KR0131136B1 - Novel bacteriocin producing enterococcus and preservation methkimchi - Google Patents

Abstract

Enterococus fercium generating a bacteriocin strain is produced by mixing pickles, sugar and salt in the medium of cabbage juice. Therefore, the produced lactic ferments has the characteristics of KCTC 8256P to extend the time when kimchi is stored.

Description

[Name of invention]

Lactic acid bacteria producing bacteriocin and long-term preservation method of kimchi using the same

Detailed description of the invention

[Industrial use]

The present invention relates to a novel microorganism producing bacteriocin and a long-term preservation method of kimchi using the novel microorganism.

[Prior art]

Kimchi is an indispensable food at the table of Koreans. Kimchi has a unique scent when it is matured with various vegetables and spices. Especially, recently, the nutritional and functionalities of kimchi are being revealed one after another. It is known as a representative fermented food. As a result, the number of foreigners who prefer kimchi is increasing.

In addition, kimchi has traditionally been immersed in the home, but as women's social participation and the lifestyle of apartments have spread recently, more and more families buy and eat kimchi without immersing it in their homes. As the sales of commodity kimchi increases, problems that have not been important in the past are emerging. The most urgent problem to be solved is Kimchi's storage.

To date, various methods have been tried to improve the preservation of kimchi. Typical examples are heat treatment method (quotation reference: Korean Patent Publication No. 67-273, 87-22), preservative addition method (quotation reference: Korean Patent Publication No. 89-4895, 90-939, 90- No. 1001, No. 90-1002, No. 90-1003, No. 90-2396, No. 90-3009, No. 90-4271, No. 93-12196), Irradiation method (quotation cited: Republic of Korea Patent Publication No. 91-5282) and the method of using the said process together (quotation quotation: Republic of Korea Patent Publication No. 90-3010) etc. are mentioned.

However, when the heat treatment is performed, the tissue of the kimchi becomes soft and heat odor is generated, and the fresh taste of the kimchi is lost.In the case of the addition of the preservative, the preservative may cause a strong objection from the consumer due to the harmfulness to the human body. In the radiation irradiation method, there are problems in the treatment technology and safety problems, and therefore, they are not actually used. In addition, the method of using them together can compensate for some of the disadvantages of the treatment alone, but it is not a fundamental solution and has the problem that the above-mentioned side effects may appear in combination. In addition, the low temperature distribution method currently used has the disadvantage that the cost is expensive because the refrigeration or freezing facilities are essential, and the refrigeration is not properly performed during the distribution process due to inadvertent storage, which is also a problem.

[PROBLEMS TO BE SOLVED BY THE INVENTION]

The present invention is to solve the problem of the long-term preservation method of the conventional kimchi as described above to provide a method for safe preservation of kimchi for a long time to increase the period of kimchi decorating.

Means for Solving the Problems of the Invention

In order to solve the above problems of the present invention, the present invention provides a novel strain having the characteristics of KCTC 8625P.

The present invention provides a novel strain Enterococcus fasium DU 0267, which produces bacteriocins with strong antibacterial activity against kimchi lactic acid bacteria.

The present invention also provides a method for producing kimchi, which extends the kimchi decorating period by using a bacteriocin producing strain as a starter.

In the kimchi production method of the present invention, the starter is selected from enterococcus fascium, and the enterococcus fascium comprises the bacteriocin-producing strain Enterococcus fascium DU 0267 of the present invention.

In addition, in the kimchi production method of the present invention, the bacteriocin producing strain is added to the kimchi immersion, the starter of the present invention is cultured in a kimchi medium made of the same composition as the composition of kimchi to be added to the starter.

In the present invention, the bacteriocin producing strain Enterococcus fascium is pre-cultured in the cabbage juice medium, the cabbage juice medium is cabbage juice: milk: sugar: salt 99.3% to 85% by weight: 5% by weight to 0.1 weight%: 5 weight%-0.1 weight%: It manufactures by mixing in 5 weight%-0.5 weight% ratio.

In the kimchi production method of the present invention, the kimchi includes all the existing kimchi including Chinese cabbage kimchi, white kimchi, yeolmu kimchi, mustard kimchi, leek kimchi, green kimchi, diced, Dongchimi, clogged kimchi, gojikkimchi and water kimchi and the like. It is selected from the group consisting of.

