KR100211529B1 - A novel lactobacillus reuterii which inhibits harmful microorganism and probiotics for livestock - Google Patents

Abstract

The present invention relates to a novel acid-resistant Lactobacillus genus microorganism having an inhibitory activity against harmful microorganisms, and according to the present invention, Lactobacillus reuterii anaerobicly isolated from domestic sows feces (Lactobacillus reuterii) has acid resistance and bile resistance, As a novel lactic acid bacterium that is resistant to antibiotics and exhibits the property of inhibiting the growth of harmful microorganisms such as pathogenic bacteria, the Lactobacillus flora in feces is maintained high while E. coli flora is decreased over time when administered to livestock such as piglets. Smaller feeds can also be increased enough to be useful as livestock bioactive agents.

Description

Novel acid-resistant Lactobacillus microorganisms with harmful microbial inhibitory activity and bioactive agents for livestock containing the same

The present invention relates to a novel acid-resistant Lactobacillus sp. Microorganism having an inhibitory activity against harmful microorganisms, and more particularly, as a novel lactic acid bacterium anaerobicly isolated from porcine feces, having acid resistance, bile resistance, and resistance to antibiotics and pathogenicity. It relates to a novel microorganism Lactobacillus reuterii that can be used as a livestock bioactive agent by showing the property of inhibiting the growth of harmful microorganisms such as bacteria.

Probiotics are medicines or animal drugs formulated with beneficial microorganisms that live in the intestines of humans or animals, and treat or ameliorate all the symptoms caused by abnormal fermentation of the intestinal flora. Antibiotics are substances that kill microorganisms, but probiotics have the effect of activating microorganisms. Microorganisms used in the probiotic active agent is administered alive as aerobic group, anaerobic group, lactic acid bacteria, yeast and the like. When applied to livestock such as pigs, cattle, etc., and poultry such as chickens (hereinafter referred to collectively as livestock), it is referred to as livestock.

When the weather is impure or the feed changes, or when the livestock moves, harmful microorganisms such as Escherichia coli, Salmonella and Staphylococcus are multiplied abnormally, and the balance of the intestinal microorganisms is broken. It can be fatal. Antibiotics are used beforehand to prevent this. Therefore, the microorganism to be used as a probiotic must pass gastric acid, which is a strong acid, so that it is acid resistant (obtained by screening under a condition of pH 3 or lower) and bile after passing through the stomach. Screened in medium added), while resistance to antibiotics is required. Accordingly, the present applicant is lactic acid bacteria KCTC 8457P (Korean Patent Publication No. 91-4366), which shows resistance to antibiotics in addition to acid resistance and bile resistance, and lactic acid bacteria KCTC 8447P (Korean Patent Publication 91-7817), which produces antibiotics in addition to acid resistance and bile resistance. Has been developed.

However, growth hormones are used in animal husbandry, harmful microbes that are resistant to antibiotics have been isolated, and residual antibiotics have been detected in meat products, increasing the need for safer meat foods. 28 (1995). Therefore, from this point of view, it is absolutely necessary to use probiotic in livestock, and the function of microorganisms used as probiotic should be further strengthened.

In terms of enhancing the function of the microorganisms, it is necessary to isolate the microorganisms in an anaerobic manner. In other words, livestock intestines are anaerobic without oxygen, so even though anaerobic microbes dominate most of the intestinal microflora (Michuoka, the world of intestinal bacteria (1980)), the existing screening methods were carried out in aerobic conditions. It was not possible to isolate anaerobic microorganisms.

The present inventors searched for more functionalized microorganisms, which can be usefully used as probiotic agents, and as a result, were able to isolate 22 species of lactic acid bacteria having anaerobic acid resistance, bile resistance and harmful microbial inhibitory activity in pig feces. Among them, the lactic acid bacteria strains having the strongest activity were isolated and ingested in young pigs (hereinafter referred to as piglets), which was sufficiently increased by a smaller amount of feed, thereby confirming its effectiveness as a probiotic active agent, thereby completing the present invention.

