KR100457002B1 - Novel microorganism inhibiting the growth of harmful bacteria and microbial preparation containing same as an effective ingredient - Google Patents

Abstract

The present invention relates to a novel microorganism that inhibits the growth of harmful bacteria and a microbial additive comprising the same as an active ingredient. Specifically, the present invention produces organic acids and derivatives thereof that inhibit the growth of harmful bacteria and exhibits excellent acid resistance, bile resistance and antibacterial activity. Novel microorganisms Lactobacillus pentosus K34 and a microbial additive containing the same as an active ingredient. The Lactobacillus pentosus K34 of the present invention produces phenylorganic acid, indole organic acid and derivatives thereof and effectively inhibits the growth of harmful bacteria causing disease in animal tissues, so that it can be usefully used as an additive in feed, food, cosmetics, medicines, etc. have.

Description

Novel microorganisms that inhibit the growth of harmful bacteria and microbial additives comprising the same as active ingredients {NOVEL MICROORGANISM INHIBITING THE GROWTH OF HARMFUL BACTERIA AND MICROBIAL PREPARATION CONTAINING SAME AS AN EFFECTIVE INGREDIENT}

The present invention relates to a novel microorganism that inhibits the growth of harmful bacteria and a microbial additive comprising the same as an active ingredient. Specifically, the present invention produces organic acids and derivatives thereof that inhibit the growth of harmful bacteria and exhibits excellent acid resistance, bile resistance and antibacterial activity. Novel microorganisms Lactobacillus pentosus K34 and a microbial additive containing the same as an active ingredient.

Lactobacillus is a kind of microorganisms that humans have been using for a long time, and is widely used for kimchi in Korea and dairy fermentation in the West. Lactic acid bacteria are also commonly referred to as lactic acid bacteria and refers to bacteria that produce a significant amount of lactic acid as the final metabolite. In recent years, research on lactic acid bacteria has been actively conducted, and as a result, its application range has been expanded, such as being used as health food and medicine as well as general foods. Among these bacteria, Streptococcus sp. And Pediococcus sp. .), Leuconostoc sp., Lactobacillus sp., Sporolactobacilus sp. And Bifidobacterium sp.

These bacteria are gram-positive bacteria that have anaerobic or uni-aerobic properties. They live in the intestines of animals, decompose nutrients and fiber ingested by the animals, use them as energy sources, and produce lactic acid and antibiotics to produce harmful bacteria in the intestines. It is known to play an important role in maintaining intestinal health by inhibiting development.

Food preservation by lactic acid bacteria is primarily due to the production of lactic acid and acetic acid due to sugar fermentation, and by antibacterial substances such as hydrogen peroxide (H ₂ O ₂ ), diacetyl (diacetyl), bacteriocin (bacteriocin). Among these, bacteriocin produced by lactic acid bacteria was recognized as an antimicrobial peptide (peptide) that contributes to the preservation of fermented food as a natural antibiotic that is distinguished from common antibiotics, and many bacteriocins have been separated from lactic acid bacteria.

Among the bacteriocins that occupy an important position in the industry, a representative one is nisin, which is used as a food additive that suppresses the growth of gram-positive rot bacteria (Baekyoungsin, Bioindustry 8 (2): 26, 1995).

In addition to the antibacterial substances produced by lactic acid bacteria, formic acid, lactic acid, acetic acid, propionic acid, butyric acid are used to inhibit the growth of harmful bacteria and to store food and feed for a long time. butyric acid and benzoic acid have been used (Tamime, AY, In Dairy Microbiology vol. 2 RK Robinson (Ed), Applied Science Publishers, GB, p. 113, 1981). It has been reported that formic acid, acetic acid, lactic acid and propionic acid inhibit the growth of harmful bacteria by the low pH and high concentration of hydrogen ions acting on harmful bacteria and disturbing the potential difference of cell membranes (Kang, Kee-Hee, Biology and Industry 12 (2): 16, 1999).

However, such an organic acid can be easily combined with salt ions such as calcium or sodium, but when the organic acid is chlorinated, there is a problem of rapidly degrading harmful bacterium growth inhibitory effect. In addition, bacteriocin is a proteinaceous antimicrobial material having a molecular weight greater than that of organic acids and a lower tissue absorption rate, and is easily decomposed by proteolytic enzymes and easily inactivated by physical factors such as heat. Therefore, bacteriocin is used only as a food preservative or growth inhibitory agent of harmful bacteria in the digestive tract.

