KR101781211B1 - A composition comprised of novel Lactobacillus salivarius KCTC18458P or the culture medium thereof - Google Patents

Abstract

The present invention relates to a probiotic composition comprising a Lactobacillus salivarius KCTC18458P strain or a culture medium thereof deposited in Korea Research Institute of Bioscience and Biotechnology, wherein the strain promotes the growth of pigs and improves diseases resistance by having the properties: (a) when the strain is cultured in a pig intestine imitation model designed with a mouth environment, a stomach environment, and an intestine environment are composed, viability is superior to that of L. rhamnosus GG (LGG), wherein the mouth environment has pH of 6.50.2, a culturing time of 5 minutes, and mucin of 0.5 gl^(-1), the stomach environment has pH of 4.40.2, a culturing time is 2 hours, and mucin of 55 gl^(-1), and the intestinal environment has a pH of 6.50.2 and a culturing time of 5 hours; and (b) when the strain is mixed and cultured with E. coli in pig splenocytes (PSI), an TEER change value is positive.

Description

[0001] The present invention relates to a novel composition comprising Lactobacillus salivarius KCTC18458P or a culture medium thereof,

The present invention relates to a new strain of Lactobacillus salivarius KCTC 18458P isolated from swine feces or a culture thereof, a pig feed additive using the same, and a growth method. Experiments to confirm intestinal fitness of lactic acid bacteria used in feeds have shown that acidic and brittle experiments reflecting extremely limited environments such as acidic environment by acid and acidic environment by bile acid after feeding of lactic acid bacteria are minimized in laboratory scale However, the present invention has been made in view of the fact that the porcine intestinal strains selected from the feces of pigs are used in a complex stress environment, that is, a porcine intestinal model simulating the environment of the actual pigs, (PSI), which is not an experiment of sex, etc., and developed new probiotics and feed additives optimized for pigs through the selection of new strains.

Conventional livestock breeding greatly increases the stress of the target livestock, and it has been pointed out that the immune force is also lowered and the livestock is easily exposed to various diseases and infectious diseases. In order to solve this problem, antibiotics abuse often causes another problem of antibiotics-resistant bacteria, so that the antibiotics are no longer effective as an effective insecticide for rearing livestock. It is known that it does not become a control method. At this point, Korea has also banned the use of antibiotics in 2011, and the demand for substitutes for antibiotics has increased rapidly.

Probiotics such as probiotics have started to attract attention recently as an alternative to these antibiotics. However, until now, the probiotics used as feed additives have been lacking in information on their safety and functionality, and the actual effects have not been confirmed in many cases. The specific proliferative agents developed for gut microbiota of host animals The problem was also pointed out that the effects were not clearly understood either. The screening tests for the probiotic candidate strains used were based on the physiological immunological characteristics of humans, and it was therefore true that there was a lack of consideration for livestock suitability, especially for the inherent characteristics of major livestock.

However, the probiotics have already been widely known to humans for a long time, and in recent years, probiotics have been used to improve the gut microbiota status of the intestinal tract by metagenomics, The results are also reported to be positive. Positive effects of these microorganisms on the host's health include stabilization of intestinal microflora, improvement of digestion, prevention of diarrhea and improvement of immunity (horizon scientific press, 1999), and inhibition of pathogen fixation and abnormal proliferation (Pro Nutr Soc, 2007) have also been reported. Although the scientific basis for livestock fitness has not been established, the livestock industry is paying attention to the efficacy of these probiotics as feed additive. In particular, application of a probiotic agent having an antimicrobial activity has attracted attention as an antibiotic substitute as an environmentally friendly material.

Lactobacillus, Enterococcus, Bacillus, and Saccharomyces are among the most frequently used microorganisms to be added to feeds. Among them, Lactobacillus is a feed additive, and the risk related to the safety of the strain is reported , And it was only presented as a QPS (qualified presumption of safe) as GRAS (generally recognized as safe) (EFSA, 2007). According to data from the Korea Animal Pharmaceutical Association, antibiotics sales data for each season from 2003 to 2010 showed that the use rate of pigs was 54 ~ 57%, which is the highest level of antibiotic dependency. In case of pigs, It is easy to cause infectious diseases accompanied by diarrhea and lowered immunity due to enemy stress, which affects the initial productivity of pigs.

Therefore, the administration of Lactobacillus probiotics, which have a formal and antimicrobial activity, is expected to be a solution to the reduction of the productivity of cultivated finishing piglets as well as young piglets by inhibiting the outbreak of infectious diseases and protecting the digestive organs (Micoecol Ther, 1997; Res Vet Sci, 1999; Korean Society of Animal Resources and Science, 2004). As a feed additive using Lactobacillus , Korean Patent Registration No. 10-1098946 discloses a feed additive composition containing Lactobacillus salivarius G1-1 as an active ingredient. However, the feed additive composition has been developed by paying attention to antimicrobial activity, acid resistance and brittle bacterium of Lactobacillus salivarius G1-1 strain, but it is insufficient to obtain excellent effect as a feed additive.

For example, the addition of probiotic culture byproducts and types of lactic acid bacteria in the nursery feeds affect the growth performance, feed digestibility and diarrhea of the Lactobacillus sp. (1986), Food Engineering Progress, 14 (1), 65-74 (2010.2) have also reported the use of Lactobacillus sp. Strain as a feed additive. Although these feed additives are encouraging in that they use the unique antimicrobial activity of the Lactobacillus sp. Strain, they do not reach high feed additives that can replace the existing antibiotics and promote livestock resistance and growth.