Hereinafter, the present invention will be described in more detail.

The present invention is a completely new approach, different from existing methods for long-term storage of kimchi, in which a novel strain producing bacteriocin that has the property of inhibiting the growth of fermented microorganisms in kimchi is isolated from kimchi: the isolated new strain By inhibiting the proliferation of fermentation microorganisms in kimchi by using a method of extending the kimchi period. Bacteriocin is an antimicrobial substance produced by bacteria. It is mainly composed of proteins or complex proteins, and when absorbed by the human body, it is decomposed and absorbed into the body as a nutrient source. The main difference between bacteriocins and general antibiotics is that the antibacterial activity of bacteriocins is very specific. In other words, unlike ordinary antibiotics, bacteriocins often exhibit specific antimicrobial activity only to the species of the strain that produces them.

There are many lactic acid bacteria known to produce bacteriocins, a representative of which is lactobacillus lactis that produces nisin. However, lactobacillus lactis, which produces nisin, is a lactic acid bacterium derived from dairy products and has a disadvantage in that it is difficult to grow in low-salt foods such as kimchi because of its low salt resistance.

The major lactic acid bacteria involved in fermentation of kimchi are known as leukonostock mesenteroides, Lactobacillus plantarum, Lactobacillus brevis, Enterococcus pecalis, Pidiococcus pentosaceus, etc. It is being separated and identified.

In the present invention, among the various lactic acid bacteria isolated from kimchi, while showing a high antibacterial activity against kimchi lactic acid bacteria, at the same time, a relatively weak lactic acid bacteria were isolated. When the lactic acid bacterium of the present invention is added to about 0.5 to 3% by weight in the production of kimchi, the amount of gas generated is reduced to about 60% compared to when the microorganism of the present invention is not added. Period) was also extended about 4 times at 10 ℃.

The reason why the kimchi can be extended for a long time is that the strain of the present invention, which has a relatively weak acid producing ability, grows actively and simultaneously produces a large amount of bacteriocin, resulting in gaseous bacteria such as leukonostockmethenoteroides. This is because it inhibits the growth of the causative bacterium Lactobacillus plantarum.

The novel strain producing the bacteriocin of the present invention was isolated and identified as follows.

[Separation of strains]

The kimchi prepared by a conventional method is cooked well with acidity of 0.55 to 0.65%, finely ground and then filtered with sterile gauze. The filtered solution is diluted 109 times with sterile water, 0.1 ml of the diluted solution is prepared in MRS agar medium prepared in advance, evenly smeared with a smeared rod, and then incubated at 30 ° C. After 2 to 3 days, the colonies produced on the medium were taken in a loop and inoculated in a prepared MRS liquid medium to purely isolate the strain of the present invention. At this time, the composition of the MRS liquid medium is to remove agar from the MRS agar medium described below.

[Sympathy of strain]

The lactic acid bacteria of the present invention isolated from kimchi by the above method were identified according to their morphological, cultural and physiological properties based on the manual of buggy. The results are as follows.

(1) form of cells: aureus

(2) cell size: 0.5-1.0 탆

(3) Gram dyeing: Positive

(4) Mobility: None

(5) spore forming ability: none

(6) Formation of Gases from Glucose:-

(7) catalase:-

(8) Reaction in litmus milk

1) Reduction: +

2) peptonation:-

3) Acid curd formation: +

(9) Formation of Ammonia from Arginine: +

(10) Hydrolysis of Gelatin:-

(11) Hydrolysis of Escalin:-

(12) Hydrolysis of Arginine: +

(13) Hydrolysis of Hypurate:-

(14) reduction of nitrate:-

(15) Tellurite Web Reduction:-

(16) reduction of tetrazolium: +

(17) Dextran Production:-

(18) Growth at 10 ° C .: +

(19) Growth at 15 ° C .: +

(20) Growth at 40 ° C .: +

(21) Growth at 45 ° C .: +

(22) Growth at 50 ℃:-

(23) Growth at pH 3.6:-

(24) Growth at pH 3.9:-

(25) Growth at pH 4.2:-

(26) Growth at pH 4.8:-

(27) Growth at pH 8.6: +

(28) Growth at pH 9.6: +

(29) Growth at salt concentration of 6.5%: +

(30) presence or absence of DAP in peptidoglycan:-

(31) Isomers of Lactic Acid: L (+)