For this reason, organic acid has long been used to suppress the growth of harmful bacteria causing animal disease. However, organic acids have a neutral pH, which is easily neutralized by chloride reaction, and the effect of inhibiting growth of harmful bacteria is reduced. In particular, since it is absorbed in large quantities in gastrointestinal epithelial cells, and some of them are easily converted into amino acids and other substances by enzymatic reactions, it was difficult to inhibit the growth of harmful bacteria causing the disease in animals using organic acids.

Therefore, the present inventors earnestly researched to develop a growth inhibitor of harmful bacteria that can cause the disease in the tissue of the animal, and produce phenyl organic acid, indole organic acid and derivatives thereof that inhibit the growth of harmful bacteria, and excellent acid resistance and bile resistance And Lactobacillus pentosus K34, a microorganism that exhibits antimicrobial activity, is useful for additives such as feed, food, cosmetics, and medicines by effectively inhibiting the growth of harmful bacteria causing disease in animal tissues. The present invention has been completed by confirming that it can be used.

An object of the present invention is to provide a microbial additive that can effectively inhibit the growth of harmful bacteria in animal tissues.

Figure 1 shows the result of identifying the K34 strain isolated in the present invention by 16s rRNA sequence analysis.

In accordance with the above object, the present invention provides a novel microorganism Lactobacillus pentosus K34 which produces organic acids and derivatives thereof that inhibit the growth of harmful bacteria and exhibits excellent acid resistance, bile resistance and antibacterial activity.

The present invention also provides a microbial additive for inhibiting the growth of harmful bacteria containing the microorganism as an active ingredient.

Hereinafter, the present invention will be described.

The present invention provides a novel microorganism Lactobacillus pentosus K34, which produces organic acids and derivatives thereof that inhibit the growth of harmful bacteria and exhibits excellent acid resistance, bile resistance and antimicrobial activity.

The present inventors inhibit the growth of harmful bacteria such as Salmonella , Propionibacterium acnes , athlete's foot, E. coli , Staphylococcus aureus , etc. Microorganisms producing phenol organic acids and their derivatives, indole organic acids and their derivatives were isolated from the small intestine of native chickens.

The microorganisms were identified by API kit, which is a biochemical identification method, and 16s rRNA sequencing, which is a molecular biological identification method. As a result, it was confirmed that Lactobacillus sp. It was identified as a bacterium close to Bacillus plantarum ( Lactobacillus plantarum ). On the other hand, 16s rRNA sequence analysis showed more than 95% homology with Lactobacillus pentosus . The microorganism of the present invention is located between the Lactobacillus plantarum and Lactobacillus pentosus in terms of taxonomy, but more preferably because of its high homogeneity with Lactobacillus pentosus, the present inventors named the microorganism as Lactobacillus pentosus K34. It was deposited on November 12, 2001 to the Center for Microbial Conservation (KCCM) (Accession No .: KCCM-10331).

Lactobacillus pentosus K34, which inhibits the growth of harmful bacteria isolated and identified in the present invention, is a Gram-positive bacterium, grown at a condition of 42 ° C. or lower, pH 3.0 to 8.5, heterofermented, and as shown in Table 1 below . Biochemical characteristics.

temperament Fermentation ability temperament Fermentation ability temperament Fermentation ability temperament Fermentation ability temperament Fermentation ability Glycerol - Erythritol - D-Arabinose - L-Arabinose + Ribose + D-Xylose - L-Xylose - Adonitol - β-methyl-xyloxide - Galactose + D-glucose + D-fructose + D-Mannose + L-sorbos - Rhamnos ± Dulcitol - Inositol - Mannitol + Sorbitol + α-methyl-D-mannoside + α-methyl-D-glucoside - N-acetyl-glucosamine + Amigdalin + Arbutin + Esculin + Salinity + Cellobiose + Maltose + Lactose + Melibiose + Sakaros + Trehalose + insulin - Melezitos + D-Raffinose + Amidodon - Glycogen - Xylitol - β-gentiobiose + D-Turanos + D-Resource - D-tagatose - D-fucose - L-fucose - D-Arabitol - L-Arabitol - Gluconate ± 2-ceto-gluconate - 5-ceto-gluconate -

The Lactobacillus pentosus K34 is not degraded by proteolytic enzymes, exhibits superior antimicrobial activity than lactic acid, and is used in antibiotics such as colistin, streptomycin, ciprofloxacin, and gentamycin. It is resistant and exhibits excellent acid and bile resistance as well as an excellent intestinal fixation effect (see Tables 2 to 6 ).

