited thereto. The fermented feed may be prepared by fermenting organic matter by adding the strain of the present invention and various microbial bacteria or enzymes, and the compounded feed may be prepared by mixing various types of general feed and the Lactobacillus strain of the present invention. . The pellet-type feed may be prepared by formulating the fermented feed or the blended feed into a pellet machine, and the silage may be prepared by fermenting the blueberry 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 intestinal pathogenic microbial infectious diseases, the intestinal pathogenic microorganism is Enterohemorrhagic Escherichia coli ; EHEC), Escherichia coli O157:H7, Escherichia coli O78, Escherichia coli O26, Salmonella enteritidis ( Salmonella enteritidis ), Salmonella typhimurium ( Salmonella typhimurium ), Salmonella cholerasuis ( Salmonella 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, abdominal distension, cholera, and food poisoning, but is not limited thereto.

The composition for the treatment or prevention of intestinal pathogenic microbial infectious diseases can be administered orally or parenterally during clinical administration, and one or two or more pharmaceutically acceptable compositions for the effective amount of the Lactobacillus strain or their culture as the main component A conventional carrier or one or two or more additives may be selected to prepare a composition of a conventional pharmaceutical formulation. The carrier can be used by selecting one or two or more of diluents, lubricants, binders, disintegrants, sweeteners, stabilizers, and preservatives, and one or two or more of flavors, vitamins, and antioxidants can be selected as additives. Can be used.

The novel microorganisms in the genus Lactobacillus of the present invention are probiotics that are acid-resistant and bile-resistant, and that inhibit harmful pathogenic bacteria in the intestine.When administered to livestock by replacing existing antibiotics, it stabilizes the intestinal microflora and occurs due to abnormal fermentation of harmful microorganisms in the intestine. Possible symptoms can be treated or prevented in advance, and health conditions can be improved, and effects such as increase in weight gain, improvement in meat quality, increase in milk production and increase in immunity can be obtained.

1 is a result of confirming the adhesion pattern of strains PL9028, PL9031, PL9033, and PL9034 to IPI-21 cells, which are pig intestinal cell lines, through Gram staining.
2 is a result of confirming the adhesion pattern of strains PL9028, PL9031, PL9033 and PL9034 to Caco-2 cell lines, which are human intestinal cell lines, through Gram staining.
FIG. 3 is a diagram of TNF-a in the macrophage cell line RAW 264.7 in the untreated control group (cell control), LPS alone treatment group (LPS control), lactic acid bacteria alone treatment group (gray bar), and LPS and lactic acid bacteria treatment group (white bar). It shows the degree of secretion.

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

Example 1. Collection of intestinal substances in pigs and separation of lactic acid bacteria

1-1. Collection of intestinal substances in pigs

Lactobacillus 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, cecum and rectum, and can be separated from porcine feces. Accordingly, 1120 pig intestinal material was collected 8 times in 2004 and 2006 from healthy pigs slaughtered in a slaughterhouse located in Gyeonggi-do. In 2004, 572 pig feces samples were collected 4 times in 2004, and 548 pig feces samples were collected 4 times in 2006, and a total of 1,120 pig feces samples were collected from pig intestinal material.

1-2. Isolation of presumed lactic acid bacteria

For the primary isolation of lactic acid bacteria, samples collected on De Man Rogosa (hereinafter MRS, Difco, Becton, Dickinson and Company sparks, MD, U.S.A) medium with 0.002% Bromophenol blue were smeared and incubated for 48 hours in an incubator at 37°C. Colonies that appear in the form of lactic acid bacteria were collected and passed through the first passage. Cultured colonies were selected for negative reactions through catalase reaction experiments, and then Gram-stained and Gram-positive strains and rod-form strains were subjected to M μltiplex PCR (Yu-Li Song, et.al., FEMS Microbiology Letters, 187: 167-173, 2000).

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

Example 2. Pathogen Inhibitory ability Test ( antimicrobial activity test Selection of lactic acid bacteria through)

2-1. Pathogen Inhibitory ability Test

A well test was performed to select a strain capable of inhibiting pig diarrhea-causing bacteria from among the 432 lactic acid bacteria first selected in Example 1. First of all, E. coli as a bacteria that causes pig diarrhea (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) swine), S. typhimurium DT104 (CCARM125, human), S. cholerasuis ( CCARM43, swine) and S. derby ( CCARM42, swine) were selected. The lactic acid bacteria selected in Example 1 were each cultured in 100 ml MRS medium at 37° C. for 24 hours, and then centrifuged (10,000 xg, 15 min) supernatant was mixed with the same amount of ethyl acetate. After shaking vigorously for 1 hour, the ethyl acetate layer was picked and concentrated to 1/100 using an evaporator. Pig diarrhea-causing bacteria were inoculated in M µller-Hington (hereinafter referred to as MH) solid medium with MacFarland 0.5, and 100 µl of lactic acid bacteria concentrate was injected by making a hole with a sterile Pasteur pipette. As a negative control, MRS broth was extracted and used. After incubation at 35° C. overnight, the presence and size of the inhibitory ring were checked, and a total of 4 types of lactic acid bacteria were finally selected.