An object of the present invention is to provide a novel lactic acid bacteria strain that can be used as a probiotic for livestock by showing resistance to antibiotics as well as acid resistance, bile resistance, harmful microbial inhibitory activity.

In order to achieve the above object, in the present invention, anaerobic isolated from feces of domestic sows, novel acid and bile lactic acid bacteria strain Lactobacillus reuterii having an antimicrobial inhibitory activity and resistance to antibiotics and the same Provided is a bioactive agent composition for livestock.

Hereinafter, the present invention will be described in detail.

Isolation and identification of novel lactic acid bacteria strains having acid resistance and bile resistance and resistance to antibiotics as well as resistance to antibiotics of the present invention are as follows.

Pig feces were collected using an anaerobic bag (Gas Pack pouch, BBL) from 11-month-old sows bred in a barn in Daejeon, and diluted with an anaerobic dilution adjusted to pH 2 and shaken with a shaker. 1 ml of shake was spread on BL solid medium (Youngyeon Chemical Co., Ltd.) to which 0.2% of Oxgal (Diphco Co.) was added and incubated at 37 ° C. for 48 hours. The resulting colonies were observed under a microscope to select 144 rod type microorganisms. For selected hepatitis microorganisms, we compared the growth inhibition ability of 13 harmful microorganisms used in the evaluation of antibiotics with Kuroiwa et al. (Kuroiwa et al., Journal of Infectious Diseases, 64, 257 (1990)). One strain of lactic acid bacteria showing inhibitory activity against all 13 harmful microorganisms was finally selected.

As a result of screening and separating the lactic acid bacteria strain as described above, the microbiological characteristics were examined using API 32A and CHL kit (Biomerio), and when cultured in BL solid medium, they were stained gram positive and cultured in BL solid medium for 48 hours. The lower surface was raised to 0.4-1.0 mm milk brown bacilli, forming an opaque and smooth colony. The colony morphology was milky in MRS solid medium and the rest of the characteristics were consistent with incubation in BL solid medium. MRS solid medium showed a cell size of 0.5-1.5 μm. Based on these culture characteristics and Bergy's manual, it was isolated into Lactobacillus. In addition, it was identified as Lactobacillus reuterii as a result of investigating various physiological and biochemical properties, carbon source requirements, analysis of cell fatty acids and sequencing of 16SrRNA (ribosomal RNA).

Novel acid resistant lactic acid bacteria strain Lactobacillus reuteri isolated from feces of domestic sows in accordance with the present invention and resistant to antibiotics, dated February 12, 1997, in the Gene Bank of the Institute of Biotechnology, affiliated with the Korea Institute of Science and Technology Deposited as Accession No. KCTC 0315BP.

The novel lactic acid bacterium Lactobacillus reuteri of the present invention was cultured in large quantities and added to pig feed and then ingested in piglets for two weeks and observed feed intake, weight gain, frequency of diarrhea, intestinal bacteria and coliform bacteria, E. coli was reduced over time, and the E. coli was reduced over time, and it was confirmed that it had an effect as a probiotic activator because it was sufficiently increased with a smaller amount of feed.

Lactobacillus reuteri (KCTC 0315BP) of the present invention can be used as a probiotic for livestock in addition to lactic acid bacteria preparations, fermented feed, silage feed and the like.

Hereinafter, the present invention will be described in more detail based on the following examples. However, these Examples are only for illustrating the present invention, the present invention is not limited to these.

Experimental operation in the present invention was carried out in an anaerobic system (Anaerobic system, Forma Scientific Co., Ltd.) supplying a gas mixed with a carbonic acid: hydrogen: nitrogen in a ratio of 5: 7: 88 unless otherwise indicated.