(32) positive blood hemolysis:

(33) Production of Acids from Sugars

(1) imgdalin: +

(2) arabinose: +

(3) arbutin: +

(4) Cellobioses: +

(5) Escalin: +

(6) Fructose: +

(7) galactose: +

(8) Glucose: +

(9) Gluconate: +

(10) Glycerol: +

(11) lactose: +

(12) Maltose: +

(13) Malto Trioise: +

(14) Mannitol: +

(15) Mannose: +

(16) Melegitos:-

(17) Melibiose: +

(18) Raffinose:-

(19) Rhamnose:-

(20) Live Bosses: +

(21) He raised: +

(22) Sorbitol:-

(23) Solvoise: =

(24) Sucrose: +

(25) Trehalose: +

(26) Xylose: +

[Check the strain]

In order to confirm the identification of the strain of the present invention, the fatty acid composition of the cells was examined by gas chromatography. The freeze-dried powdered strain was treated with hydrochloric acid solution containing 5% methanol to methylate the fatty acids of the cells, extracted with hexane, and analyzed under the following conditions.

(1) Gas chromatography: GC-8A gas large chromatography system (Shimadzu)

(2) Analyzer: Chromatopac C-RC1B (Shimadzu)

(3) Detector: Flame ionized detector (FID)

(4) Column: 15% DRGS coated on Chromosorv WAW DMCS (100/120 mesh)

(5) Temperature: 1) Injector: 240 ℃

2) Detector: 240 ℃

3) Column: 195 ℃

(6) Carrier gas: Nitrogen (flow rate, 0.4 ml / min))

Each fatty acid was identified and quantified by comparing the retention time of each peak of the chromatogram obtained as a result of the gas chromatography performed under the above conditions with that of the standard mixture. The quantitative results of each fatty acid are reported in Table 1 below.

In the subscript, the number before the colon in the subscript indicates the number of carbon atoms in the fatty acid, the number after the colon indicates the number of double bonds, Cy means cyclopropinic acid, and the number represents the proportion of each fatty acid in total fatty acids. Means (%).

From the table, it can be seen that the bacteriocin producing strain of the present invention contains the highest ratio of C and C linear acids, and then contains C linear cycloacids and C cyclopropinic acids in high ratios.

This fatty acid composition is identical to the fatty acid composition of the microorganism of the microorganism of ATCC 19434, the standard strain of Enterococcus fascium, and is consistent with the identification results according to the morphological, cultural, and physiological test results.

[Investigation of the characteristics of the strain product of the present invention]

In order to investigate the characteristics of the bacteriocin produced by the strain of the present invention, a series of experiments were conducted.

I. Antimicrobial Spectrum Investigation on Various Microorganisms

Agar Well Diffuseion Assay was performed to investigate the antimicrobial spectrum of various microorganisms of bacteriocin produced by the microorganism of the present invention. The medium used for the analysis is MRS (De Man, Rogosa, Sharpe) medium, the composition is as follows.

10 g of bacto proteose peptone

10 g bacto beef extract

5 g of bacto yeast extract

20 g of bacto dextrose

Twin 801g ammonium citrate 2g

Sodium Acetate 5g Magnesium Sulfate 0.1g

Manganese sulfate 0.05g dipotassium phosphate 2g

Agar 15g Distilled Water 1L

After sterilization of the MRS medium having the composition described above, it was poured into a Petri dish and hardened. Then, the MRS medium having the same composition of which the agar content was reduced to 0.7% was finally diluted to a concentration of 107 colony forming unit (CFU). Each indicator microbial suspension was added and then layered and hardened on an already prepared MRS medium. The MRS medium thus prepared was punched with a cork bore of 6 mm in diameter and then the bottom was sealed by dropping one or two drops of sterile agar. The Enterococcus fascium DU-0267 strain was incubated for 18 hours at 30 ° C. in MRS liquid medium, and then the culture solution was centrifuged (5,000 × g, 15 minutes), neutralized with 3N NaOH, and the mixture was neutralized with 0.2 μm membrane filter. Sterilized by passing. The filtrate thus obtained was diluted in each of the two-fold steps and dropped 0.1 ml into the hole of the MRS medium for the antimicrobial test, and the reciprocal at the lowest concentration at which the low concentration of the indicator bacteria can be seen at 30 ° C. for 18 to 24 hours. Shown as titer (AU). The results are shown in Table 2 below.