In particular, most of the pig feed additives developed so far have been verified and tested for human cells, so whether or not they are appropriate for the intestinal environment of pigs and whether they can function normally in various stress environments of pigs Since there is not much, it is urgent to develop a probiotic feed preparation customized for pigs. Caco-2 is a colon adenocarcinoma cell, the first cell isolated from a 72-year-old Caucasian male, and Caco-2 is a colon cancer cell. Therefore, in the intestinal in vitro studies, Caco-2 is the most commonly used colon cancer cell line. The problem is that it has the disadvantage of not reflecting the environment and it has the tumorigenic characteristic. Most studies to date on the function of enterocytes have used human cancer cells or mouse cells as such and there has been considerable difficulty in seeing and using the results as being equivalent to the pig intestinal environment. Because the immune system as well as the intestinal morphology of pigs are very different from those of humans or mice (Berl Munch Tierarztl Wochenschr, 2007; Immunology, 1996; Physiol Genomics, 2006). In addition, the results of pathogenic E. coli studies have shown that there is a significant difference in the cell adhesion capacity of human and porcine epithelial cells (Appl Environ Microbiol, 2005). Accordingly, it is necessary to improve the problems of the conventional limit in the field of the art, and to develop a full-fledge-specific probiotic agent.

Korean Patent Publication No. 10-1098946 Korean Patent Publication No. 10-0796963

Master's thesis, Kangwon National University graduate school, Democratic Hong (2016.2)) Food Engineering Progress, 14 (1), 65-74 (2010.2)

SUMMARY OF THE INVENTION The present invention has been made to overcome the problems of the prior art as described above, and it is an object of the present invention to develop a probiotic agent that examines the selection and stress environment of authentic porcine customized probiotics. For this purpose, in order to accurately reflect the intestinal environment of pigs, a strain in the intestines of pigs is selected from pig feces.

In addition, for the safety test of whether there is no problem in human body and pig, antibiotic resistance, bio-aminogenesis, hemolysis, gelatinase activity and BSH activity were confirmed, and E. coli K88, which causes diarrhea, We investigated the adhesion to porcine small intestine cells and the function of probiotics through the change of TEER during mixed culture.

In the above-mentioned processes, the vertebrate environment of the actual pig is modeled, and various experiments for verifying whether or not the functionality as the probiotics in the actual feeding environment are maintained by experimentally reproducing the oral environment, stomach environment, and small intestine To develop commercially viable strains.

For this purpose, PSI (Pig small intestine) cells isolated from adult small intestine cells were mixed with Escherichia coli to verify their functionality. Particularly, a feed additive capable of promoting the growth of pigs and improving disease resistance is selected by selecting strains having excellent ability to inhibit the activity of E. coli, which is the most problematic in breeding pigs, and ability to adhere to small intestine cells themselves The purpose of that is to do.

The present invention confirm the viability in the pig model simulated secretary raised bariwooseu Lactobacillus (Lactobacillus salivarius) provide a probiotic composition comprising at KCTC18458P novel strain or culture fluid thereof. The porcine intestine model is composed of (a) MOUTH environment, STOMACH environment, and INTESTINE environment, the pH of the oral environment is 6.5 ± 0.2, the incubation time is 5 minutes, and Mucin 0.5 g · l ^-1 , the pH of the gastric environment is 4.4 ± 0.2, the incubation time is 2 hours, the mucin is 55 g · l ^-1 , the intestinal environment is 6.5 ± 0.2 and the culture time is 5 hours by said Lactobacillus bacteria raised superior to bariwooseu (Lactobacillus salivarius) viability is Lactobacillus ramno suspension (L. rhamnosus) GG (LGG) in KCTC18458P novel strain and, (b) E. coli in the swine small intestine cell culture (PSI) ( E. coli) and mixed TEER value changes even when the culture is shown as a positive value Lactobacillus ramno suspension (L. rhamnosus) GG (adhesion of the cells from pig small intestine compared to the LGG) or E. coli Growth inhibition effect is more excellent probiotic or Feed additives.

The prophylactic composition comprising Lactobacillus salivarius KCTC 18458P new strain or culture thereof according to the present invention showed better antioxidative effect on E. coli than porcine intestinal environment, Commercialization of pig feed additives or probiotics is expected to contribute considerably to animal husbandry. It is anticipated that it will be possible to dramatically improve the initial survival and growth deterioration problems caused by frequent diarrhea of piglets when pigs are raised by suppressing the growth of E. coli, which is considered to be the main cause of invention of livestock, by replacing existing antibiotics. In addition, as intestinal microflora of an individual is stabilized as a beneficial microorganism, the incidence of individual and collective diseases will be largely lowered, and productivity is expected to be greatly increased.