Table 1 shows the results of measuring the size of the inhibitory rings of the negative control and final selection strains PL9028, PL9031, PL9033 and PL9034. There were no diarrhea-causing bacteria showing inhibitory activity in the control MRS broth extract, but lactic acid bacteria PL9028, PL9031, PL9033 and PL9034 showed excellent inhibitory ability against all eight pathogens. Accordingly, lactic acid bacteria PL9028, PL9031, PL9033, and PL9034 were given accession numbers KACC91678P, KACC91679P, KACC91680P and KACC91681P from the Agricultural Genetic Resource Center of the National Academy of Agricultural Sciences, respectively.

Pathogen Size of suppression ring (mm) Control 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 Inhibitory ability

In order to exclude the inhibitory ability according to the acidity of the lactic acid bacteria, the pH of the culture medium for lactic acid bacteria was raised to weak acidity (pH 5.5 to 6.0), and then the inhibitory ability was examined. Lactobacillus was inoculated in MRS liquid medium and cultured at 35°C for 24 hours, centrifuged (10,000 xg, 4°C for 15 minutes) to remove the cells, and the same amount as the lactic acid bacteria culture supernatant, Mueller-Hinton (hereinafter MH (2X)) After mixing with the medium, the pH was adjusted to pH 6, 5.75, and 5.5 with saturated TRIS, and filtered through a 0.45 μm filter. The diarrhea-causing bacteria used in Example 2-1 were ^{inoculated to a concentration of 10 6} CFU/ml, 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 lactic acid bacteria culture supernatant.

Table 2 shows the growth inhibition rate (%) of diarrhea-causing bacteria of each lactic acid bacteria culture medium according to pH. There were no diarrhea-causing bacteria that showed inhibitory activity in the control MRS broth extract, but lactic acid bacteria PL9028, PL9031, PL9033 and PL9034 all showed an inhibition rate of 20% or more, regardless of pH, for all eight pathogens. Each lactic acid bacteria inhibited a lot of diarrhea-causing bacteria at pH 5.5.

Control 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 the culture supernatant
² : Growth inhibition rate (%) = absorbance of MRS
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, the test bacteria were inoculated on blood agar medium, cultured at 37°C for 24 hours, and the type and type of hemolysis were observed and recorded.

To test the gelatin liquefaction reaction, the test bacteria were inoculated in MRS gelatin medium (0.3 g beef extract, 0.5 g peptone, 12 g gelatin, 100 ml MRS broth) and tested at 35° C. for 6 weeks. After incubation, it was confirmed after cooling for 4 hours at 4°C together with the non-inoculated control group. If the medium did not harden even after refrigeration, it was considered a positive reaction.

To test for the generation of hazardous substances, it was examined whether or not harmful substances such as ammonia, indole, and phenylpyruvic acid were produced.

In order to confirm harmful enzymes, 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 the ability to invade cells, and the ability to break down proteins is required for cell invasion. In the gelatin liquefaction experiment, which is a method of measuring protein resolution, all four strains were negative. In addition, ammonia, which is a harmful metabolite, was not produced in all four strains, and indole and phenylpyruvate were not produced. In addition, among the enzymes known as harmful enzymes produced by some intestinal microorganisms, it was confirmed that not all of the enzymes produced were beta-glucuronidase and beta-glucosidase.

Inspection items PL9028 PL9031 PL9033 PL9034 Hemolysis α α - - Gelatin liquefaction reaction - - - - Urease production - - - - Indole creation - - - - Phenylpyruvate production - - - - Beta-glucuronidase production - - - - Beta-glucosidase production - - - -

3-2. Acid resistance And Bile resistance inspection

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

Bile resistance experiments were performed in artificial bile juice containing 0.3% bile (pork bile extract, Sigma) and 1000 U/ml trypsin in a pH 7.0 medium. . After centrifugation of the artificial gastric juice-treated culture, the same amount of artificial bile as the supernatant was added to react for an additional 90 minutes, followed by diluting compared to the control and spreading on an MRS plate to count the number of viable cells to check the degree of bile resistance.