In addition, after the growth of the microorganisms, the culture solution thereof was concentrated or extracted with an organic solvent. As a result, the culture medium of Lactobacillus pentosus K34 contained not only general organic acids but also volatile fatty acids, and phenyl organic acids (phenyl) organic acid (POA), derivative phenyl organic acid (DPOA), indole organic acid (IOA) and derivative indole organic acid (DIOA) were detected, especially phenyl lactic acid and phenyl hydroxide. Lactic acid, indole lactate, etc. were detected in large quantities.

In the case of general organic acids, while the antimicrobial ability is rapidly decreased at a pH of weak acidity (for example, pH 5.5 to 6.0), organic acids and derivatives thereof produced by Lactobacillus pentosus K34, a harmful bacterium growth inhibitory microorganism of the present invention, In particular, phenyl lactic acid, indole lactic acid and derivatives thereof maintain high antibacterial activity even at weak acidic pH.

The present invention also provides a microbial additive for inhibiting the growth of harmful bacteria containing the microorganism as an active ingredient.

As confirmed above, Lactobacillus pentosus K34 isolated and identified in the present invention inhibits the growth of harmful bacteria such as Salmonella, Acne, Athlete's foot, Escherichia coli, Staphylococcus, etc. It produces phenol organic acid, indole organic acid and derivatives thereof, shows excellent acid resistance and bile resistance, and has excellent intestinal fixation effect, so it can be useful as a microbial additive for inhibiting the growth of harmful bacteria in feed, food, cosmetics, pharmaceuticals, etc. .

The microbial additive of the present invention may be prepared by mixing the cells obtained by culturing the Lactobacillus pentosus K34 with an excipient, or recovering the cells to heat- or lyophilized probiotic. In addition, the microbial additive of the present invention may be prepared as a concentrate containing a large amount of organic acids and derivatives thereof obtained by drying the concentrate of Lactobacillus pentosus K34 or by extracting the culture solution with a solvent and then concentrating the extract under vacuum. Can be.

The microbial additive prepared as described above contains 1 × 10 ⁶ to 1 × 10 ¹² cells / g of Lactobacillus pentosus K34, which inhibits the growth of harmful bacteria, and preferably 1 × 10 ⁶ to 1 × 10 ⁹ cells / g. It is preferable to contain in the content of g. The probiotics and concentrates may be used in amounts of 0.1 to 0.3% by weight in various livestock feeds such as pigs, chickens and cattle, and fermented feeds, silage feeds, powdered feeds and pellets, regardless of the type or type of feed. It can be applied to both types of feed.

In addition, the microbial additive of the present invention can be used as additives such as food, cosmetics, pharmaceuticals to inhibit the growth of harmful bacteria such as Salmonella, E. coli, Staphylococcus, Acne, Athlete's foot.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Isolation of Strains

In order to isolate microorganisms that inhibit the growth of pathogenic bacteria in animal tissues, such as Salmonella gallinarum , Escherichia coli , Staphyloccus aureus , and Propionibacterium acnes Was finely ground in a blender and used as a source of separation of the target microorganism. Prepare the supernatant by diluting the prepared sample with physiological saline solution, and serially diluting it again, and then placed the disc containing the dilution on the nutrient agar medium in which Salmonella gallinarum (ATCC 9184) was layered and incubated for 72 hours in an anaerobic incubator at 37 ° C. It was. From this, lactic acid bacteria that inhibit the growth of Salmonella gallinarum to form an inhibition zone (inhibition zone) was first isolated. Among them, agar coated with Salmonella gallinarum (ATCC 9184), Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 6538) and Propionibacterium acnes (ATCC 6921) in order to obtain a strain that effectively inhibits the growth of harmful bacteria. Selected strains were cultured by inoculating the medium. After culturing for 3 days, seven strains that form a large inhibition zone of 15 mm or more were selected and named as BD14, BD16, BD22, BD33, K34, B36 and BL, respectively.

Example 2 Stability Study for Protease

In order to investigate whether the active material produced by the seven strains isolated in Example 1 is stable against proteolytic enzymes, each strain was cultured in Lactobacillus MRS medium (Difco) for 3 days at 37 ° C., and the culture supernatant. After obtaining the protease 0.1% (W / V) trypsin (Sigma) and Permizyme (Fermizyme ^R , Vision Biochem Co., Ltd.) and each strain culture solution in a medium layer of Salmonella gallinarum bacteria 200 μl was aliquoted to examine the antimicrobial activity.