FIG. 1 is a photograph showing the result of hemolytic activity test, showing that Staphylococcus aureus ATCC 25923 (left) and Lactobacillus salivarius LS6 (right).
2 shows LSH (1), LS 3 (2), LS 4 (3), LS 6 (4) and LS 8 (5) counterclockwise as a result of BSH activity of Lactobacillus salivarius strain derived from pig feces. Respectively.
3 is a graph showing the survivability in the porcine parasitic model of the present invention.
Figure 4 is a standard growth curve of E. coli (Ε.coli) K88.
FIG. 5 is a graph showing the change of TEER at the initial 6 hours after the microbial inoculation in Transwell culture.
Figure 6 is a graph showing the LAB and E. coli (Ε.coli) mixed culture of K88 (co-culture) TEER compared to the initial value of the amount of change in time of 6 hours after PSI cells (%).
7 is a LAB and E. coli (Ε.coli) of K88 mixed cultures (co-culture) when a graph showing E. coli (Ε.coli) K88 and LAB each check adhesion performance results for PSI cells, E. coli (Ε.coli (%) (Left ) of the K88, and the degree of attachment (%) of the LAB (right).

Hereinafter, the present invention will be described in detail with reference to examples.

The strain LS6 of the present invention is Lactobacillus salivarius LDTM7702 and is a novel strain deposited with the Korea Biotechnology Research Institute under the deposit number KCTC18458P, which is characterized by being isolated from pig feces. In addition, the pig feed additive of the present invention comprises the Lactobacillus salivarius KCTC18458P new strain or a culture thereof as an active ingredient, and is particularly characterized in that it is added to a feedstuff.

1. Identification and selection of microorganisms

1) Collecting microorganisms

The microorganisms used in this study were isolated from the feces of the finishing pigs.

2) Identification of microorganisms (16s rRNA  sequencing, API 50 CHL  kit assay)

The isolated microorganisms were subjected to 16s rRNA sequencing analysis on Macrogen. For the analysis, the 16s rRNA gene was amplified by PCR using 27F and 1492R primers, and the nucleotide sequences of the primers 785F and 907R of about 1300 bp or more were obtained.

Table 1 shows the information of the primers used.

Primer Target Sequence 27F / 1492R Universal Primer forward 5'-GGATTAGATACCCTGGTA-3 '
reverse 5'-CCGTCAATTCMTTTRAGTTT-3 ' 785F / 907R Universal Primer forward 5'-AgAgTTTgATCMTGGCTCAg-3 '
reverse 5'-TACggYTACCTTgTTACgACTT-3 '

The obtained nucleotide sequence was compared with the nucleotide database registered in GenBank to complete identification of the microorganism (Accession numbers: AB911506 for L. salivarius , and CP006011 for L. reuteri ). The results of the identification of 16s rRNA are shown in Table 2.

Lab isolates Sequencing result LR1 Lactobacillus reuteri I5007, complete genome LR2 Lactobacillus reuteri I5007, complete genome LS1 Lactobacillus salivarius gene for 16S rRNA, partial sequence, strain: JCM 8662 LS2 Lactobacillus salivarius gene for 16S rRNA, partial sequence, strain: JCM 8662 LS3 Lactobacillus salivarius strain JCM 1231 16S ribosomal RNA gene, partial sequence LS4 Lactobacillus salivarius strain Probio-37 16S ribosomal RNA gene, partial sequence LS5 Lactobacillus salivarius gene for 16S rRNA, partial sequence, strain: JCM 8662 LS6 Lactobacillus salivarius strain Probio-37 16S ribosomal RNA gene, partial sequence LS7 Lactobacillus salivarius gene for 16S rRNA, partial sequence, strain: JCM 8662 LS8 Lactobacillus salivarius strain Probio-37 16S ribosomal RNA gene, partial sequence

The sequence of the 16s rRNA of the LS6 strain is shown in FIG.

In addition, biochemical sugar fermentation characteristics of microorganisms were examined using API50 CHL kit (bioMerieux, France) for accurate microbial identification. The results are shown in Table 3.

Lab isolates % identity identification LR1 99.9 Lactobacillus reuteri LR2 99.9 Lactobacillus reuteri LS1 99.9 Lactobacillus salivarius LS2 99.9 Lactobacillus salivarius LS3 99.9 Lactobacillus salivarius LS4 99.9 Lactobacillus salivarius LS5 99.9 Lactobacillus salivarius LS6 99.9 Lactobacillus salivarius LS7 99.9 Lactobacillus salivarius LS8 99.9 Lactobacillus salivarius

3) Selection of laboratory microorganisms

The function of lactic acid bacteria is known to be strain-specific, such as survival in the intestinal tract, adhesion to enterocytes, and antimicrobial activity against pathogenic microorganisms. Therefore, in order to confirm the function of microorganisms in the porcine intestinal model and the porcine intestinal cell model proposed by the present inventor, it is judged that it is correct to compare with the same strain, and five Lactobacillus salivarius strains which occupy a relatively large pool are used as test samples Were randomly selected. The selected strains and the microorganisms used in this study are shown in Table 4.

