KR100273742B1 - Lactococcus lactis microorganism producing natural antibacterial substances (KFCC 11047) - Google Patents

Abstract

(1) The technical field to which the invention described in the claims belongs

It relates to a lactococcus lactis microorganism producing a natural antibacterial substance.

(2) The technical problem to be solved by the invention

It is an object of the present invention to provide a Lactococcus lactis subsp. Lactis A164 (KFCC-11047) microorganism that produces a pure bacteriosin of molecular weight molecular weight 5.0-5.5 KDa having antimicrobial activity against most lactic acid bacteria and pathogenic microorganisms.

(3) Summary of the solution method of the invention

The present invention is to isolate and purify the bacteriocin from lactic acid bacteria lactococcus lactis A164, which produces a broad bacteriocin, which has a high antibacterial activity, and the bacteriocin has an inhibitory effect on most lactic acid bacteria and various pathogenic bacteria. It is stable to pH and is characterized by producing a bacteriocin having a molecular weight of 5.0-5.5 KDa.

(4) important terms of the invention

The strain according to the present invention has an inhibitory effect on most lactic acid bacteria and pathogenic microorganisms, and has a characteristic of bacteriocin that is stable to acid and heat, so it is used as a food additive or a raw material of pharmaceuticals to show extended shelf life and an antimicrobial effect. Competitive advantage can be competitive.

Description

Lactococcus lactis microorganism producing natural antibacterial substances (KFCC 11047)

FIG. 1 is a photograph showing the whole cell protein format of several lactic acid bacteria by SD polyacrylamide gel electrophoresis.

1. Standard Molecular Weight Protein

2. Whole Cell Protein of Lactobacillus Brevis ATCC 13648

3. Whole Cell Protein of Lactobacillus Brevis ATCC 14869

4. Whole Cell Proteins of Lactococcus Lactis Subspices Lactis ATCC 11454

5. Whole Cell Protein of LAB-A164

6. Whole Cell Protein of Enterococcus picalis ATCC 19433

2 is a diagram showing the cell growth and bacteriocin activity by time while culturing the strain LAB-A164 in M17 liquid medium.

3 is a photograph showing the inhibition of Lactobacillus to the petri dish by partial purification of the strain.

A. Untreated Partial Purifying Liquid

B. Partial Purified Solution Treated with α-chymotrypsin

C. Partial Purified Solution Treated with Pronase E

D. Partial Purified Solution Treated at 100 ° C for 30 Minutes

Figure 4 shows the pH stability of partially purified bacteriocins.

FIG. 5 is a photograph showing the BD polyacrylamide gel electrophoresis of purified bacteriocin (A) and growth inhibition (B) to Lactobacillus by purified bacteriocin.

1. Low Molecular Weight Standard Protein

2. Purified Bacteriocin

[Purpose of invention]

[Technical field to which the invention belongs and the prior art in that field]

The present invention relates to bacteriocin, a natural antimicrobial material that can be used as a food preservation or biocontrol agent. The bacteriocin is a similar protein by several researchers since Davis et al. Discovered a protein substance that inhibits the growth of microorganisms from Escherichia coli in 1925 and named this protein colicin. Sexual material has been reported. These bacteriocins consistently have a narrow antimicrobial range limited to strains that are closely related to the antimicrobial producing strain itself and to lineage and lineage, and have similar characteristics to antibiotics.

Because bacteriocin molecules are composed of proteins, they are decomposed by protein hydrolase in the digestive system as soon as they are ingested in the human body. Therefore, they are expected to be useful as new bio-preservatives for foods or biocontrol agents for fermented foods. It is becoming. Until now, physical methods such as heating, freezing and drying and chemical methods such as acidification, chemical synthesis preservatives, pickling and smoking using salt or sugar have been used to prevent food from being deteriorated by chemical, microbiological or enzymatic changes. Has been used. However, these methods impair the natural quality of food, such as loss of nutrients, changes in texture and taste.

Lactic acid bacteria are microorganisms that produce lactic acid by anaerobic carbohydrates and are widely distributed in nature. This microorganism has been applied as an important preservation means of foods in both East and West by suppressing the growth of decaying microorganisms that grow well in neutral and alkaline, especially in the processing of milk, dairy products, meat products, vegetables and various salted fish. come. Lactic acid bacteria improve the preservation of foods by reducing the pH by their various metabolites, organic acids, because organic acids such as lactic acid and acetic acid have bactericidal action against most microorganisms. In addition to organic acids, hydrogen peroxide and the bacteriocins they produce are also involved in conservation. Nisin, the most widely known bacteriocin produced by lactic acid bacteria, has long been allowed for use due to its broad antimicrobial range, and in the United States, the Food and Drug Safety Administration (FDA) in 1988 identified a food additive as Grass (GRAS). Admitted to.