Specifically, the antimicrobial activity was measured by a bio-assay method using penicillin (Lee Chae-young et al., Journal of Industrial Microbiology 24: 534-539, 1996; Ichikawa, T., et al., Folia Microbiol . 16 : 218-224, 1971), and the results are shown in Table 2 below.

Strain No treatment Permime treatment Trypsin treatment Control 0 0 0 BD14 28 24 20 BD16 25 27 24 BD22 24 30 25 BD33 27 27 23 K34 27 28 24 B63 28 23 23 BL 22 15 25

As shown in Table 2 , the strains isolated in Example 1 did not show a rapid decrease in antimicrobial activity against trypsin or permezyme, which is a protease, thus confirming that they are very stable against protease.

Example 3 Comparison of Antimicrobial Activity

In order to investigate the antimicrobial activity of the strains isolated in Example 1 was compared with the antimicrobial activity of lactic acid. To this end, the culture supernatant of each of the strains BD14, BD16, BD22, BD33, K34, B63, BL in a buffered medium (0.6% yeast extract, 1% peptone, 1% glucose, hydrochloric acid) adjusted to Salmonella gallinalum bacteria After 100 μl of lactic acid at a concentration of 0 to 2.0%, the antimicrobial activity was measured in the same manner as in Example 2, and the results are shown in Table 3 below. At this time, the pH of each strain culture ranged from 3.46 to 4.5, acidity (acidity) ranged from 1.08 to 1.80.

Lactic acid (%) Diameter of Suppression Zone (mm) Strain Diameter of Suppression Zone (mm) 0.0 0 BD14 10 0.5 - BD16 9 1.0 - BD22 11 1.2 - BD33 11 1.4 - K34 10 1.6 - B63 11 1.8 9 BL 11 2.0 10 - -

As shown in Table 3, it was confirmed that the strains isolated from the present invention than lactic acid effectively inhibit the growth of Salmonella gallinarum bacteria and excellent antibacterial activity.

Example 4 Antibiotic Resistance Survey

In order to examine the antibiotic resistance of the strains isolated in Example 1, each microorganism was laminated on the Lactobacillus MRS medium, and then loaded with a disk containing various antibiotics, and the diameter of the inhibitory zone was measured.

Antibiotic BD14 BD16 BD22 BD33 K34 B63 BL Neomycin 7.0 8.0 7.0 15.0 7.0 12.0 17.0 Erythromycin 25.0 25.0 23.0 23.0 24.0 25.0 27.0 Cephalosporins 25.0 26.0 25.0 25.0 24.0 26.0 30.0 Amocillin Clavulanic Acid 24.0 47.0 45.0 40.0 42.0 25.0 33.0 Colistin 0.0 0.0 0.0 0.0 0.0 0.0 7.5 Streptomycin 0.0 0.0 0.0 0.0 0.0 0.0 7.0 Ciprofloxacin 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Ampicillin 22.0 46.0 46.0 40.0 38.0 20.0 30.0 Gentamicin 0.0 0.0 7.0 10.0 0.0 8.5 14.0 Oxtetracycline 21.0 25.0 25.0 26.0 26.0 22.0 15.0

As shown in Table 4 above, among the seven isolates, BD14, BD16 and K34 have antibiotic resistance to gentamicin, streptimycin, ciprofloxacin and colistin. Confirmed.

Example 5 Acid Resistance Investigation

In order to measure the resistance to acidic pH of the microorganisms isolated in Example 1, each microbial cell obtained by centrifugation of the culture was inoculated at 1 × 10 ⁸ cells / ml in Lactobacillus MRS medium containing 0.3% hydrochloric acid. After incubation for 6 hours, the viability of each strain was examined. The viability of each strain was determined by comparing the absorbance values measured after 6 hours of incubation based on the absorbance values measured immediately after inoculation.

Strain Viability (%) BD14 Not detected BD16 17.8 BD22 Not detected BD33 2.92 K34 27.3 B63 6.56 BL 0.18

As shown in Table 5 , K34 bacteria showed the best acid resistance.