Strain purpose of use Culture conditions Lactobacillus salivarius LS1 테스트 전압 37 C, MRS (Difco) Lactobacillus salivarius LS3 테스트 전압 37 C, MRS (Difco) Lactobacillus salivarius LS4 테스트 전압 37 C, MRS (Difco) Lactobacillus salivarius LS6 테스트 전압 37 C, MRS (Difco) Lactobacillus salivarius LS8 테스트 전압 37 C, MRS (Difco) Lactobacillus rhamnosus GG 정정 제어 37 C, MRS (Difco) Escherichia coli K88 기준
antimicrobial activity test 37 ° C, BHI (Difco) Enterococcus faecalis ATCC 29212 - positive control for biogenic
amine production gelatinase
activity test
- reference strain for
antimicrobial activity test 37 ° C, BHI (Difco) Listeria innocua ATCC 33090 기준
antimicrobial activity test 37 ° C, BHI (Difco) Listeria monocytogenes KCTC 13064 기준
antimicrobial activity test 37 ° C, BHI (Difco) Staphylococcus aureus ATCC 25923 positive control for
haemolytic test 37 ° C, BHI (Difco)

2. Safety check

1) Investigation of antibiotic resistance

The minimum inhibitory concentration (MIC) of the test strains was determined using the Lactobacillus spp . Assay of the National Committee for Clinical and Laboratory Standards Institute guidelines (NCCLS, 2010) . Of broth microdilution susceptibility methods. The strain cultured at 37 ° C for 18 hours was adjusted to a 600 nm absorbance of 0.3 on double concentrated MRS media. The applied antibiotics were diluted in serial two-folds in a 96-well microplate at twice the concentration. Antibiotic resistance cut-off values (mg / l) of microorganisms were used according to the European Food Safety Authority guidelines (EFSA, 2012). The results of the experiment are shown in Table 5. In Table 5, AMP, VAN, GEN, CHL, STR, ERY and CLI are ampicillin, vancomycin, gentamycin, chloramphenicol, streptomycin, erythromycin and clindamycin, respectively.

AMP VAN GEN CHL STR ERY CLI L. salivarius LS1 2 > 512 64 2 128 One 2 LS3 2 > 512 64 4 256 > 8 > 8 LS4 One > 512 128 4 512 > 8 > 8 LS6  One > 512 128 2 256 One 0.5 LS8  2 > 512 128 2 256 One 0.25 Suggested breakpoints for L. salivarius CLSI = 16 = 16 = 8 = 2 EFSA 4 n.r. 16 4 64 One One SCAN 2 4 One 16 16 4 Danielsen & Wind 4 4 256 16 > 256 One 2

The criteria for determining the antibiotic resistance of Lactobacillus are currently proposed by CLSI, EFSA, and SCAN, but those that have been validated and standardized are pathogenic microorganisms, and the criteria for antibiotic resistance compartment points to Lactobacillus There is a controversy about the research on the. Therefore, the criteria proposed by Danielsen & Wind (2003) are also included in this study.

In the case of vancomycin, according to EFSA, Lactobacillus spp . Was suggested to be basically acquiring tolerance to vancomycin, so it was considered appropriate to have a high MIC. Resistance to erythromycin and clindamycin was confirmed in LS3 and LS4 strains.

In other words, by combining the above four MIC breakpoints, it was confirmed that LS6 and LS8 have safety that can be used as feed additive probiotics.

2) Amine  Investigation of creation

Biogenic amines (BA) are usually produced by the microbial substrate-specific decarboxylase enzymatic reaction to produce histamine, tyramine, putrescine, cadaverine, . The high concentration of these amino acid products is toxic and is known to cause unpleasant odors as well as allergic reactions, sympathetic nervous system promotion, and vasodilation in the host. Therefore, it is one of the criteria to judge the safety of the lactic acid bacteria (LAB). The BA broth and agar composition used in this experiment were performed according to the procedure proposed by Bover-Cid & Holzapfel (1999).

Five L. salivarius Experimental strains were subcultured daily at 1% inoculum for 5 days. At the same time, a positive control, Enterococcus faecalis ATCC 29212, proceeded simultaneously.

After 5 days, the bacteria were streaked into BA agar prepared in advance and incubated at 37 ° C in an aerobic and anaerobic incubator (Coy Laboratory Products, Ann Arbor, Mich .; 5% CO2, 10% H2, 85% Lt; / RTI >

(AE) and anaerobic (ANAE) culture conditions in the experimental group except for the positive control (Table 6).

positive
control LS1 LS3 LS4 LS6 LS8 AE ANAE AE ANAE AE ANAE AE ANAE AE ANAE AE ANAE Bio-amines + + - - - - - - - - - - haemolytic + + - - - - - - - - - - gelatinase 1.1 One 0.85 One 0.9 0.8 0.8 0.8 0.8 0.8 0.7 0.7

3) Hemolytic ( Haemolytic ) And gelatinase gelatinase ) Activity investigation

The haemolytic activity and gelatinase activity of microorganisms were examined by Birri et al. (2013). ≪ / RTI > Blood agar base (Oxoid) medium supplemented with 7% sheep blood was prepared, and the microorganisms cultured for 18 hours after 1% inoculation were streaked and cultured in an aerobic and anaerobic incubator at 37 ° C for 2 days . Staphylococcus aureus ATCC 25923 was used as a positive control.

Staphylococcus aureus ATCC 25923 (left) and Lactobacillus salivarius The results of haemolytic activity of LS6 (right) showed that LS6 was not haemolytic in both aerobic and anaerobic culture conditions.

To investigate the gelatinase activity of microorganisms, a Todd-Hewitt (Oxoid) medium supplemented with 3% bovine gelatin was prepared, and the microorganisms cultured for 18 hours after 1% inoculation were diluted with 0.85% NaCl solution at a 600 nm absorbance of 1.0. After incubation for 2 days at 37 ℃ aerobic and anaerobic incubator, a spot was taken by using a needle in the medium. The cells were placed in a refrigerator at 4 ℃ for 5 hours before observing the zone formed around the spot. Positive controls include Enterococcus faecalis ATCC 29212 was used.