Therefore, in Korea, which is rich in fermented foods traditionally made with natural microorganisms such as kimchi, jangjang, and salted fish, these foods constitute the main basic food that affects the health of the people, so the development of scientific and industrial application technologies for bacteriocin is urgent and important. Do.

[Technical problem to be achieved]

Therefore, it is an object of the present invention to provide a lactic acid bacterium that produces a bacteriocin having a broad antibacterial range that can be used as a natural food preservative.

[Configuration and Function of Invention]

[Sample Used for Invention]

Samples used in the present invention were used while preserving kimchi collected from commercial restaurants, kimchi sold on the market and kimchi prepared by the traditional method at home at 4 ℃.

[Isolation of Lactic Acid Bacteria and Screening of Antimicrobial Production Strains]

Kimchi samples were suspended in sterile physiological saline, allowed to stand for 5 minutes to precipitate the kimchi suspension, and then diluted in physiological saline sequentially and plated on MRS agar medium and incubated at 30 ° C. for 1-2 days. The colonies formed were transplanted into a new MRS agar medium with sterile toothpicks and incubated at 30 ° C. until colonies were formed, and then MRS soft agar medium containing Locobacillus plantarum, known as rancid microorganism of kimchi, was used as an indicator. 5 ml of the layer was incubated until the lawn was formed to examine the formation of inhibitory rings around the colonies. The strain showing the inhibitory ring was first selected as a bacteriocin producing strain.

Example 1

The first selected strains were selected second by observing the activity in the liquid medium. Primary strains were inoculated in MRS liquid medium and incubated overnight, followed by centrifugation to remove the cells, and only the supernatant was removed. The remaining cells were completely removed with a 0.22 μm filter, and then placed on a Petri dish. 5 ml of MRS soft agar medium containing the indicator bacteria specified above was laid and a hole was sterilized with a sterilized tip. 100 μl of supernatant was added to the prepared hole and cultured to form a lawn at 30 ° C. Were selected, and LAB-A164, the largest inhibitory ring, was selected.

[Identification of Selected Strains]

LAB-A164, a selected strain, was identified through morphological, physiological and biochemical experiments.

Example 2

The results of morphological, physiological and biochemical experiments on LAB-A164 were shown in Table 1, and from the results shown in Table 1, the selected strains were considered as lactic acid bacteria.

Example 3

This selection strain, which is considered to be Lactobacillus, is used to express the whole intracellular protein type with representative strains of existing spawn organs.

TABLE 1

Compared with SDS-polyacrylamide gel electrophoresis, as shown in FIG. 1, it was found to be consistent with the representative strain of Lactococcus lactis subsp. A164 was identified as Lactococcus lactis subsp. Lactis.

Measurement of Antimicrobial Activity

Antimicrobial activity of bacteriocin was measured using the spot-on-lawn method. The supernatant was collected by centrifugation of the bacteriocin producing strain at 10,000 rpm for 10 minutes, and the remaining strain was completely removed with a 0.22 μm membrane, and then dipped into a previously poured solid medium as described above. Dried for about 30 minutes. In order to quantitatively measure the antimicrobial activity, the supernatant was diluted 2 times sequentially using a new sterilizing liquid medium, and then the antimicrobial activity was measured after dropping on a solid medium. The activity unit (AU) showed antimicrobial activity per 1ml. Inverse of the maximum dilution multiples.

Example 4

[Measurement of Antibacterial Range]

The antimicrobial range of the bacteriocin produced by the strain LAB-A164 of the present invention was carried out by a well-diffusion method against various kinds of lactic acid bacteria, food-derived pathogenic bacteria and rot bacteria. The bacteriocin exhibited broad antimicrobial activity against all lactic acid bacteria including Lactobacillus, Leuconostoc, Pediococcus and Carnobacterium genus, and Bacillus cereus cereus), Staphylococcus aureus, and Listeria monocytogenes, such as Listeria monocytogenes was also shown activity against the pathogenic microorganisms (Table 2).

Example 5

[Establishment of Optimal Growth Conditions for Bacteriocin Production]

In order to establish the optimum conditions for the production of bacteriocin produced by the strain Lactococcus lactis subsp. Lactis A164, various kinds of synthetic media, incubation temperature, incubation time and initial pH of the media were changed.

TABLE 2

By culturing the strain, the cell weight and bacteriocin activity were measured. M17 medium showed the highest cell growth and bacteriocin activity. The strain of the present invention was inoculated in M17 liquid medium, and the result of stationary culture at 30 ° C was the same as that of FIG. Cell growth increased rapidly from 4 hours after inoculation and entered stationary phase after 16 hours. Lactate production was highest between 4-16 hours and rarely after 20 hours. Bacteriocin production was produced with cell growth after inoculation, and showed a maximum yield in 16 hours of culture, but gradually decreased after 20 hours.