Example 1 Isolation and Identification of Microorganisms

(Step 1) Isolation of Acid-Resistant, Bile-resistant Bacterial Microorganisms from Porcine Feces

Feces were collected anaerobicly using an anaerobic bag (Gas Pack pouch, BBL) from an 11-month-old healthy sow bred in a barn in Daejeon. 1 g of the collected pig feces was diluted to 1:10 with an anaerobic dilution adjusted to pH 2 and shaken for 120 minutes using a shaker. 1 ml of shake was plated in BL solid medium to which 0.2% of Oxgal (Dipco) was added and incubated at 37 ° C. for 48 hours. The colonies that emerged were selected from 144 microbial microorganisms using a microscope, transferred to GAM semi-agar medium, and cultured, and then stored at -80 ° C.

In the above, the anaerobic dilution solution was 0.78% K ₂ HPO ₄ , 37.5 ml of salt mixture, 0.5 g of L-cysteine, 2 ml of 25% L-ascorbic acid, 50 ml of 8% Na ₂ CO ₃ , 0.1% resazurin, 1 mL of Sigma) was dissolved in 860 mL of distilled water, and 0.5 g of agar was added thereto. The salt mixture consists of 0.47% K ₂ HPO ₄ , 1.18% NaCl, 1.20% (NH ₄ ) ₂ SO ₄ , 0.12% CaCl ₂ , 0.25% MgSO ₄ . The composition of the BL solid medium was 2.4 g of Lab-lemco powder, 10 g of Proteose peptone No. 3, 5 g of Trypticase, 3 g of Phytone peptone (BBL), yeast extract 5 g, 150 ml of liver extract, 10 g of glucose, 0.5 g of soluble starch, 10 ml of Solution A, and 5 ml of Solution B were dissolved in 1 L of distilled water and 15 g of agar was added. The composition of Solution A is a mixture of 10% KH ₂ PO ₄ and 10% K ₂ HPO _4. Solution B consists of 10 g of magnesium sulfate, 0.5 g of iron sulfate, 0.5 g of salt, and 0.337 g of manganese sulfate in 250 ml of distilled water. Dissolved in. The composition of GAM semi-agar medium was 10 g of peptone, 3 g of soy peptone, 10 g of proteose peptone No. 3, 13.5 g of peptonized whale serum, 5 g of yeast extract, 2.2 g of bee extract, liver extract 1.2 g, 2.5 g KH ₂ PO ₄ , 3 g NaCl, 0.3 g L-cysteine, 0.3 g sodium thioglycolate and 2 g glucose were dissolved in 1 L of distilled water and 1.5 g of agar was added.

(Step 2) Determination of harmful microbial inhibitory activity of isolated microorganism

Comparison of growth inhibition capacity against 13 harmful microorganisms used in antibiotic evaluations with Kuroiwa et al. (Kuroiwa et al., Journal of Infectious Diseases, 64, 257 (1990)) for the bacteriostatic microorganisms selected in stage 1 It was. Strains used as harmful microorganisms in the experiment are shown in Table 1.

First, 0.1 ml each of the harmful microorganisms cultured anaerobicly for 18 hours in the MRS culture medium was taken, and plated on a MRS solid medium prepared in a Petri dish (Falcon) having a diameter of 150 mm, and a filter paper having a diameter of 8 mm 4) put 40 pieces per plate. MRS liquid medium is 10 g peptone, 10 g bee extract, 5 g yeast extract, 30 g glucose, 1 g of Tween 80, 2 g ammonium citrate, 5 g sodium acetate, 0.1 g MgSO ₄ , 0.05 g MnSO ₄ 2 g of KH ₂ PO ₄ was dissolved in 1 L of distilled water. In the case of a solid medium, 1.5% of bacto agar was added.

Microorganisms isolated from porcine feces in step 1 were inoculated into the paper disk by 30 µl of the test solution obtained by anaerobic culture for 18 hours in MRS culture medium, and Petri dishes with a diameter of 150 mm were incubated for 48 hours. After the culture, the diameter of the inhibitory ring was measured and compared with the control group. As a result, one species of lactic acid bacterium having an inhibitory effect on all 13 harmful microorganisms was selected. The diameter (mm) of the growth inhibitory ring of the harmful microorganism by the selected lactic acid bacteria is shown in Table 1.