Example 6 Bile Resistance Research

In order to measure the resistance to bile acids of the strains isolated in Example 1, each microbial cell obtained by centrifugation of the culture was inoculated at a concentration of 1 × 10 ⁸ cells / ml in Lactobacillus MRS medium containing 0.3% bile acid. After incubation for 24 hours, the viability of each strain was examined. The viability of each strain was determined by comparing the absorbance values of the culture solution measured after 24 hours incubation based on the absorbance value of the culture solution immediately after inoculation.

Strain Viability (%) BD14 34.2 BD16 21.1 BD22 87.8 BD33 100.0 K34 96.1 B63 5.2 BL 25.

As a result, as shown in Table 6 , BD33 showed a very strong resistance to bile acids, K34 and BD22 also showed excellent viability.

Example 7 Biochemical and Molecular Biology Identification

From the results of Examples 2 to 6, API kit which is a biochemical identification mechanism to effectively inhibit the growth of harmful bacteria, select K34 strains excellent in acid resistance and bile resistance, and have antibiotic resistance, and identify them (bioMerieux) and molecular biological identification method 16s rRNA sequence analysis (Prober, JM, et a;., Science 238: 336-341, 1987; Fred, R. Rurangirwa, et al., International Journal of Systematic and Evolutionary Microbiology 30: 759-765, 2000). Specifically, the culture of the K34 strain was suspended in saline dilution, and then inoculated into the API kit to test the fermentation ability of the substrate in the kit for 36 to 60 hours, and the results obtained therefrom were entered into the API-PLUS identification program (Note 16M rRNA sequencing was performed by separating 16S rRNAs from lactic acid bacteria and analyzing the sequencing according to the polymerase chain reaction. Finally, it was identified.

As a result, K34 having excellent inhibitory ability against harmful bacteria showed 98% homology with Lactobacillus plantarum by biochemical identification method using API kit. On the other hand, 16s rRNA sequencing, a molecular biological identification method, confirmed that K34 consists of 1,509 nucleotide sequences described as SEQ ID No. 1 showing 95% homology with Lactobacillus pentosus . However, as shown in FIG . 1 , as a result of examining the taxonomy, since the K34 strain is more closely related to Lactobacillus pentosus than Lactobacillus plantarum, the strain was named Lactobacillus pentosus K34 and the Korean microorganism preservation center was used. Deposited November 12, 2001 (Accession No .: KCCM-10331).

Example 8 Organic Acid Analysis

The culture solution obtained by incubating the K34 strain identified as Lactobacillus pentosus in Example 8 in a medium containing 0.6% yeast extract, 1% peptone, and 1% glucose for 48 hours was centrifuged to separate only the supernatant. The supernatant was diluted with 2.8 ml of distilled water in a vial containing 0.2 ml of the filtrate, filtered through a 0.45 µm syringe filter, and then 50 ㎍ (1 mg / ml methanol solution) of pentadecanoic acid used as an internal standard. ) Was added. 10 mg of methoxyamine hydrochloride was added thereto, and the pH was raised to 12 or more using 5 M NaOH, followed by methoxylation at 60 ° C. for 1 hour. Then, the pH was adjusted to 9 or more, extracted twice with 3 ml of diethyl ether, and water was removed with anhydrous Na ₂ SO ₄ . The resulting extract was completely dried under nitrogen stream, and then derivatized with TBDMS (tertiary-butydimethylsilylation) at 60 ° C. for 30 minutes using MTBSTFA (N-methyl-N- (tert.-butyldimehylsily) -trifluoroactamide) and GC-MSD. The analysis was carried out. GC-MSD used HP 6890 Gas Chromatography (Hewlett-Packard) equipped with 5973 mass selective detector, the column is Ultra-2 capillary, 25 m × 200 μm ID, 0.11 μm d _f ), and were carried out according to the following analysis conditions; Ionization: EI at 70 eV; Interface and Ion source Temp .: 300 ° C. and 230 ° C., respectively; Injection mode: Split mode (10: 1); Flow mode: constant flow mode (0.5 ml / min); Carrier gas: helium (He); Injection volume: 1 μl. At this time, the control group used a culture medium in which the microorganisms were not cultured.