Example 6

[Influence on Degrading Enzyme Treatment and Heat Treatment]

This experiment was carried out with a partial purification of LAB-A164, a strain of the present invention. LAB-A164 partial purified solution was inoculated to 100% M17 liquid medium at 1%, incubated for 16 hours at 30 ° C, centrifuged at 8,000 rpm for 10 minutes to remove cells, and 35% saturated ammonium sulfate was added to the supernatant. After standing at 4 ° C. overnight, centrifugation was carried out at 12,000 rpm for 20 minutes to recover only the precipitate, suspended in distilled water, and then dialyzed. Dialysis was used in the experiment while partially purified with a distilled water every 12 hours using a dialysis membrane having a molecular weight cutoff of 3.5 kDa and stored at -20 ℃. Proteolytic enzymes trypsin, pepsin, α, β -chymotrypsin, papain, pronase E and glycolytic enzyme α-amylase were added to the final concentration of 1 mg / ml to 2, at 37 ° C As a result of measuring the remaining vitality after the reaction by incubation for a time, the activity by trypsin, pepesin, papain and α-amylase as shown in Table 3

TABLE 3

There was no change in activity, but the activity was partially lost by α, β -chymotrypsin and all activity was lost by pronase E, a bacterial protease. In addition, the results of comparing the antimicrobial activity on the agar medium also showed the same results (Figure 3).

Table 4 shows the results of measuring the remaining vitality by heat treatment of the partially purified bacteriocin solution at pH 3.0, 5.0 and 7.0. When heat-treated at 100 ° C. for 30 minutes, there was no change in activity at pH 3.0, whereas pH 5.0 and

TABLE 4

In 7.0, the activity was partially lost. After heat treatment at 121 ° C. for 15 minutes, activity was partially lost at pH 3.0 and 5.0, and at pH 7.0, all activity was lost. In conclusion, the bacteriocin was found to be stable against heat under acidic conditions.

Example 7

[Stability of pH of Bacteriocin]

Partially purified bacteriocin solution was mixed 1: 1 with a buffer solution from pH 2.0 to 10.0 and left at 37 ° C. for 3 hours to stabilize the pH and then measure the activity. As shown in FIG. 4, bacteriocin activity was shown from pH 2.0 to 10.0, but the highest activity was found at pH 6.0, 7.0, and 8.0. Therefore, this bacteriocin was found to have the highest activity near neutral pH rather than acidic pH.

Example 8

[Purification of Bacteriocin]

50 ml of 50 mM sodium acetate buffer (pH 5.0) was charged with 50 ml of a partially purified liquid sample collected by ammonium sulfate precipitation, 10 ml of a strong cation exchange resin, S-sepharose fast flow resin, in a column of 1.5 × 50 cm. The pH of the column was equilibrated with. Then, 50 ml of partial purification solution was applied to the column to wash unadsorbed protein with 50 mM sodium acetate buffer (pH 5.0), and then eluted with 50 ml of sodium acetate buffer containing 50 ml of 0.5 M sodium chloride. Was collected and passed through ultrafiltration with a molecular weight of 10 kDa to conduct a final purification experiment. As shown in FIG. 5, pure bacteriocin was obtained, and the activity was measured through direct polyacrylamide gel electrophoresis. The molecular weight was found to be approximately 5.0-5.5 kDa.

Example 9

[Effects of Nutrients on Bacteriocin Production]

In order to establish the optimum production medium composition of the bacteriocin produced by the strain of LAB-A164 of the present invention, after culturing the strain by adding various carbon sources and nitrogen sources, the cell weight and bacteriocin activity were measured. First, sucrose (lactose) and glucose (glucose) affected cell growth, and bacteriocin production was significantly higher in lactose (Table 5). As the next nitrogen source, soytone, yeast extract, and proteose peptone were found to be effective in cell growth, and bacteriocin production was highest in yeast extract (Table 6). ). The optimal bacteriocin production concentration of lactose as a carbon source was 0.5-1.0%, and growth of cell mass and production of bacteriocin could not be observed at 15-20% (Table 7). The production of bacteriocin was the highest at 15-20% according to the concentration of yeast extract as a nitrogen source, and the inhibition of bacteriocin production was also observed at the concentration of 1.0-2.0% (Table 8).

TABLE 5

TABLE 6

TABLE 7

TABLE 8

Claims (1)

Isolated from traditional fermented food kimchi against most lactic acid species, including Lactobacillus, Leukonostock, Pidiococcus and Canobacterium genus, and pathogenic microorganisms such as Bacillus cereus, Staphylococcus oreus and Listeria monocytogenes Lactococcus lactis subsp. Lactis A164 (KFCC-11047) strain, characterized by producing a pure natural antibacterial bacteriocin having a broad antimicrobial activity of 5.0-5.5 KDa.