TABLE 1

Harmful microorganisms Restraint ring (mm) E. coli KCTC 2441 12 Escherichia coli KCTC 2571 15 Klebsiella pneumoniae 14 Staphylococcus aureus (KCTC 1621) 13 Staphylococcus epidermidis (KCTC 1917) 16 Salmonella enteritidis Kim sp 14 14 Shigella flexneri (KCTC 2008) 13 Proteus vulgaris (KCTC 2579) 13 Enterobacter cloacea (KCTC 2361) 16 Enterococcus lactis (KCTC 1913) 13 Serratia marscense (KCTC 2172) 14 Citrobacter freundii (KCTC 2006) 14 Bacillus subtilis (KCTC 1021) 14

(Step 3) Investigate the characteristics of microorganisms

The lactic acid bacteria strains isolated and isolated in steps 1 and 2 were stained gram positive upon incubation in BL solid medium, and were 0.4-1.0 mm in milk brown color when raised in BL solid medium for 48 hours, and were raised, opaque and smooth. Formed a colony of. The colony morphology was milky in MRS solid medium and the remaining properties were consistent with those in BL medium. The size of cells incubated in MRS solid medium was 0.5-1.5 μm. The culture was inoculated with API 32A and CHL kit (Biomerio) used for anaerobic bacteria.

Biochemical properties of the lactic acid bacteria strains are shown in Table 2.

TABLE 2

characteristic Responsive Aerotolerance + Catalase - Hemolysis - OF test F motility - Gas generation - Spore formation - Lipase - Recitase - Protein resolution - Alkaline phosphatase - Urease + Nitrate reducing ability + Arginine Dihydrolase - Arginine arylamidase + Proline arylamidase - Louisyl glycine arylamidase + Leucine arylamidase + Phenylalanine arylamidase + Tyrosine arylamidase + Alanine arylamidase + Glycine arylamidase + Histidine arylamidase + Serine Arylamidase + Glutamic Acid Decarboxylase - Pyroglutamic Acid Arylamidase - Glutamine Glutamic Acid Arylamidase - Alpha-galactosidase + Beta-galactosidase + Hexaphosphate beta-galactosidase - Alpha-glucosidase + Beta-glucosidase - Alpha-arabinosidase - Beta-glucuronidase - Beta-N-acetyl-glucosaminidase - Alpha-Fulcosidase - Mannose fermentation + Raffinose fermentation + Bile resistance More than 5%

From the above results, a novel lactic acid bacteria strain isolated from pig feces according to the present invention and showing inhibitory activity against 13 harmful microorganisms was identified as Lactobacillus genus.

(Step 4) 16S rRNA sequence determination and microorganism identification

For more accurate identification of the lactic acid bacteria of the present invention having the above microbiological characteristics, the base sequence of 16S rRNA known to be very effective for the identification of microorganisms in the genome (genom) DNA of the bacteria was determined. The 16s rRNA base sequence is as shown in SEQ ID NO: 1.

From the above results, the strain of the present invention was identified as Lactobacillus reuterii, and was deposited with the accession No. KCTC 0315BP dated February 12, 1997 to the Gene Bank in the Biotechnology Research Institute of Korea Institute of Science and Technology.

Example 2 Antibiotic Susceptibility Testing of Lactobacillus Reuteri (KCTC 0315BP)

The susceptibility of the lactic acid bacteria of the present invention to 13 antibiotics was tested according to the method of [Microbiology procedures handbook vol. 1, Henry D. Isenberg, ASM] and Korean Patent Publication No. 91-4366. The antibiotics used in the experiment were ceparexin (Cephalexin), erythromycin (Erythromycin, Sigma), flumequinine (Dong-A Biotech Co.), furazolidine (Great microbial history), and gentamicin ( Gentamycin (Dong-A Biotech Co.)), penicillin (Procaine Penicilline G. Antibioticos), Norflaxacine (Norflaxacine), Spectinomycine (Great Microbiology), Tetracycline (Tetracycline) As bites (Tiamuline, macrobiological death), neomycin (Neomycine, Sigma), chloramphenicol (Chloramphenicol, Sigma), and kanamycin (Kanamycin, Sigma), these were dissolved in an appropriate solvent and used.

