<i>Leuconostoc mesenteroides </i>CJLM181 strain producing reduced amount of gas, and kimchi production method using same

Abstract

The present application relates to a Leuconostoc mesenteroides CJLM181 strain (KCTC 13042BP) producing decreased amounts of gas, a fermentation starter composition comprising the same, and a method for preparing kimchi using the strain.

Claims

1. A fermentation starter composition comprising: Leuconostoc mesenteroides CJLM181 strain KCTC 13042BP or culture thereof; and one or more cryoprotectants; wherein the Leuconostoc mesenteroides CJLM181 strain KCTC 13042BP comprises 16s rRNA nucleotide sequence encoded by the nucleotide sequence of SEQ ID NO:1.

2. The fermentation starter composition of claim 1, comprising the Leuconostoc mesenteroides CJLM181 strain KCTC 13042BP at a concentration of 10.sup.7 cfu/ml or more.

3. Kimchi prepared by using the fermentation starter composition of claim 1, wherein the fermentation starter composition comprises the Leuconostoc mesenteroides CJLM181 strain KCTC 13042BP at a concentration of 10.sup.7 cfu/ml or more.

4. A method for preparing kimchi, comprising a step of bringing the Leuconostoc mesenteroides CJLM181 strain KCTC 13042BP into contact with a material to be fermented; wherein the Leuconostoc mesenteroides CJLM181 strain KCTC 13042BP comprises 16s rRNA nucleotide sequence encoded by the nucleotide sequence of SEQ ID NO:1.

5. Kimchi prepared by using the fermentation starter composition of claim 2.

6. A method for preparing kimchi, comprising a step of bringing the fermentation starter composition of claim 1 into contact with a material to be fermented.

7. A method for preparing kimchi, comprising a step of bringing the fermentation starter composition of claim 2 into contact with a material to be fermented.

Description

MODE FOR INVENTION

(1) Hereinafter, the present application will be described in further detail with reference to examples. It is to be understood, however, that these examples are provided for better understanding of the present application and are not intended to limit the scope of the present application in any way.

Example 1: Isolation of Strain Producing Decreased Amounts of Gas

(2) 1-1) Strain Isolation and Identification

(3) Various kinds of kimchi purchased from supermarkets were aged at a low temperature of 5° C., and kimchi that reached a pH ranging from 3.8 to 4.5 was used as a kimchi sample. The kimchi sample was diluted 10-fold with 0.85% saline, inoculated onto a PES agar medium (phenyl ethyl alcohol sucrose agar; per liter of distilled water, 5 g of trypton, 0.5 g of yeast extract, 20 g of sucrose, 2 g of ammonium sulfate, 1 g of potassium phosphate dibasic, 0.244 g of magnesium sulfate, 2.5 ml of phenyl ethyl alcohol, and 15 g of agar) plate, and spread using a spreader. Next, the plate was incubated in an incubator at 25° C. for 24 hours, and then each produced colony was streaked onto a separate agar plate and separated into single colonies.

(4) 1-2) Selection of Strains Producing Decreased Amounts of Gas

(5) Each of the strain colonies separated in Example 1-1 above was inoculated into 10 ml of MRS broth (Difco MRS broth; 10 g of bacto peptone, 10 g of beef extract, 5 g of yeast extract, 20 g of glucose, 1 g of Tween 80, 2 g of ammonium citrate, 2 g of potassium phosphate dibasic, 5 g of sodium acetate, 0.1 g of manganese sulfate, 0.05 g of magnesium sulfate, and 1 L of distilled water) in a test tube comprising a 30 mm-height Durham tube, and then was stationary-cultured at 25° C. for 24 hours. The height of gas trapped in the Durham tube was measured to determine the production of gas, and strains showing a gas production of 10 mm or lower were selected.