Organic acid Area ratio MRS (control) K34 Formic acid 0.492 0.254 Acetic acid 0.920 1.083 Propionic acid 0.029 0.027 Isobutyric acid 0.032 0.027 Butyric acid 0.138 0.052 Isovaleric acid 0.010 0.006 Valeric acid 0.007 0.004 Lactic acid 3.976 15.414 Glycolic acid 0.072 0.077 2OH-butyric acid 0.007 0.027 2OH-isovaleric acid 0.007 0.149 2OH-isopropionic acid 0.000 0.378 2OH-3-methyl valeric acid 0.000 0.185 Succinic acid 0.408 0.515 Phenyl Lactic Acid 0.002 0.913 4OH-phenyl lactic acid 0.001 0.105 Indole-3-lactic acid 0.000 0.051 Citric acid 0.167 0.102

As a result, as shown in Table 7 , the main component of the organic acid produced from Lactobacillus pentosus K34 was confirmed that the lactic acid and a large amount of organic acid derivatives such as phenyl lactic acid, phenyl hydroxide lactic acid and indole lactic acid are produced.

Example 9 Acid Resistance Study of Organic Acid and Its Derivatives

In order to investigate the acid resistance of various organic acids and derivatives thereof produced from Lactobacillus pentosus K34, which inhibit the growth of harmful bacteria, Salmonella gallinarum , a pathogen causing disease in animal tissues, was coated in a medium of 0.05 M. The disk containing acetic acid, lactic acid, formic acid, and phenyl lactic acid was placed and the antibacterial activity was examined without adjusting the pH or adjusting the pH to 5.0.

Concentration of various organic acids pH Size of suppression area (mm) Acetic acid Lactic acid Formic acid Phenyl Lactic Acid 0.05 M Unregulated 16.0 17.0 14.0 17.0 5.0 9.0 8.0 8.0 18.0

As a result, as shown in Table 8 , when treated without pH adjustment, the antibacterial activity was similar in all acetic acid, lactic acid, formic acid, phenyl lactic acid, but when the pH was adjusted to 5.0, acetic acid, lactic acid, formic acid, etc. Silver antimicrobial activity decreased relatively rapidly, while phenyl lactic acid effectively inhibited the growth of Salmonella gallinarum regardless of pH change.

Example 10 Antimicrobial Spectrum Investigation of Phenyl Lactic Acid

In order to investigate the antimicrobial spectrum of phenyl lactic acid showing excellent antimicrobial activity under acidic conditions in Example 9, Salmonella gallinarum, which is a harmful bacterium (S. gallinarum, ATCC 9184), Staphylococcus aureus (S. aureus, ATCC 6538), Helicobacter pylori (H. pylori, ATCC 43504), Lactobacillus rhamnosus (Lactobacillus rhamnosusATCC 7469), Escherichia coli (E. coli, ATCC 25922), Saccharomyces cerevisiae (S. cerevisiae, ATCC 9080) and Fungal Reophorus Stronifer (Rhizopus stronifer, The antimicrobial activity against ATCC 6227b) was investigated. The strains were put on a disk containing 0.2 to 5.0 M phenyl lactic acid in a multi-layered medium to measure the growth inhibitory activity.

Phenyl Lactate Concentration (%) Salmonella Galinarum Staphylococcus aureus Helicobacter pylori Lactobacillus rhamnosus Escherichia coli Saccharomyces cerevisiae Reapers Stronifer 0.2 9.0 7.0 10.0 0.0 0.0 0.0 0.0 0.4 10.5 8.0 13.0 0.0 0.0 0.0 0.0 0.6 18.0 11.0 14.0 0.0 0.0 0.0 0.0 1.0 23.0 14.0 19.0 0.0 8.0 0.0 0.0 1.5 N.D. 15.0 27.0 0.0 8.5 0.0 0.0 3.0 N.D. 15.0 31.0 0.0 12.0 0.0 0.0 5.0 N.D. 25.0 36.0 13.0 13.0 8.0 8.0

As a result, as shown in Table 9 , phenyl lactic acid showed very good growth inhibitory activity against the pathogenic harmful bacteria Salmonella, Helicobacter, E. coli, etc., and showed a killing effect at high concentrations for lactic acid bacteria and fungi.

The microbial additive of the present invention for inhibiting the growth of harmful bacteria using Lactobacillus pentosus K34, which exhibits excellent antibacterial activity by generating organic acids and derivatives thereof that inhibit the growth of harmful bacteria, can be prepared by the following method. However, the following preparation example only illustrates the microbial additive of this invention, and this invention is not limited to the following preparation example.