The strain culture of the present invention was Brucella agar (BBL, 10 g of pancreatic digest of casein of casein, 10 g of peptin digest of animal tissue, 1 g of dextrose, yeast) 2 g extract, 5 g sodium chloride, 0.1 g sodium bisulfate, 15 g agar) 5% sheep blood, Vit. McFaland No. was added to a 150 mm diameter Petri dish prepared by adding 0.1% K1. Inoculated with a turbidity equal to 0.5, and seven filter papers having a diameter of 8 mm were placed per dish. 30 μl of the antibiotic solution prepared in advance was inoculated into the paper disc, and the diameter of the inhibitory ring which appeared after the incubation for 24 hours anaerobicly after 1 hour at 4 ° C. was measured. As a result, the minimum inhibitory concentration (µg / ml) for each antibiotic is shown in Table 3 below.

TABLE 3

Antibiotic Minimum inhibitory concentration (㎍ / ml) Separexin 75 Erythromycin 3 Flumequinine 5,000 Furazolidine 75 Gentamicin 100 penicillin 5 Nofloxacin 1,000 Spectinomycin 3,000 Tetracycline 1,500 Tiamulin 300 Neomycin 100 Chloramphenicol 200 Kanamycin 3,000

Example 3 Determination of the Effect of Lactobacillus Reuteri (KCTC 0315BP) on Swine Administration

The Lactobacillus reuteri (KCTC 0315BP) of the present invention was anaerobicly cultured in MRS liquid medium at 37 ° C. for 18 hours, followed by centrifugation to recover the microorganisms, dispersed in 10-fold 10% skim milk, and lyophilized. Then, the mixture was mixed and dispersed in powder (unusual powder of Daehan Sugar). When administered to piglets, it was mixed with livestock feed.

Lactobacillus KCTC 0315BP of the present invention was administered to piglets of 1 month old for 5 days, divided into 5 groups of 5 animals per group in Gyeongnam-do breeding ground located in Jinju, Gyeongsangnam-do. In this case, group 1 is antibiotic-free feed group, group 2 is antibiotic-free feed group, group 3 is antibiotic-free feed + probiotic (2 × 10 ⁹ cells per head), and group 4 is antibiotic-free feed + probiotics (2 × 10 per head) ¹⁰ cells), and group 5 is the antibiotic-added feed + probiotic (2 × 10 ¹⁰ cells per head daily). The basic feed used in the present invention is a livestock feed, and the antibiotic-added feed was added to the livestock feed so that 13.2% of oxytetracycline (macromicrobial history) and 1.1% of thiamuline (macromicrobial history) were added.

After 14 days of administration, diarrhea often occurred due to good breeding conditions, but soon recovered, and no piglets died. 1 g of the feces of the piglets were diluted with an anaerobic dilution solution, and cultured for 48 hours at 37 ° C. using a selective medium to determine the total change of bacteria in fecal coliform and lactic acid bacteria. E. coli was cultured in aerobic state. For E. coli, DHL medium (Yungyeon Chemical Co.) and LBS medium (BBL company) were used. E. coli and Lactobacillus bacteria were measured from individual volume, feed intake and feces from 8 days before the probiotic was administered. 14 days of dosing continued until 7 days after discontinuation.

Table 4 below shows the mean value of E. coli flora by the date of the piglets in which diarrhea occurred (unit = × 1000), Table 5 shows the mean value of the Lactobacillus flora by the date of piglets from the diarrhea occurred (unit = × 10 ⁶ ).

