KR960015891B1 - Lactococcus sp. bl1 and new bacteriocin, and processing of new bacteriocin - Google Patents

Abstract

The novel bacteriocin which is a kind of antibiotics, termed KOFRIJIN-1, is prepared by culturing Lactococcus SP. BL1(KFCC-10790), warming the culture media up in a double boiler, cooling, centrifuging, removing a precipitation, removing a precipitation 2-3 times after adding a solvent in the supernatant, dissolving an active fraction of a final precipitation in HCl solution, freeze-drying, and homogenizing to obtain the final product. The KOFRIJIN-1 shows a strong antibiotic effect to gram positive microorganisms, and don't shows any harmful effect to eukaryotes. Its titer is 2.2=G105IU/g and a molecular weight is 5,600 dalton.

Description

Novel antimicrobial substance (Coprizine-I) and Lactococcus SP.BL1 to produce the same and method for producing the same

Figure 1 is a photograph showing the degree of inhibition of the test bacteria (Lactobacillus plantarum) of the present invention antimicrobial material (KOFRIJIN-I).

Figure 2 is a micrograph of the antibacterial substance producing bacterium Lactococcus SP.BL1 of the present invention.

The present invention relates to a novel antimicrobial material (hereinafter referred to as "KOFRIJIN-I"), and microbial Lactococcus SP.BL1 (Lactococcus SP.BL1) for producing the same and a method for producing the same.

Bacteriocin in the form of protein, a kind of antimicrobial substance, is relatively low in toxicity and is easily decomposed by various proteolytic enzymes when ingested with food, and is widely used as a food preservative.

Bacteriocin is produced by a variety of bacteria, most of which are composed of low molecular weight proteins, and are known to act only on similar species.

The types of bacteriocins known until recently are colicins, alveicins, cartovoricins, arizonacins, cloacins, marcescins, nu Pneumocins, aerocins, pyocins, fluocins, megacins, monocins, cerecins, enterococcins, enterococcins Staphylococcins, epidermidins, etc. exist, and these bacteriocins are known to produce different types of substances even if they are produced by the same family of fungi.

Describing the inhibitory function and action of the above-mentioned antimicrobial substances, the first step is the adsorption of an antimicrobial substance, ie, bacteriocin, to a specific receptor on the surface of the cell. Will die.

The bacteriocin-induced cell damage is classified into two types, one of which damages DNA or ribosomes in a cell, and damages DNA or ribosomes by acting on DNAse or RNAse. In addition, bacteriocins directly damage membranes, such as inhibiting polymer synthesis, inhibiting active transport of potassium and amino acids, and inhibiting ATP levels.

The bacteriocin is found to be produced by a variety of bacteria, typical antimicrobial agents and their production are shown in Table 1.

Table-1 Representative Antimicrobial Substances and Producing Bacteria

As described above, bacteriocin can be used as a food preservative that can replace chemical preservatives, and can be used as an antimicrobial substance produced by the microorganisms of the genus Lactococcus, although it can improve the effective shelf life of foods without causing the consumer to avoid. Ninisin is known only.

Accordingly, an object of the present invention is to provide an antimicrobial substance (coprizin-I (KOFRIJIN-I)) which can be easily degraded by proteolytic enzymes and used as a food preservative, and the Lactococcus microorganism producing the same and a method for producing the same.

In order to achieve the above object, in the present invention, strain Lactococcus SP.BL1 (KFCC-10790) which isolates edible lactic acid bacteria in nature and produces an antimicrobial substance was selected and cultured in a medium containing glucose and casein hydrolyzate as a main component. Thereafter, the culture solution was separated and purified using a solvent method, a membrane filter method, and a lyophilization method, thereby obtaining a new antimicrobial substance (coprizin-I) in high yield. Same as

[Strain separation process]

Strains producing antimicrobial substances were isolated from nature based on known methods.

In other words, by using plate culture method, the microorganism is purely separated from commercial milk and the microorganism having antimicrobial activity is selected, and then Lactobacillus plantarum IFO 3070 (ATCC 8014) is used as a test bacterium to obtain microorganisms having excellent antimicrobial activity. Deposited to the spawn association (Accession No. KFCC-10790).

[Production process of antibacterial substance]

15 to 25 g of glucose, 12 to 18 g of caseinate hydrolysate, 3 to 7 g of yeast extract, 1 to 3 g of ammonium citrate, 1.5 to 4.5 g of sodium acetate, 0.05 to 0.2 g of magnesium sulfate, 1 to 3 g of phosphate, Mix 0.03 to 0.07 g of manganese sulfate to a pH of 6.2 and add to fermenter. Subsequently, sterilization was performed at a temperature of 110 to 130 ° C. for 12 to 18 minutes, followed by deposit with Lactococcus SP.BL1 (microbial accession no. KFCC-10790 to the Korean spawn association) as shown in FIG. Microorganisms) are inoculated and fermented at 30 to 40 ° C. for 10 to 14 hours to produce an antimicrobial substance.