As shown in Table 6, the gelatinase activity of Lactobacillus salivarius strain was found to be lower than that of positive control.

As a result, Lactobacillus salivarius All of the experimental strains were judged to have secured the basic safety.

3. Functional Review

In order to verify functionality as a potential probiotic agent, it is necessary to confirm the result that the host has a beneficial function. Thus, the present invention additionally confirmed the inhibition of intestinal adhesion of the pathogens in the new porcine intestine model and the porcine small intestinal cell model and additional selection criteria.

In the functional review process, the control strain is a third-generation Lactobacillus, and probiotic function is already known in L. rhamnosus GG (LGG) was used, and L. salivarius spp . Respectively. However, the above-mentioned LGG is a proven probiotic in vitro and in vivo with various functions that have been germs isolated from healthy persons and have been generally regarded as safe (GRAS). In addition, it is one of the most widely distributed microorganisms in the world as a probiotic for humans in general, and is often used as a comparative group when examining the probiotic function of newly isolated microorganisms.

1) Bile salt hydrolase (BSH) activity test

BSH activity of microorganisms detoxifying bile salts in the body is known to have a function to lower cholesterol during the growth of pigs (J Food Prot, 2001). Therefore, in the present invention, it was verified whether or not the BSH activity of the experimental strains isolated from pig feces could be confirmed to ensure healthy growth at the time of feeding.

The medium used was 0.5% (w / v) sodium salt of taurodeoxycholic acid (Sigma, USA) and 0.37 g · l ^-1 of CaCl ₂ . All of the experimental strains were prepared by inoculating 1% of the culture solution for 18 hours and then diluting the culture with 0.85% NaCl solution at an absorbance of 600 nm. A blank disk was placed on a plate with a disk dispenser (Oxoid) in the medium, and 10 μl of the prepared microbial dilution was inoculated. The cells were incubated in an anaerobic incubator at 37 ° C. for 3 days. As a result, LS 1 All L. salivarius strains showed high BSH activity, such as LS 2 (4), LS 4 (3), LS 6 (4) and LS 8 (5).

2) Antimicrobial activity test

The L. salivarius of the present invention The strain was developed as an alternative agent for AGPs (Antibiotics as growth promoter), and it is required to have antimicrobial properties against pathogenic microorganisms. In addition, the antibacterial properties of microorganisms should be established as probiotics. Therefore, the characteristics of selected strains were verified.

The pathogens used were obtained by diluting BHI broth with Mcfarland 1 to 2 OD ₆₀₀ = 0.3, plated on a MRS agar plate with a sterilized cotton swab, and prepared by placing a blank disk on a plate with a disk dispenser (Oxoid). L. salivarius The strains were washed by centrifugation (4 ° C, 10,000 rpm, 3 minutes), incubated for 18 hours after 1% inoculation, diluted in 1 × PBS solution at 600 nm absorbance at 1.0, For 24 hours (E. coli, E. faecalis group) and 48 hours ( Listeria spp. Group) in an incubator.

Experimental strains were E. coli K88 and L. innocua ATCC33090 showed antimicrobial activity in both aerobic and anaerobic conditions. However, E. faecalis The antimicrobial activity against ATCC 29212 was not observed in the anaerobic state, and only the LS1 and LS8 strains were active in the aerobic phase.

L. monocytogenes The antimicrobial activity against KCTC13064 was not observed in anaerobic condition, but all showed high antimicrobial activity in aerobic condition. As a whole, L. rhamnosus GG.

E. coli and L. innocua , which showed antimicrobial activity of L. salivarius strains in anaerobic environments, were noticed in pigs' intestinal environment, E. coli associated with mortality K88 was used as a target pathogen for the inhibition of the adherence of pathogenic microorganisms to porcine small intestinal cells.

Table 7 shows the test results of the antimicrobial activity against pathogenic bacteria of L. salivarius strain. In Table 7, growth inhibition diameter was classified as more than 3 cm (+++), more than 2 cm (++), less than 1 cm (+), and a diameter is unclear (-). AE, and ANAE are aerobic and anaerobic conditions at 37 ℃, Ec, Ef, Lm and Li are E. coli K88, E. faecalis ATCC29212, L. monocytogenes KCTC13064 and L.innocua ATCC33090.

Strain E.c E.f L.m. L.i AE ANAE AE ANAE AE ANAE AE ANAE L. salivarius LS1 + ++ (+) - ++ - ++ +++ LS3 + + - - ++ - + ++ LS4 + ++ - - ++ - ++ ++ LS6 + ++ - - ++ - +++ ++ LS8 + ++ (+) - ++ - ++ ++ L. rhamnosus GG + ++ - - ++ - ++ ++

3) Enzymatic profile

Enzymes possessed by L. salivarius strains derived from pig feces were analyzed using API ZYM kit (bioMerieux, France). Experimental strains were diluted with Mcfarland 5 ~ 6 and inoculated on API ZYM strips and incubated at 37 ° C for 4 hours. After that, the ZYM reagents A and B were dropped one by one in order, and after 5 minutes, the color change was observed and the results were obtained. As a result, Table 8 shows the result of enzyme profile of L. salivarius strain and classified into (+) when there is color change and (-) without color change.