II. Enzyme Influence Investigation

In order to characterize the bacteriocin produced by the strain of the present invention, the effect of various enzymes on the antimicrobial activity was investigated.

After culturing the strains as in the antimicrobial spectrum investigation results for the various microorganisms described above, the sterilized culture solution was immersed in ice, cooled, and 75% saturated solution was added by adding ammonium phosphate little by little, followed by stirring for 30 minutes. . The precipitate was dissolved in a 5 Mm phosphate buffer solution with a pH of 1/20 of Ph 7.0, dialyzed at 5 ° C. for 12 hours with the same buffer solution, and then lyophilized to obtain a crude bacteriocin. The prepared bacteriocin was dissolved in the buffer as described above to a concentration of 1 mg / ml and used in the following experiment.

Trypsin and alpha chymotrypsin were dissolved in the same buffer solution, and pepsin was dissolved in 0.2 N hydrochloric acid to a concentration of 1 mg / ml.

The prepared bacteriocin solution and the enzyme-treated solution were filtered through membrane filters, respectively, and sterilized and treated at 37 ° C. for 1 hour. Before testing the antimicrobial activity was neutralized and boiled again for 5 minutes to eliminate the activity of the enzyme and the remaining antimicrobial activity was measured by the same method as the test for the antimicrobial activity against the microorganisms using Lactobacillus sake JCM 1157 as an indicator microorganism. The results are shown in Table 3 below.

The figures in the table are values (%) comparing the antimicrobial activity after treatment to the antimicrobial activity of bacteriocin before the enzyme treatment.

III. Investigate the effects of temperature

In the same manner as above, the bacteriocin was partially purified to prepare a prepared bacteriocin solution at a concentration of 1 mg / ml. After heat treatment at 100 ° C. for 10 minutes, 100 ° C. for 30 minutes, 100 ° C. for 60 minutes, and 121 ° C. for 10 minutes, the antimicrobial activity was measured using Lactobacillus sake JCM 1157 as an indicator microorganism. The results are shown in Table 4 below.

The figures in the table above are the values (%) comparing the antimicrobial activity after treatment to the antibacterial activity of bacteriocin before the enzyme treatment.

Ⅳ. Comparison of bacteriocins with conventional bacteriocins by strains of the invention.

In order to clarify the difference between the bacteriocin produced by the known bacteriocin producing strain and the bacteriocin produced by the strain of the present invention, the characteristics of the two were compared. Strains reported to produce conventional bacteriocins include Enterococcus fascium strains, Enterococcus fascium DTC 1146, Enterococcus fascium EI, Enterococcus fascium-100, Enterococcus fascium JBL 1061, Enterococcus fascium Such as ATCC 27273. The characteristics of the bacteriocins produced by the bacteriocins and the strains of the present invention shown in the results of Experiments I to III were compared by the previously reported Enterococcus fascium strains. And the results are shown in Table 5 below.

※ R (Rrsistan): Not affected by enzyme at all.

I (Insensitive): intermediate between Resistant and Sensitive, slightly lost activity by enzyme

S (Sensitive): Activity is completely lost by enzyme.

As can be seen in Table 5, the bacteriocin produced by the strain of the present invention is a bacteriocin of a new bacterium that is completely different from the bacteriocins produced by the existing E. fascium strains. It was found that the new strain. Therefore, on October 11, 1994, the strain of the present invention was deposited with the Genetic Bank, Genetic Engineering Center, Korea Institute of Science and Technology (Accession No. KCTC 8625P).

Example 1.

Hereinafter, preferred embodiments of the present invention will be described. However, the examples described below are not limited to the present invention as an example of a method of extending the preservation period of kimchi using the strain of the present invention.

Example 1.