The acid resistance and bile resistance test results are shown in Table 4. The PL9028, PL9031 and PL9033 strains survived almost 100% even after treatment with artificial gastric juice and artificial bile juice, and PL9034 decreased 1/100 CFU, but also showed acid resistance and bile resistance.

(Unit: CFU) 0 minutes In ^a simulated gastric fluid
90 minutes In artificial bile fluid ^b
90 minutes 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 artificial gastric juice
^b : After treatment of artificial gastric juice, artificial bile Processed

Example 4. Antibiotic susceptibility test of selected lactic acid bacteria

Antimicrobial susceptibility tests were conducted according to the method of the Clinical and Laboratory Standards Institute (CLSI, M45-A, Vol. 26, No. 19, 2006), followed by Cation-adjusted Mueller-Hinton broth with lysed horse blood (hereinafter. CAMHB 2.5 ~ 5%) medium was used to investigate by broth microdilution method. Briefly, the selected lactic acid bacteria were inoculated into MRS agar and _{cultured overnight in a 5% CO 2} incubator, and the cells were in sterile saline (0.85% NaCl) by adjusting the turbidity to a MacFarland value of 0.5 to a final 5 x 10 ⁵ CFU/ µl. It was inoculated in the medium to which antibiotics were added. Imipenem was obtained and used by the Korea Research Institute of Chemical Technology (KRICT), and all other antibiotics were purchased from Sigma (St. Louis, Mo., USA). The concentration of antibiotics was used from 0.25 to 128 mg/ml. ATCC25922 and ATCC49619 were used as QCs for antibiotics, and the results were analyzed according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS).

Table 5 shows the results of the antibiotic resistance test of each lactic acid bacteria. All of the selected lactic acid bacteria showed a high degree of resistance of 128 or more to vancomycin and tetracycline. PL9028 and PL9033 showed resistance to penicillin. In addition, all four strains showed sensitivity 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 ;
*: Based on MIC according to the guidelines set by CLSI, S stands for susceptible, I stands for moderate sensitivity, and R stands for resistant.

Example 5. For intestinal cells Adhesion Experiment

5-1. For pig intestinal cells Adhesion

Pig intestinal cell line IPI-21 cells (Boar miniature male ileum, ECACC) were heat-inactivated with 10% Fetal bovine serum (hereinafter, FBS) and 0.024 IU/ml of insulin (from bovine pancreas, Sigma, St. Louis, Mo). ., USA) added DEME medium (D μl becco's modified minimal essential medium, Gibco, BRL. USA), ^{inoculated into 6 well plates at 1.0 X 10 5} increments, and then confirmed the post-confluence state while incubating. The experiment was carried out. Washing was performed three times with 10 mM phosphate buffer saline (hereinafter, referred to as PBS), and a new DMEM medium was dispensed. Lactobacillus passaged in MRS (about 1 X 10 ⁸ CFU/ml) was washed three times with PBS, inoculated into a 6-well plate, and cultured in a _{37° C. CO 2 incubator for 1 hour.} After the cultured cells were washed three times with 10 mM PBS, each non-adherent lactic acid bacteria was removed, fixed with 4% fixative, Gram stained, and observed with an optical microscope (Nikon, eclipse80i, JAPAN). I did. In addition, for accurate counting, the plate washed with PBS above was treated with 0.1% TritonX-100 to recover cells and lactic acid bacteria, subjected to serial dilution, and then put 100 µl each into an agar plate and spreading the plate. Counting was performed after culturing in an incubator at 37°C for 2 days. The control group was made into an untreated group.

The number of each lactic acid bacteria attached to the intestinal cells is shown in Table 6, and the adhesion pattern through Gram staining is shown in FIG. 1. PL9034 was most often attached at about 3.37 X 10 ⁴ , and PL9028, PL9031 and PL9033 were also attached at about ^{10 4 respectively.} In addition, as shown in the Gram stained photos, PL9034 was confirmed to be enterococci with different properties from other selected lactic acid bacteria.

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 Adhesion

Caco-2 cells (Human intestinal cell line, KCLB), a human intestinal cell line, ^{were inoculated into a 6 well plate at 1.0 X 10 5} cells/well and cultured. After confirming the post-confluence state, the experiment was conducted. The culture medium was used by adding 20% inactivated FBS (Gibco, BRL. USA) to DMEM (Gibco, BRL. USA), and was cultured 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 made into an untreated group.

Table 7 shows the number of each lactic acid bacteria attached to intestinal cells, and Fig. 2 shows the adhesion pattern through Gram staining. PL9034 was most often attached at about 1.79 X 10 ⁵ , and PL9028, PL9031 and PL9033 were also attached at about ^{10 4 respectively.} In addition, as shown in the Gram stained photos, PL9034 was confirmed to be enterococci with different properties from other selected lactic acid bacteria.