(6) 1-3) Selection of Strains Producing Decreased Amounts of Acid

(7) Each of the strain colonies selected in Example 1-2 above was inoculated onto 10 ml of MRS broth (Difco MRS broth; 10 g of bacto peptone, 10 g of beef extract, 5 g of yeast extract, 20 g of glucose, 1 g of Tween 80, 2 g of ammonium citrate, 2 g of potassium phosphate dibasic, 5 g of sodium acetate, 0.1 g of manganese sulfate, 0.05 g of magnesium sulfate, and 1 L of distilled water). For measurement of acid production, each colony was stationary-cultured at 25° C. for 24 hours, and then measured for its pH using a pH meter (SevenCompact/Ion S220, Mettler Toledo), and strains having a pH of 4.39 or higher were selected.

(8) 1-4) Selection of Strain Producing Increased Amounts of Mannitol

(9) Each of the strain colonies selected in Example 1-3 above was inoculated into 10 ml of minimal medium (10 g of bacto peptone, 20 g of fructose, 1 g of Tween 80, 2 g of ammonium citrate, 2 g of potassium phosphate dibasic, 5 g of sodium acetate, 0.1 g of manganese sulfate, 0.05 g of magnesium sulfate, and 1 L of distilled water) containing 2% fructose, and was stationary-cultured at 25° C. for 24 hours, followed by measurement of the amount of mannitol produced. The amount of mannitol produced was measured by HPLC, and a strain showing a mannitol production of 16,000 mg/L or more was selected.

(10) 1-5) Identification of Selected Strain

(11) The strain selected in Example 1-4 above was named “CJLM181”, and the 16s rRNA nucleotide sequence thereof (SEQ ID NO: 1) was analyzed. As a result, it could be seen that the 16s rRNA nucleotide sequence of the CJLM181 strain was 99% identical to the 16s rRNA nucleotide sequence of Leuconostoc mesenteroides subsp. Mesenteroides CCMMB1121 (SEQ ID NO: 2). Accordingly, the CJLM181 strain was named “Leuconostoc mesenteroides CJLM181”, and deposited in the Korean Collection for Type Cultures at the Korea Research Institute of Bioscience and Biotechnology on Jun. 10, 2016 under accession number KCTC 13042BP.

Example 2: Comparison of Gas Production

(12) In order to compare the production of gas by Leuconostoc mesenteroides CJLM181 selected in Example 1 with those of other Leuconostoc mesenteroides strains, Leuconostoc mesenteroides KCTC3100 and KCTC3722 that are Leuconostoc mesenteroides standard strains were used as control strains to measure gas production. Each strain was inoculated into 10 ml of MRS broth in a test tube comprising a 30 mm-height Durham tube, and was cultured at 25° C. for 24 hours, after which the height of gas trapped in the Durham tube was measured and gas production was compared. Gas generation was rated according to the following criteria: “−”=no gas production; “+”=the height of gas trapped in the Durham tube is 1 to 5 mm; “++”=the height of gas is 6 to 10 mm; “+++”=the height of gas is 11 to 15 mm; “++++”=the height of gas is 16 to 25 mm; and “+++++”=the height of gas is higher than 25 mm.

(13) As a result, it was shown that the gas production of Leuconostoc mesenteroides CJLM181 was “++”, which was significantly lower than the gas production of the control strains (“++++” or “+++”) (Table 1).

(14) TABLE-US-00001 TABLE 1 Leuconostoc Leuconostoc Leuconostoc mesenteroides mesenteroides mesenteroides CJLM181 KCTC3100 KCTC3722 Gas production ++ ++++ +++

Example 3: Comparison of Acid Production

(15) The production of acid by Leuconostoc mesenteroides CJLM181 selected in Example 1 was compared with those of Leuconostoc mesenteroides KCTC3100 and KCTC3722 which are control strains. Each of the strains was inoculated into 10 ml of MRS broth. For measurement of acid production, each strain was stationary-cultured at 25° C. for 24 hours, and then measured for its pH using a pH meter (SevenCompact/Ion S220, Mettler Toledo).

(16) As a result, it was shown that the acid production of 10 Leuconostoc mesenteroides CJLM181 was significantly lower than that of the control strains (Table 2).