Preparation Example 1 Preparation of Microbial Additives 1

Lactobacillus pentosus K34, which produces phenylorganic and indole organic acids, which inhibit the growth of harmful germs that cause disease in animal tissues, was added to 1% yeast extract, 2% glucose, 0.2% KH ₂ PO ₄ , Incubated at 37 ° C. for 3 days in a medium containing 0.2% sodium acetate, 0.1% Tween 80, 0.5% peptone, and the like. The culture solution was simply mixed with degreasing steel, soybean meal, jade powder and the like in a ratio of 1: 9 to 1: 7, respectively, and dried at a temperature of 40 ° C. or below, and then ground to prepare a probiotic for feed addition. The probiotics thus prepared contain 1.0 × 10 ⁸ cfu / g or more of Lactobacillus pentosus K34.

Preparation Example 2 Preparation of Microbial Additives 2

Lactobacillus pentosus K34 was incubated at 37 ° C. for 3 days in a medium containing 1% yeast extract, 2% glucose, 0.2% KH ₂ PO ₄ , 0.2% sodium acetate, 0.1% Tween 80, 0.5% peptone and the like. The culture solution was inoculated at 10% concentration in a solid fermenter containing grains containing soybean meal, soybean meal, jade powder, octave and molasses in a ratio of 3: 1: 1: 1, and then water was added to the water content by adding water at 45 to 60%. Adjusted to%. This was a solid fermentation with stirring for 3 days at 40 ℃ to prepare a probiotic for feed addition. Probiotics thus prepared contain more than 3.0 × 10 ⁷ cfu / g of Lactobacillus pentosus K34.

Preparation Example 3 Preparation of Microbial Additives 3

Lactobacillus pentosus K34 was incubated at 37 ° C. for 3 days in a medium containing 1% yeast extract, 2% glucose, 0.2% KH ₂ PO ₄ , 0.2% sodium acetate, 0.1% Tween 80, 0.5% peptone and the like. Milk powder or skim milk (skim milk) and the same amount of starch or lactose were added to the culture solution, and then frozen at −70 ° C. or lower, frozen for 24 hours, and freeze-dried for 36 hours. At this time, the temperature of the hot plate was 30 ℃, freeze-drying temperature was -65 ℃. The probiotic thus prepared contains Lactobacillus pentosus K34 at least 2.0 × 10 ¹⁰ cfu / g.

Preparation Example 4 Preparation of Microbial Additives 4

Lactobacillus pentosus K34 was incubated at 37 ° C. for 3 days in a medium containing 1% yeast extract, 2% glucose, 0.2% KH ₂ PO ₄ , 0.2% sodium acetate, 0.1% Tween 80, 0.5% peptone and the like. After mixing milk powder or lactose into the culture solution, the mixture was spray dried at an inlet temperature of 150 ° C. and an outlet temperature of 110 ° C. The spray-dried preparation in this way does not survive the Lactobacillus pentosus K34 strain, but contains a large amount of phenyl organic acid and indole organic acid that inhibit the growth of harmful bacteria causing disease in animal tissues, and thus exhibit antimicrobial activity.

Preparation Example 5 Preparation of Microbial Additives 5

Lactobacillus pentosus K34 was incubated at 37 ° C. for 3 days in a medium containing 1% yeast extract, 2% glucose, 0.2% KH ₂ PO ₄ , 0.2% sodium acetate, 0.1% Tween 80, 0.5% peptone and the like. After adjusting the pH of the culture solution to 3.5 and mixing the same amount with ethyl acetate, hexane and ether, 500 ml of the solvent layer recovered therefrom was evaporated and concentrated using a vacuum concentrator at 60 ° C. for 0.2 hours.

Test Example 1 Investigation of Antimicrobial Activity of Microbial Additives

Lactobacillus pentosus K34 prepared as a feed additive probiotic according to the above preparation was mixed with 0.3% of a microbial additive containing 1.0 × 10 ⁸ cfu / g or more in laying hens feed, infected with poultry typhoid, and Salmonella bacteria in feces 1.0 ×. Salmonella was not detected after 30 days of feeding on laying hens that detected more than 10 ⁶ cfu / g. In addition, the microbial additive prepared by spray drying and the microbial additive prepared by dissolving in distilled water after solvent extraction obtained the same result, and furthermore, the microbial additive of the present invention is not only antibiotic resistant staphylococci but also skin acne bacteria, dandruff bacteria It also showed strong growth inhibitory effect against athlete's foot.