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

Macrophage cell line RAW 264.7 (mouse monocyte; macrophage from KCLB (Korean Cell Line Bank)) was added to RPMI1640 with 10% FBS (Gibco BRL, Grand island, NYUSA) and 1% antibiotic-antimycotics (Gibco BRL, Grand island, NYUSA) added. (Roswell Park Memorial Institute-1640, Gibco BRL, Grand island, NYUSA) Cultured in medium, 1.0 X 10 ⁵ /well was inoculated into 96 wells, and cells were stimulated with 1 ug/ml LPS (lipopolysaccaride). Afterwards, each lactic acid bacteria subcultured in MRS broth was washed three times with PBS, ^{and then suspended in RPMI1640 so that the final number of bacteria became 1.0 X 10 8} CFU/well, dispensed into a plate, and incubated in _{a CO 2 incubator at 37°C for 24 hours.} I did. The supernatant of LPS-only reaction, lactic acid bacteria-only reaction, and LPS and lactic acid bacteria-reacted supernatant were collected, respectively, to make a cell free state, and then stored at -70°C and used for immune enhancement-related cytokine experiments. TNF-α Cytokine kit (Biosource, 542Flynn Road Camarillo, CA 93012, USA) was used. 50 µl of the cell culture supernatant was triple analyzed according to the manufacturer's protocol. Optical density (OD) was read at an absorbance of 450 nm with a SPECTRAmax 250 Microplate Spectrophotometer (Molec μLar Devices, Sunnyvale, California, USA). The standard of each cytokine for determining the concentration of cytokine in the sample was generalized to the manufacturer's standard, and the OD value by absorbance was the concentration of cytokine using SOFTmaxPRO ELISA (Molec μLar Devices, USA). Change was measured.

The cytokine values of the immuno-enhancement experiment were tested by triple, and the mean, standard deviation, and significance of each were analyzed by PASW Statistics 17 version (spss Inc. USA), and a one-way ANOVA test was used. The significance was calculated by comparing and analyzing the control group and the experimental group.

TNF-α, a proinflammatory cytokine, is secreted when non-pathogenic bacteria (lactic acid bacteria) stimulate macrophage cells, thereby increasing cytokine mRNA levels and secreting IL-10. In the present invention, as a result of measuring the concentration of TNF-α secreted from the macrophage cell line (Raw 264.7) by the above method, when only lactic acid bacteria were treated, all of the TNF-α involved in cellular immunity increased as compared to the control group that was not treated. I did. In addition, when stimulating the macrophage cell line with LPS, more than 2000 pg/ml of TNF-α was secreted. When the macrophage line was stimulated with LPS and treated with lactic acid bacteria, PL9031 and PL9034 secreted TNF-α more significantly than when LPS alone was treated. Decreased to (Fig. 3). As a result of this, first of all, all 4 strains induced TNF-α when treated with only lactic acid bacteria, thus having an immunity enhancing effect.In particular, PL9031 and PL9034 also showed a function of lowering the secretion of excessive TNF-α by LPS, so bacterial infection It is believed that it has a function to minimize septic shock induced by LPS.

Example 7. Lactobacillus resistance transfer gene test

7-1. Identification of the resistance gene of lactic acid bacteria

Genomic DNA of lactic acid bacteria was performed by modifying the Cetyltrimethylammonium bromide extraction method (hereinafter, CTAB, Murray and Thompson, 1980). Lactic acid bacteria were inoculated into 5 ml of MRS medium and cultured overnight. Tris-EDTA (hereinafter referred to as TE) buffer was washed, and the pellet was mixed with 567 µl TE buffer, 5 µl RNaseA, 15 µl 10% SDS, and 20 units mutanolysin, and pipetting was repeated to prepare a suspension. The suspension was incubated at 37°C for 1 hour while mixing well, 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 at 37° C. for 1 hour while mixing well until lysed. 100 µl of a 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 dose of isopropanol, centrifuged, and resuspended in 50 µl TE buffer.

To investigate the antibiotic resistance gene of lactic acid bacteria, well-known determinants were confirmed by PCR. Tetracycline (tet genes), erythromycin (erm genes), clindamycin (lnu (A) gene) and beta-lactam antibiotics (bla gene) resistance-related genes were confirmed by PCR amplification method, and PCR conditions were performed as described in Table 8. I did. As a result, the antibiotic resistance genes possessed by each lactic acid bacteria are shown in Table 9.