(17) TABLE-US-00002 TABLE 2 Leuconostoc Leuconostoc Leuconostoc mesenteroides mesenteroides mesenteroides CJLM181 KCTC3100 KCTC3722 Ph 4.39 4.33 4.37

Example 4: Comparison of Mannitol Production

(18) The production of mannitol by Leuconostoc mesenteroides CJLM181 selected in Example 1 was compared with those of Leuconostoc mesenteroides KCTC3100 and KCTC3722 which are control strains. Each of the strains was inoculated into 10 ml of minimal medium (10 g of bacto peptone, 20 g of fructose, 1 g of Tween 80, 2 g of ammonium citrate, 2 g of potassium phosphate dibasic, 5 g of sodium acetate, 0.1 g of manganese sulfate, 0.05 g of magnesium sulfate, and 1 L of distilled water) containing 2% fructose, and was cultured at 25° C. for 24 hours, after which the content of mannitol in the supernatant was measured by HPLC.

(19) As a result, it was shown that the production of mannitol by Leuconostoc mesenteroides CJLM181 increased 146% and 185% compared to those of the control strains, respectively (Table 3).

(20) TABLE-US-00003 TABLE 3 Leuconostoc Leuconostoc Leuconostoc mesenteroides mesenteroides mesenteroides CJLM181 KCTC3100 KCTC3722 Mannitol 16336.34 11224.70 8815.10 production (mg/L)

Example 5: Evaluation of Safety of Strain

(21) In order to examine whether or not Leuconostoc mesenteroides CJLM181 selected in Example 1 would be used as a starter in preparation of kimchi, the safety of the strain was analyzed. Specifically, according to the safety evaluation testing methods proposed in the Korean Bio Venture Association Standards, hemolysis, gelatin liquefaction, toxic metabolic (ammonia) production and phenylalanine deaminase tests were performed.

(22) As a result, it was shown that the Leuconostoc mesenteroides CJLM181 strain showed negative results in all the hemolysis, gelatin liquefaction, toxic metabolic (ammonia) production and phenylalanine deaminase tests, indicating that it is a safe strain that may be administered to the human body and may be used in the preparation of food (Table 4).

(23) TABLE-US-00004 TABLE 4 Gelatin Phenylalanine deaminase Ammonia liquefaction test test Hemolysis test production Negative Negative γ Negative *γ (gamma-hemolysis): no hemolysis.

Example 6: Preparation of Kimchi

(24) 6-1) Preparation of Kimchi Using Leuconostoc mesenteroides CJLM181

(25) A medium was prepared by mixing 2.5 g of sucrose, 1.0 g of trisodium citrate, 1.5 g of peptone, 1.0 g of glucose, 1.0 g of yeast extract, 0.5 g of fructose, 0.5 g of sodium acetate and 1 L of distilled water, followed by sterilization. About 10.sup.9 CFU/ml of the Leuconostoc mesenteroides CJLM181 strain was inoculated into the medium in an amount of 1 wt % based on the total weight of the medium, and cultured at 25° C. for 24 hours, thereby preparing a strain culture. Next, the prepared strain culture was added to a general kimchi seasoning (obtained by mixing red pepper powder (2.5 wt %), garlic (2 wt %), ginger (0.4 wt %), green onion (1 wt %), radish (18 wt %) and fish sauce (5 wt %)) in an amount of 0.1 wt % based on the total weight of kimchi to prepare a seasoning. Then, the prepared seasoning was mixed with salted Chinese cabbage, thereby preparing kimchi (Experimental Example 1).

(26) 6-2) Preparation of Kimchi Using Standard Strain Culture

(27) Kimchi was prepared in the same manner as described in Example 6-1, except that a culture of each of Leuconostoc mesenteroides standard strains KCTC3100 and KCTC3722 was used (use of KCTC3100 culture: Comparative Example 1; use of KCTC3722 culture: Comparative Example 2).