The leeks, garlic, ginger, etc. were selected and washed by a conventional method, and the cabbage was pickled with sun salt. Prepared kimchi at the ratio of 1kg cabbage, 20kg garlic, 20g leek, 20g red pepper powder, 5g ginger, 100g anchovy, and then crushed and centrifuged to use the supernatant as kimchi medium to produce the bacteriocin producing strain of the present invention at 30 ° C 24. Time incubation. This culture solution was added at a concentration of 1% (v / w) when preparing kimchi with the material prepared in the above composition. The salt concentration after kimchi was made to be 2.5-3.0%. Kimchi prepared in this way as shown in Tables 6 and 7 when the ripening time at 10 ℃ compared to the control time 4 times, 20 times at 5 ℃ extended. In addition, when the amount of gas produced was measured while sealing the prepared kimchi and aged at 10 ° C., the amount of generated gas also decreased by about 1/2 compared to the control (Table 8).

Example 2

The leeks, garlic, ginger and the like are selected and washed by a conventional method, and the cabbage is pickled with sun salt. Unsalted cabbages were ground and centrifuged to obtain supernatant. Chinese cabbage juice: salted salt: sugar: salt = 87: 7: 3: 3: 3 in the ratio (w / w / w / w) to prepare a cabbage juice medium, and then inoculated with the production strain of bacteriocin of the present invention 24 at 30 ℃ Time incubation. The culture solution was added at a concentration of 1% (v / w) when preparing kimchi at the ratio of 1 kg of Chinese cabbage, 20 kg of garlic, 20 g of green onions, 20 g of red pepper powder, 5 g of ginger, and 100 g of anchovy broth. The salt concentration after kimchi was made to be 2.5-3.0%.

The kimchi prepared in this way was aged at 10 and 20, and as shown in the following Tables 6 and 7, the ate period was extended three times and four times, respectively, compared to the control. It was also found that the amount of gas produced was reduced by almost half (Table 8).

In the table, acidity is the value converted from% of lactic acid.

Example 3

Radish, scallion, garlic, ginger, fresh, green pepper, etc. were selected and washed by a conventional method. Pickle radish with sun salt. The green peppers were pre-fried in brine. The prepared ingredients were mixed in the ratio of 1 pickled radish (1kg), fresh 100g, 5 shredded red peppers, 100g green onion, 20g garlic, 5g ginger, and poured brine (2.5% to 3.5%) to prepare Dongchimi as usual. . After grinding the prepared Dongchimi, original separation (5,000 × g, 15 minutes) and the supernatant was used as kimchi medium, the bacteriocin producing strain of the invention was incubated at 30 ℃ for 15 hours. This culture solution was added at a concentration of 1% (v / w) when preparing copper chisel with the material prepared in the above composition. The salt concentration after the preparation of the copper tooth was set to be 2.5 to 3.0%. As a result of aging the prepared Dongchimi at 10 ℃ and 20 ℃ it can be seen that as shown in Tables 9 and 10, the decorative period was extended three times and four times, respectively, compared to the control.

Acidity is a value converted into% of lactic acid.

Acidity is a value converted into% of lactic acid.

effect

The characteristics of the bacteriocin produced by the new strain of the present invention is significantly different from the bacteriocin produced by the known strains, and unlike the other bacteriocin-producing lactic acid bacteria, the production strain itself grows properly even at high salt concentrations such as kimchi. can do. Therefore, as can be seen in the above embodiment, the addition of the strain of the present invention during the production of kimchi significantly reduces the gas production rate in kimchi, and has the effect of significantly extending the preservation period and the decorating period of kimchi by inhibiting the ripening of kimchi.

Claims (7)

Novel strains with the characteristics of KCTC 8625P. A method for preserving kimchi, which extends the kimchi period by using the strain of claim 1 as a bacteriocin producing strain as a starter. The method of claim 2, wherein the bacteriocin producing strain is added when the kimchi is immersed. The method of claim 2, wherein the starter is cultured in a kimchi medium having the same composition as the composition of the kimchi to which the starter is added. The method of claim 2, wherein the bacteriocin producing strain is pre-cultured in cabbage juice medium. 6. The cabbage juice medium according to claim 5, wherein the cabbage juice medium: cabbage juice: salt: sugar: salt 99.3% to 85% by weight: 5% to 0.1% by weight: 5% to 0.1% by weight: 5% to 0.5% by weight Preservation method of kimchi which is prepared by mixing in the ratio of%. The method of claim 2, wherein the kimchi is selected from the group consisting of Chinese cabbage kimchi, white kimchi, yeolmu kimchi, gad kimchi, leek kimchi, green kimchi, kakdugi, dongchimi, nabak kimchi, minced kimchi, and water kimchi.