Here, the production pattern of the antimicrobial material according to the fermentation time is shown in Table 2.

Table 2 Production Pattern of Antimicrobial Agents by Lactococcus SP.BL1

From Table 2 it can be seen that when the fermentation time is about 12 hours or more, the antimicrobial substance exhibits a titer of 3894 IU / m, which is the maximum.

[Isolation and Preparation Process of Antibacterial Substances]

In the production process of the antimicrobial material, the fermentation broth is finished to pH 2 with hydrochloric acid, and then cooled by boiling water for 3 to 7 minutes and centrifuged at 5,500 to 6,500 rpm for 10 to 30 minutes to remove the precipitate. By making a supernatant. Subsequently, the same amount of methanol as the supernatant was added to the supernatant to remove the precipitate, and 2 times the cold acetone was added to the supernatant to remove the formed precipitate, and then 4 times acetone was added to finally remove the active fraction in the precipitate. Take it.

Next, the active fraction is dissolved in 0.04-0.06 normal hydrochloric acid solution, lyophilized and ground to obtain an antimicrobial substance.

The amount of antimicrobial material thus obtained can be obtained from about 16 to 17 g (titer 2.2 × 15 ⁵ IU / g) per fermentation broth.

Hereinafter, the manufacturing method of this invention antimicrobial substance (KOFRIJIN-I) is demonstrated in detail as an Example.

Example 1

1 g of distilled water, 20 g of glucose, 15 g of caseinic acid hydrolyzate, 5 g of yeast extract, 2 g of ammonium citrate, 3 g of sodium acetate, 0.1 g of magnesium sulfate, 2 g of potassium phosphate, and 0.05 g of manganese sulfate were mixed to a pH of 6.2 and placed in a fermenter. .

Next, the temperature of the fermenter was 121 ° C. and sterilized for 15 minutes, and inoculated with Lactococcus SP.BL1 (microorganisms deposited as microorganism accession no. KFCC-10790) at 35 ° C. incubated in the same medium for 18 hours. The fermentation was carried out at 12 hours to produce an antimicrobial substance.

Example 2

The fermentation broth was fermented in Example 1 was adjusted to pH 2 with hydrochloric acid, and then bathed in boiling water for 5 minutes, and after cooling, the precipitate was removed by centrifugation at 6,000 rpm for 20 minutes.

Next, the same amount of methanol was added to the supernatant to remove the precipitate, and then, twice the amount of acetone was added to the supernatant to finally remove the precipitate.

Subsequently, four times the amount of acetone of the supernatant was added to the supernatant to finally obtain an active fraction in the precipitate.

Thereafter, the obtained active fraction was dissolved in 0.05 normal hydrochloric acid solution, lyophilized, and ground to obtain Koprijin-I (KOFRIJIN-I), an antimicrobial substance as shown in FIG.

The amount of coprizin-I thus obtained was about 17 g (titer 2.2 × 10 ⁵ IU / g) per fermentation broth.

Example 3

The fermentation broth obtained in Example 1 was pretreated by hydrochloric acid and heat treatment, and then filtered through a membrane filter (Microsart 0.45 μm) to remove cell debris.

Subsequently, the filtrate was continuously fractionated and purified by a membrane filter (Ultrasart Cut-off = 10,000: 5,000) and lyophilized to obtain an antimicrobial substance, Coprizin-I (KOFRIJIN-I) as shown in FIG. .

Next, the characteristics of the production strain producing the antimicrobial substance in the form of protein and the antimicrobial substance KOPRRIJ-I (KOFRIJIN-I) as an experimental example.

Experimental Example 1

As a result of reviewing the morphological and physiological characteristics of the producing strain producing the antimicrobial substance, it was identified as Lactococcus SP.BL1 (microorganism deposited as KFCC-10790 with accession to the Korean spawn association).

[One. Morphological properties]

Strains of Lactococcus SP.BL1 form pairs or streptococci that form off-white colonies on agar media. Cells are elongated, positive for Gram staining, and lacking motility.

[2. Determination of Culture]

Strains of Lactococcus SP.BL1 show catalase, gas production, escurin degradation, acetoin production negative, anaerobic growth, hypurate degradation, and anichinase positive. Specifically, the results of various biochemical or physiological tests Was shown in Table 3.

[Table 3 Biochemical Test Results of Antibacterial Substances Producing Strain Lactococcus SP.BL1]

Lactococcus SP.BL1, the antimicrobial producing strain, has a growth temperature of 35 ° C and an optimal pH of 6.2, enters the growth plateau after about 9 hours, and has a non-base rate of 0.92H ^-1 in glucose medium. Lavin and pentothenic acid were found to be required.