division LS1 LS3 LS4 LS6 LS8 Control - - - - - Alkaline 포스화제 - - - - - Esterase (C4) - - - - - Esterase Lipase (C8) - - - - - Lipase (C14) - - - - - Leucine arylamidase + + + +   + Valine arylamidase - - (+) - - Crystine arylamidase - - - (+) - Trypsin - - - - - a- chymotrypsin - - - - - Acid phospatase - - - (+) (+) Naphtol-AS-BI-phosphohydrolase  - - - - - α- 가alat 시드세제 - - - - - β- 가alat 시드세제 + + + + + β- glucuronidase - - - - - α- glucosidase - - - - - β- glucosidase - - - - -

4) Pig pig  In the simulated model Survivability

It is clear that the intestinal stress environment of pigs is obviously different from that of humans, and the pH of the pigs varies according to the kind and amount of food. Human stomach pH has been reported to range from 2.0-4.0 depending on the study (Biopharm Drug Dispos, 1995), and the pH in the small intestine of the pigs has been reported to be 6.1-6.7 and lower than the human small intestine pH 6.4-7.3. Based on the results of this study, it was found that, in order to establish the intestinal stress environment of pigs and imitate physical environment of pigs, anaerobic culture condition, passage time setting of culture mixture according to the length of each organ, After establishing the model by studying the rpm setting, the viability of the fecal - derived strains was confirmed.

The in vitro pig gastrointestinal tract (GIT) simulation model consists of three digestive systems: mouth, stomach, small intestine. In the present invention, a pig GIT model is described by Oomen et al. (2003) modified the human GIT simulation model according to the physiological anatomical environment of pigs (Kararli 1995; Murdan et al. 2011), the pH, the residence time, and the amount of mucin.

Table 9 shows the constituent reagents and concentrations applied to each fire extinguishing system in this model.

Inorganic Solution Organic Solution Digesitve Constituents Human & Pig factors


Mouth
(saliva) KCl 89.6 g · l ^-1
NaCl 175.3 g · l ^-1
KSCN 20 g · l ^-1
NaH ₂ PO ₄ 88.8 g · l ^-1
Na ₂ SO ₄ 57 g · l ^-1
NaOH 40 g · l ^-1 urea 25 g · l ^-1 a-amylase
Uric acid Total pH: 6.5 ± 0.2
Incubation time: 5 min
Mucin: 0.5 g · l ^-1



Stomach
(gastric juice)
KCl 89.6 g · l ^-1
NaCl 175.3 g · l ^-1
NaH ₂ PO ₄ 88.8 g · l ^-1
NH ₄ Cl 30.6 g · l ^-1
CaCl ₂ ? 2H ₂ O 22.2 g · l ^-1
HCl 37% g · g ^-1
urea 25 g · l ^-1
glucose 65 g · l ^-1
glucuronic acid 2 g · l ^-1
glucoseamine hydrochloride 33 g · l ^-1
Bovine serum
albumin
Pepsin
Mucin
Human
Total pH: 3.2 ± 0.2
Incubation time: 2 h
Mucin: 33 g · l ^-1 Pig
Total pH: 4.4 ± 0.2
Incubation time: 2 h
Mucin: 55 g · l ^-1





Intestine
(duodenal & even juice) KCl 89.6 g · l ^-1
NaCl 175.3 g · l ^-1
NaHCO ₃ 84.7 g · l ^-1
KH ₂ PO ₄ 8 g · l ^-1
MgCl ₂ 5 g · l ^-1
HCl 37% g · l ^-1
KCl 89.6 g · l ^-1
NaCl 175.3 g · l ^-1
NaHCO ₃ 84.7 g · l ^-1
HCl 37% g · g ^-1
urea 50 g · l ^-1
CaCl ₂ .2H ₂ O 44.4 g · l ^-1
Bovine serum
albumin
Pancreatin
Lipase
Even bovine
Human
Total pH: 6.8 ± 0.2
Incubation time: 2 h Pig
Total pH: 6.5 ± 0.2
Incubation time: 5 h

The reagents used in each digest were all Sigma-aldrich products and each digestion solution was pre-warmed to 37 ° C before mixing. To simulate the in vitro digestion process of pigs, 7 ml of L. salivarius The strains were subjected to centrifugation (4,000 rpm, room temperature, 10 minutes) at about 1.0 × 10 ⁹ cells / ml ^-1 and re-suspension was performed with 1 ml of 1 × PBS (phosphate buffered saline) solution and 6 ml saliva . After 5 min incubation at 37 ° C, 12 ml of gastric juice was added (total pH 4.4) and incubated for 2 h.

Then 12 ml of duodenum juice, 6 ml of bile acid, 2 ml of NaHCO ₃ were added (total pH 6.5) and further cultured for 5 hours. The cultures were performed in a 37 ° C anaerobic chamber (Coy Laboratory Products, Ann Arbor, Mich .; 5% CO ₂ , 10% H ₂ , 85% N ₂ atmospheric composition) and simulated intestinal peristaltic contractions And continuously mixed at 50 rpm.

In general, the number of viable cells before and after the pig GIT was counted by counting the number of viable cells before and after the pig GIT using a method of dropping on an agar plate after serial dilution as a usual method of counting the number of bacteria, and the survival rate was calculated according to the following equation One).