(28) 6-3) Preparation of Kimchi without Addition of Strain Culture

(29) Kimchi (Comparative Example 3) was prepared in the same manner as described in Example 6-1 above, except that the strain culture was not added to the kimchi seasoning.

Example 7: Analysis of Characteristics of Kimchi

(30) 7-1) Comparison of Acid Production

(31) The kimchi of each of Experimental Example 1 and Comparative Examples 1 to 3 was stored at 7° C. for 30 days, and lactic acid production in each kimchi was measured. Specifically, a predetermined amount of each kimchi was crushed, and then filtered through gauze to prepare kimchi juice. To analyze the amount of lactic acid in the kimchi juice, 3 ml of the kimchi juice was taken, centrifuged (at 10,000 g for 10 minutes), and filtered through a 0.2 μm filter, and then the components thereof were analyzed by HPLC.

(32) As a result, it could be seen that lactic acid production in the kimchi of Experimental Example 1 was 70 to 75% of lactic acid production in each of the kimchi of Comparative Example 3, prepared without using the strain culture, and of the kimchi samples of Comparative Examples 1 and 2, prepared using the standard strain cultures. This suggests that when kimchi is prepared using a culture of the Leuconostoc mesenteroides CJLM181 strain, an increase in sour taste can be inhibited to maintain the taste quality of the kimchi (Table 5).

(33) TABLE-US-00005 TABLE 5 Experimental Comparative Comparative Comparative Example 1 Example 1 Example 2 Example 3 Lactic acid 5382.4 7379.2 7198.2 7585.4 production (mg/L)

(34) 7-2) Comparison of Gas Production

(35) A predetermined amount of the kimchi of each of Experimental Example 1 and Comparative Examples 1 to 3 was placed in an A1 pouch without a gas absorbent and stored at 7° C. for 30 days, and an increase in the volume was measured to determine the gas production in each kimchi.

(36) As a result, gas production in the kimchi of Experimental Example 1 is 37 to 41% of gas production in the kimchi of Comparative Examples 1 and 2, prepared using the standard strain cultures, and was lower than the kimchi of Comparative Example 3, prepared without using any strain culture. This suggests that when kimchi is prepared using a culture of the Leuconostoc mesenteroides CJLM181 strain, gas production in the kimchi can be significantly decreased to thereby increase convenience during distribution of the kimchi (Table 6).

(37) TABLE-US-00006 TABLE 6 Experimental Comparative Comparative Comparative Example 1 Example 1 Example 2 Example 3 Gas generation 0.95 2.54 2.33 2.02 (cc/g)

(38) 7-3) Comparison of Mannitol Production

(39) The kimchi of each of Experimental Example 1 and Comparative Examples 1 to 3 was stored at 7° C. for 30 days, and mannitol production in each kimchi was measured. Specifically, a predetermined amount of each kimchi was crushed, and then filtered through gauze to prepare kimchi juice. 3 ml of the kimchi juice was taken, centrifuged (at 10,000 g for 10 minutes), and filtered through a 0.2 μm filter, and then the components thereof were analyzed by HPLC.

(40) As a result, it could be seen that mannitol production in the kimchi of Experimental Example 1 was 172 to 176% of mannitol production in the kimchi of Comparative Example 3, prepared without using any strain culture, and the kimchi of Comparative Examples 1 and 2, prepared using the standard strain cultures. This suggests that when kimchi is prepared using a culture of the Leuconostoc mesenteroides CJLM181 strain, the prepared kimchi may contain a large amount of mannitol, and thus have excellent storage stability and taste quality (Table 7).

(41) TABLE-US-00007 TABLE 7 Experimental Comparative Comparative Comparative Example 1 Example 1 Example 2 Example 3 Mannitol 16553.4 9418.8 9628.7 8099.8 production (mg/L)

(42) Accession Number

(43) Name of Depositary Institution: Korea Research Institute of Bioscience and Biotechnology;

(44) Accession Number: KCTC 13042BP;

(45) Date of Deposit: Jun. 10, 2016.