Experimental Example 2

The antimicrobial spectrum of KOFRIJIN-I, an antimicrobial substance, was examined as shown in Table 4 below.

Table 4 Antimicrobial Spectra of Antibacterial Substance KOPRIZIN-I

+: Low ring <10mm + +: 10-14.9mm

+ + +: 15-19.9mm + + + +:> 20mm

As shown in Table 4, Coprizin-I antimicrobial agent not only shows a strong effect on Gram-positive bacteria such as Microcaucus luteus, but also has a weak inhibitory effect on Gram-negative bacteria such as Aeromonas hydrowheeler and Escherichia coli and inhibits on eukaryotic cells There is no substance.

Experimental Example 3

Enzyme degradability and pH safety of the coprizin-I antimicrobial were investigated and shown in Tables 5 and 6, respectively.

Table 5 Enzyme Degradability of KOFRIJIN-I Antimicrobial Substances

(-): Vitality loss, enzyme titer: 200 units / mg, antibacterial activity: 2516 units / mg

Table 6 pH Safety of KOFRIJIN-I Antimicrobial Substances

* Remaining vitality after 2 days at 25 ℃ after treatment at each pH

It can be seen from Table 5 that the coprizin-I antimicrobial material is a protein form that loses its vitality by carboxy peptidase and pronase, protease IV and alpha chymotrypsin. It was found to be a stable antimicrobial material.

Coprizine-I is also a low molecular weight material with a molecular weight of 5,600 Daltons with 60% deactivation when heated at 100 ° C. for 60 minutes.

Experimental Example 4

Amino acid analysis of the antimicrobial substance koprijin-I (KOFRIJIN-I) was performed and the results are shown in Table 7.

That is, 20 ml of 6N HCl was added to 200 mg of coprizin-I obtained in Example 2, and the resulting mixture was sealed in an ampoule and filled with nitrogen gas, and then hydrolyzed at 110 ° C. for 24 hours. 100 ml of distilled water was added to the hydrolyzed substance three times, and dried under reduced pressure to remove hydrochloric acid. The solution was dissolved in 5 ml of 0.2 N citrate buffer (pH 2.2) and passed through a membrane filter (0.22 μm co-size). Injection was performed into an analyzer (LKB4151, manufactured by Alpha Plus).

TABLE 7 Amino Acid Composition

As can be seen from Table 7, Coprizin-I of the present invention is composed of a total of 26 amino acid residues and has been shown to be different from the amino acid composition of nisin, diflocosin and subtyrin, in particular, Histidine content was much higher.

As described in the above Examples and Experimental Examples, the present invention not only has the advantage of easily producing antimicrobial substances from microorganisms, but also has an effect of being used as a food preservative by being easily degraded by proteolytic enzymes.

Claims (6)

As a strain of Streptococcus or Streptococcus, it forms gray white colonies on agar medium and is positive for gram staining, has no mobility, no amino acid requirement, but needs riboflavin and pentothenic acid, and has the ability to produce antimicrobial agents. Lactococcus SP.BL1 deposited with the association accession number KFCC-10790. It has a strong antibacterial effect on Gram-positive bacteria, has a weak antimicrobial effect on Gram-negative bacteria, has no inhibitory effect on eukaryotic cells, has a titer of 2.2 × 10 ⁵ IU / g and a molecular weight of 5,600 Daltons. Novel antimicrobial material produced from Lactococcus SP.SL1 deposited as 10790. Distilled water is mixed with main raw materials and subsidiary materials into a fermenter and sterilized. The medium is inoculated with Lactococcus SP.BL1 bacteria (Accession No. KFCC-10790, Trusted Authority: Korea spawn association) and fermented with fermented broth. After cooling, centrifugation to form a supernatant liquid from which the precipitate is removed, and a solvent is added to the supernatant solution to remove the produced precipitate two to three times, and the active fraction in the final precipitate is dissolved in hydrochloric acid solution and then lyophilized and ground. Method for producing an antimicrobial substance, coprizin-I (KOFRIJIN-I). The method of claim 3, wherein the main raw material is glucose and caseinic acid hydrolyzate. The method of claim 3, wherein the solvent is acetone and methanol. Distilled water is mixed with main raw materials and subsidiary materials into a fermentation tank and sterilized, and then fermented by inoculating L. coccus sp.BL1 bacteria (Accession No. KFCC-10790, Trusted Organization: Korean Seed Association) on the medium, and fermentation broth is pretreated by hydrochloric acid and heat treatment. And then filtration through a membrane filter to remove cell debris, and the filtrate is subjected to filtration and freeze-drying continuously, followed by freeze-drying to produce the antimicrobial substance coprizin-I.