In this experiment, L. rhamnosus The fold change in survival rate versus GG (LGG) is shown in FIG. L. salivarius LS4 and LS6 showed high survival rate compared to LGG. They showed that the tolerance to complex stress environment in GIT was superior to that of LGG strain. However, the remaining strains showed low survival rate compared to LGG .

5) Pathogenic microorganisms to pig small intestinal cells Attachment  control

The Transwell (1.12 cm seeding area, 0.4 μm pore size, Corning, USA) used in this experiment is a further developed form of the existing experimental method in which adherent cells can be cultured on the plate surface to test only apical aspects of the cells. It is a system that can simultaneously check the influence on apical aspects. In addition, PSI cells have the property of being firmly adhered to the epithelial layer, which is advantageous to apply the Transwell 3D model.

In this experiment, L. salivarius strains derived from pig feces and E. coli When K88 was mixed with PSI cells, cell adhesion inhibition of pathogenic microorganisms and inhibition of cell damage by pathogenic microorganisms were confirmed.

Pathogenic microorganism E. coli The reason why K88 was selected as an experimental microorganism is that this microorganism is a typical microorganism causing diarrhea especially in pig pig pigs and has been used as the most dangerous microorganism in swine farms and many antibiotics have been developed and used in the past, This weakness is known to be fatal to children or the elderly. Thus, E. coli It is stated that if the functionality is confirmed during mixing with K88, it can also be used as an additive for breeding mammals with intestinal environment similar to that of pigs.

On the other hand, the degree of cell damage is confirmed by the value of TEER (trans epithelial electric resistance) using a Millicell-ERS electrode (Millipore, USA). Since the TEER value reflects the permeability of intracellular tight junctions, damage, and the positive TEER value can be regarded as a basis for the probiotic function of cell protection.

PSI cells 10% foetal bovine serum (FBS, Gibco, ^{USA), L-glutamine (2mmol·l -1} ), 100㎍ streptomycin ㎖ -1, 100U penicillin ㎖ -1, 0.25㎍ amphotericin B㎖ -1 (Gibco, USA), and cultured at 37 ° C and 5% CO _{2. The} cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA). The cultured cells were inoculated on Transwell inserts 12 mm membrane at 1.0 × 10 ⁵ cells / ㎠ and cultured for about 2 weeks with changing media every day until a polarized monolayer (TEER> 2500 O / ㎠) was formed. L. salivarius strains were inoculated 1% in 10% MRS-supplemented free DMEM medium, and after 18 hours, they were washed by centrifugation (4,000 rpm, 10 min) and absorbed at 600 nm at 1.0 (approximately 1.0 × 10 ⁷ cfu / And diluted in DMEM.

E. coli K88 was inoculated 1% in free DMEM medium supplemented with 10% BHI, and after 18 hours, it was centrifuged (4,000 rpm, 10 minutes) and diluted to approximately 5.0 × 10 ⁶ cfu / Respectively. As experimental strains, LS4 and LS6 derived from pig feces and LGG were used as control strains which showed excellent viability in Pig GIT. Each microorganism was inoculated with 0.5 ml of transwell containing PSI cells in a polarized monolayer, and cultured for 6 hours at 37 ° C and 5% CO ₂ .

FIG. 5 shows the TEER change value at 6 hours after the microbial inoculation. In E. coli K88, a pathogenic microorganism, the TEER value was reduced by more than 200%, indicating that the cell damage was severe, which is due to the weakness of the tight junction between the PSI cells and increased permeability. L. salivarius and L. rhamnosus increased the amount of TEER to a positive value when treated alone, and LGG and LS4 showed excellent changes, and LS6 was also found to have no damage to the cells.

Second, the pig manure-derived L. salivarius When the strain and the pathogenic microorganism E. coli K88 were mixed with PSI cells, inhibition of cell adhesion of pathogenic microorganisms and mitigation of cellular damage by pathogenic microorganisms were confirmed. Cells and microorganism preparation conditions were prepared according to the design described above. Each L. salivarius , L. rhamnosus The strain and E.coli K88 1: 1% (v / v) ( total 0.5㎖) to the PSI cells were mixed to inoculate transwell, forming a monolayer polarised for 6 hours in 37 ℃, 5% CO ₂ conditions Mixed culture. The results of TEER measurement of PSI cells after 6 hours of mixed culture are shown in FIG. In other words, except for the treatment with K88 + LS6, the TEER change was negative in all the remaining groups, and it was confirmed that the K88 + LS4 group had greater cell damage than K88 + LS6 treatment alone. In addition, the K88 + LGG inoculation group had a negative value of TEER value relatively lower than that of the K88 + LGG inoculation group, suggesting that LGG has a relatively high cytotoxic effect. As a result, only the K88 + LS6 group had a meaningful result in which the TEER change of PSI cells was measured as a positive value. Thus, the LS6 strain showed that the effect of mitigating cell damage by E. coli would be excellent in piglets.