As described above, Lactobacillus pentosus K34 isolated and identified in the present invention inhibits the growth of harmful bacteria such as Salmonella, Acne, Athlete's foot, Escherichia coli, Staphylococcus, etc. It produces phenol organic acid, indole organic acid and derivatives thereof, shows excellent acid resistance and bile resistance, and has excellent intestinal fixation effect, so it can be useful as a microbial additive for inhibiting the growth of harmful bacteria in feed, food, cosmetics, pharmaceuticals, etc. .

<110> Biotopia Co., Ltd <120> NOVEL MICROORGANISM INHIBITING THE GROWTH OF HARMFUL BACTERIA AND MICROBIAL PREPARATION CONTAINING SAME AS AN EFFECTIVE INGREDIENT <130> FPD / 200112-0084 <160> 1 <170> KopatentIn 1.71 <210> 1 < 211> 1509 <212> DNA <213> Lactobacillus pentosus K34 <400> 1 agagtttgat cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgaac 60 gaactctggt attgattggt gcttgcatca tgatttacat ttgagtgagt ggcgaactgg 120 tgagtaacac gtgggaaacc tgcccagaag cgggggataa cacctggaaa cagatgctaa 180 taccgcataa caacttggac cgcatggtcc gagtttgaaa gatggcttcg gctatcactt 240 ttggatggtc ccgcggcgta ttagctagat ggtgaggtaa cggctcacca tggcaatgat 300 acgtagccga cctgagaggg taatcggcca cattgggact gagacacggc ccaaactcct 360 acgggaggca gcagtaggga atcttccaca atggacgaaa gtctgatgga gcaacgccgc 420 gtgagtgaag aagggtttgagactcgtgt actctgtga gca 540 gccgcggtaa tacgtaggtg gcaagcgttg tccggattta ttgggcgtaa agcgagcgca 600 ggcggttttt taagtctgat gtgaaagcct tcggctcaac cgaagaagtg catcggaaac 660 tgggaaactt gagtgcagaa gaggacagtg gaactccatg tgtagcggtg aaatgcgtag 720 atatatggaa gaacaccagt ggcgaaggcg gctgtctggt ctgtaactga cgctgaggct 780 cgaaagtatg ggtagcaaac aggattagat accctggtag tccataccgt aaacgatgaa 840 tgctaagtgt tggagggttt ccgcccttca gtgctgcagc taacgcatta agcattccgc 900 ctggggagta cggccgcaag gctgaaactc aaaggaattg acgggggccc gcacaagcgg 960 tggagcatgt ggtttaattc gaagctacgc gaagaacctt accaggtctt gacatactat 1020 gcaaatctaa gagattagac gttcccttcg gggacatgga tacaggtggt gcatggttgt 1080 cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttattatc 1140 agttgccagc attaagttgg gcactctggt gagactgccg gtgacaaacc ggaggaaggt 1200 ggggatgacg tcaaatcatc atgcccctta tgacctgggc tacacacgtg ctacaatgga 1260 tggtacaacg agttgcgaac tcgcgagagt aagctaatct cttaaagcca ttctca gttc 1320 ggattgtagg ctgcaactcg cctacatgaa gtcggaatcg ctagtaatcg cggatcagca 1380 tgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca tgagagtttg 1440 taacacccaa agtcggtggg gtaaccgtag atgta

Claims (7)

Salmonella gallinarum , Helicobacter pyroli , Lactobacillus rhamnosus , Escherichia coli , Saccharomyces cerevisiae , Resperus histropus And phenyl organic acid (POA), derivative phenyl organic acid (DPOA), indole organic acid (IODO), which inhibit the growth of harmful bacteria selected from the group consisting of Staphyloccus aureus ) And Lactobacillus pentosus K34 (Accession No .: KCCM 10331), which produces an organic acid selected from the group consisting of derivative indole organic acid (DIOA) and its derivatives. delete Microbial additive containing Lactobacillus pentosus K34 of claim 1 as an active ingredient. delete The method of claim 3, wherein A microbial additive, characterized in that the cells obtained by culturing Lactobacillus pentosus K34 are mixed with an excipient, or the cells are recovered to be produced as a thermo- or lyophilized probiotic. The method of claim 3, wherein A microbial additive, characterized in that it is prepared as a concentrate containing dried Lactobacillus pentosus K34 culture medium or a concentrate containing a large amount of organic acids and derivatives thereof obtained by extracting the culture solution with a solvent and then concentrating the extract under vacuum. The method of claim 3, wherein A microbial additive comprising Lactobacillus pentosus K34 in a content of 10 ⁶ to 10 ⁹ cells / g.