TABLE 4

1 day before administration 3 days of administration 7 days of administration 14 days of administration 3 day suspension Group 1 313.3 294.2 251.2 258.4 272.3 Group 2 311.0 160.7 64.9 34.5 26.8 Group 3 274.1 125.6 41.5 30.0 17.7 4th group 238.7 72.6 31.0 20.6 8.4 5 groups 268.8 37.1 3.0 5.9 1.6

TABLE 5

1 day before administration 3 days of administration 7 days of administration 14 days of administration 3 day suspension Group 1 13.2 22.7 23.9 21.0 9.1 Group 2 12.8 11.4 9.4 7.4 6.8 Group 3 11.0 82.3 99.7 131.0 126.9 4th group 9.5 107.5 118.1 151.3 137.2 5 groups 18.5 60.8 62.1 75.6 53.2

Table 6 below shows the average value of E. coli flora by day (unit = x 1000) of healthy piglets that did not cause diarrhea, and Table 7 shows the average value (l = 10 ⁶ ) of lactic acid bacteria flora by date.

TABLE 6

1 day before administration 3 days of administration 7 days of administration 14 days of administration 3 day suspension Group 1 328.6 233.3 194.3 256.6 266.0 Group 2 234.5 127.3 68.0 14.6 20.0 Group 3 108.6 42.1 17.2 10.8 10.0 4th group 85.3 5.3 7.2 2.0 6.6 5 groups 133.3 19.6 1.7 4.6 4.6

TABLE 7

1 day before administration 3 days of administration 7 days of administration 14 days of administration 3 day suspension Group 1 13.5 18.0 18.5 18.6 17.4 Group 2 14.6 8.1 11.3 13.3 5.9 Group 3 12.3 74.5 78.2 109.6 136.7 4th group 13.0 108.3 130.0 175.3 171.3 5 groups 8.6 60.3 56.0 77.6 54.3

As can be seen from the results of Tables 4 to 7, when the probiotic was added, the Lactobacillus bacteria were maintained and the E. coli decreased with time. The same pattern was observed in healthy piglets that did not cause diarrhea. It is noteworthy that E. coli flora was relatively low in healthy piglets.

The developmental results of piglets were measured during the experiment period, and the weight gain index and feed demand ratio were compared. The gain index and feed rate were divided into three groups and four groups, and group 5 was divided into groups 2 and the results are shown in Table 8 below.

TABLE 8

Group 1 Group 2 Group 3 4th group 5 groups Weight at start of test (g) 10840 6040 9000 8420 9440 Weight at end of test (g) 26240 17180 25220 23040 23620 Weight gain (g) 15400 11140 16220 14620 14180 Gain Index (%) 100 100 105 95 127 Feed Intake (g) 29000 20000 22800 20000 24000 Daily Feed Intake 1033 714 814 728 857 Feed demand rate (%) 100 100 75 74 90

From the above results, the piglets ingesting the Lactobacillus reuteri (KCTC 0315BP) of the present invention reduced the feed requirement and was sufficiently increased to a smaller amount of feed, and it was confirmed that the strain was effective as a probiotic.

Lcatobacillus reuterii anaerobicly isolated from pig feces according to the present invention has resistance to antibiotics while having acid resistance and bile resistance, and effectively inhibits the growth of harmful microorganisms such as pathogenic bacteria. When administered to E. coli, it reduces the total coliform bacteria and reduces the feed requirement, so it can be useful as a livestock bioactive agent.

(Sequence list)

SEQ ID NO: 1

Sequence length: 1637

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: DNA

[SEQ ID NO: 1a]

[SEQ ID NO: 1b]

Claims (3)

A novel acid- and bile-resistant lactic acid bacteria strain Lcatobacillus reuterii (KCTC 0315BP), anaerobicly isolated from feces of domestic sows, having harmful microbial inhibitory activity and resistance to antibiotics. Probiotic composition for livestock containing the microorganism of claim 1. The probiotic composition according to claim 2, which is used in addition to a lactic acid bacterium preparation, a fermented feed formulation or a silage.