Resistance gene primer Sequence number PCR conditions Size (bp) aad (E) 5'-GCAGAACAGGATGAACGTATTCG-3'
5'-ATCAGTCGGAACTATGTCCC-3' 5
6 30 seconds at 94℃
30 seconds at 55℃
At 72° C. for 30 seconds;
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℃,
45 seconds 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℃
45 seconds at 72°C;
35 cycles 300 erm (A) 5'-AAGCGGTAAACCCCTCTGA-3'
5'-TTCGCAAATCCCTTCTCAAC-3' 11
12 30 seconds at 94℃
30 seconds at 55℃
At 72° C. for 30 seconds;
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℃
At 72° C. for 30 seconds;
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℃
At 72° C. for 30 seconds;
30 cycles 352 tet (L) 5'-TGGTCCTATCTTCTACTCATTC-3'
5'-TTCCGATTTCGGCAGTAC-3' 21
22 30 seconds at 94℃
30 seconds at 53℃
At 72° C. for 30 seconds;
30 cycles 385 tet (M) 5'-GGTGAACATCATAGACACGC-3'
5'-CTTGTTCGAGTTCCAATGC-3' 23
24 30 seconds at 94℃
30 seconds at 55℃
At 72° C. for 30 seconds;
30 cycles 401 tet (O) 5'-AGCGTCAAAGGGGAATCACTATCC-3'
5'-CGGCGGGGTTGGCAAATA-3' 25
26 30 seconds at 94℃
30 seconds at 55℃
At 72° C. for 30 seconds;
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℃
At 72° C. for 30 seconds;
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 gene 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 metastasis of resistance

Receptor strain ( Enterococcus faecalis , JH2-2; Lactococcus lactis, LMG19460). Neve et al. ( J. Bacteriol . vol. 157, 833-838, 1984) was experimented with a modified filter mating method. Donor strain (lactic acid bacteria strain resistant to tetracycline) and receptor strain ( Enterococcus faecalis , JH2-2 (resistant only rifampicin), Lactococcus lactis , LMG19460 (resistant to rifampicin)) was incubated overnight in MRS broth. After incubation, a donor:recipient ratio of 10:1 was mixed. Filtered with a 0.45 μm nitrocellulose filter, the filter was placed on an MRS agar plate and mated in a 35° C. incubator for 24 hours. After mating, the filter was suspended in MRS broth, and this medium was diluted stepwise 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, it was confirmed that all 4 strains carrying the tet gene did not transfer resistance genes.

Example 8. Identification of lactic acid bacteria

In order to determine the exact species for the 4 strains of the genus Lactobacillus having an inhibitory effect on pathogenic bacteria, the 16S rRNA nucleotide sequence of the gene was compared and the strain was identified by comparing it with the nucleotide sequence of the known strains. The identification results are shown in Table 10. In the molecular phylogenetic analysis based on the 16S rRNA sequence, PL9028 was a standard strain of Lactobacillus salivarius with 99% of 16S rRNA homology (Lactobacillus salivarius strain CH-9) was identified as the strain showing the highest molecular phylogenetic relationship, and PL9031 was identified as the highest molecular phylogenetic strain in the standard strain of Lactobacillus plantarum (Lactobacillus plantarum strain MiLAB14) with 100% 16S rRNA homology. It was identified as a strain showing a related relationship. In addition, PL9033 is a standard strain of Lactobacillus plantarum with 99% of 16S rRNA homology (Lactobacillus plantarum strain BDLP0001) was identified as the strain showing the highest molecular phylogenetic relationship, and PL9034 was a standard strain of Lactobacillus amylobulin with 100% 16S rRNA homology (Lactobacillus amylovorus strain LAB16) was identified as the strain showing the highest molecular phylogenetic relationship.

Deposit 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

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

Attach electronic file of sequence list

Claims (7)

Indicating the characteristics of the following, Lactobacillus salivarius (Lactobacillus salivarius ) probiotic composition comprising a KACC91678P strain or a culture thereof:
(a) exhibits acid and bile resistance;
(b) 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 cholerasui 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 salivarius 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.
Chapter, comprising the composition of claim 1 hemorrhagic Escherichia coli (Enterohemorrhagic Escherichia coli; EHEC), E. coli O157: H7, E. coli O78, O26 E. coli, Salmonella entera ET display (Salmonella enteritidis), S. typhimurium (Salmonella typhimurium ) Salmonella cholera suis cholerasui s) and Salmonella derby (Salmonella derby) antimicrobial composition for intestinal pathogenic microorganisms is selected from the group consisting of.
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.
The method of claim 5, wherein the animal is a pig.
Lactobacillus salivarius ) KACC91678P strain.

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