This result seems to be due to the competition between LAB and pathogens (antimicrobial action, competition for nutrients, adhesion to sites, etc.). Therefore, in order to provide a basis for the interpretation of the TEER experimental results and the mechanism of action of the strain, E. coli K88, L. salivarius , L. rhamnosus GG strains were observed in PSI cells. The results are shown in Fig. 2.5 hours after the inoculation, all media of the transwell inserts were removed, washed twice with 1xPBS buffer solution, and treated with 0.5 ml of 0.05% trypsin-EDTA (Gibco, USA) to remove all cells from the membrane. After the serial dilution, microorganisms were counted by incubating at 37 ℃ for 24 hours. LAB counts were performed on MRS agar plates and E. coli K88 counts were performed on Salmonella Shigella agar plates (Difco, USA). Previous studies have shown that both media are suitable for selective counting of each colony from samples mixed with Lactobacillus and E. coli K88. The degree of attachment (%) of microorganisms was calculated according to the following formula (2).

Figure 7 shows the hypothesis for Figure 6 that the attachment of E. coli K88 to E. coli K88 was inhibited to about 20% only in the K88 + LS6 treated group (Fig. 7, left) 60%, higher than LGG (Fig. 7, right). In the case of the other treatment groups (K88 alone, K88 + LGG, K88 + LS4), the adhesion of E. coli K88 was increased to about 60%, and the adhesion of LGG and LS4 was about 40% and 30% It has been confirmed that Escherichia coli has the highest functionality and survivability when compared to the weakened activity in the small intestine.

As a result, LS6, Lactobacillus salivarius KCTC18458P is a new lactic acid bacterium, and it is estimated that it has secured the basic safety and probable use of probiotics as pig feed. In other words, Lactobacillus salivarius KCTC18458P of the present invention is expected to overcome all the stresses in the mouth, stomach and intestines of pigs, greatly inhibit the activity of E. coli in the intestines, and to significantly increase survival in the intestines as compared with other strains , It is expected that the feedstock will be able to promote the growth of pigs and greatly improve the disease resistance at the time of feeding to the livestock farmers with the probiotics or feed additives.

Therefore, the Lactobacillus salivarius KCTC18458P new strain of the present invention can be fed as a probiotic agent and may be included as an active ingredient of a conventional feed additive. In other words, feed additives can be prepared by inoculating appropriate amounts of the finishing feed and mixing them.

The embodiments described above are provided by way of example for the purpose of enabling a person skilled in the art to sufficiently transfer the technical idea of the present invention to the embodiments described above, But may be embodied in other forms without limitation.

Institution name: Korea Biotechnology Research Institute

Access number: KCTC18458P

Funding date: 2016324

<110> Seoul National University R & DB Foundation <120> A composition comprised of novel Lactobacillus salivarius          KCTC18458P or the culture medium thereof <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 982 <212> DNA <213> Lactobacillus salivarius <400> 1 ggggtgacgt ttgtcggatt attgggcgta agggaacgca ggcggtcttt taagtctgat 60 gtgaaagcct tcggcttaac cggagtagtg cattggaaac tggaagactt gagtgcagaa 120 gaggagagtg gaactccatg tgtagcggtg aaatgcgtag atatatggaa gaacaccagt 180 ggcgaaagcg gctctctggt ctgtaactga cgctgaggtt cgaaagcgtg ggtagcaaac 240 aggattagat accctggtag tccacgccgt aaacgatgaa tgctaggtgt tggagggttt 300 ccgcccttca gtgccgcagc taacgcaata agcattccgc ctggggagta cgaccgcaag 360 gttgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc 420 gaagcaacgc gaagaacctt accaggtctt gacatccttt gaccacctaa gagattaggt 480 tttcccttcg gggacaaagt gacaggtggt gcatggctgt cgtcagctcg tgtcgtgaga 540 tgttgggtta agtcccgcaa cgagcgcaac ccttgttgtc agttgccagc attaagttgg 600 gcactctggc gagactgccg gtgacaaacc ggaggaaggt ggggacgacg tcaagtcatc 660 atgcccctta tgacctgggc tacacacgtg ctacaatgga cggtacaacg agtcgcgaga 720 ccgcgaggtt tagctaatct cttaaagccg ttctcagttc ggattgtagg ctgcaactcg 780 cctacatgaa gtcggaatcg ctagtaatcg cgaatcagca tgtcgcggtg aatacgttcc 840 cgggccttgt acacaccgcc cgtcacacca tgagagtttg taacacccaa agccggtggg 900 gtaaccgcaa ggagccagcc gtctaaggtg ggacagatga ttgggtgaat tagaaaaaaa 960 ccccgcagac acaaaaaaaa ag 982

Claims (4)

A probiotic composition comprising Lactobacillus salivarius KCTC 18458P new strain or a culture thereof, exhibiting the following characteristics.
(a) MOUTH environment, STOMACH environment, and intestine environment, the pH of the oral environment is 6.5 ± 0.2, the incubation time is 5 minutes, the Mucin is 0.5 g · l ^-1 And the gastric environment was pH 4.4 ± 0.2, incubation time 2 hours, mucin 55 g · l ^-1 , intestinal environment pH 6.5 ± 0.2 and incubation time 5 hours. Excellent survival compared to L. rhamnosus GG (LGG) during culture; And
(b) positive value of TEER change in cultured pig small intestine (PSI) mixed with E. coli ; And
(c) The sequence of 16s rRNA is as follows.

An additive for pig feed comprising the composition of claim 1. A method for suppressing the growth of coliform bacteria in a small intestine of a pig by feeding the composition of claim 1 to a pig. Lactobacillus salivarius KCTC18458P New strain & lt ; RTI ID = 0.0 & gt;

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