KR20170017065A - Leuconostoc mesenteroides with an excellent salt tolerance and low temperatured growth, and Kimchi comprising the same - Google Patents
Leuconostoc mesenteroides with an excellent salt tolerance and low temperatured growth, and Kimchi comprising the same Download PDFInfo
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Abstract
Description
본 발명은 내염성 및 저온생육이 우수한 류코노스톡 메센테로이드 균주 및 이를 포함하는 김치에 관한 것으로, 좀더 상세하게는 내염성, 저온 생육도 및 내산성이 우수한 류코노스톡 메센테로이드 AFY-3 (Leuconostoc mesenteroides AFY-3) KACC 92069P 균주 및 이를 포함하는 김치에 관한 것이다.More particularly, the present invention relates to a strain of Ryukono Stokes methenetroide having excellent salt tolerance and low-temperature growth, and more particularly to a strain of Leuconostoc mesenteroides AFY-3 ( Leuconostoc mesenteroides AFY-3) KACC 92069P strain and a kimchi containing the same.
김치는 제품의 보존성과 숙성도를 확보하기 위하여 저온 발효시킨 식품으로 우리나라 고유의 대표적인 전통 발효식품이다. Kimchi is a traditional fermented food which is fermented at low temperature to ensure the preservation and aging of the product.
김치 발효에서 가장 중요한 미생물인 유산균으로 유기산과 박테리오신(bacteriocin) 등의 항균성 물질을 생산하여 김치발효 중 유해균들의 증식을 억제하고, 각종 향미성분을 생산하여, 김치에 독특한 향미를 부여하지만, 시간이 지날 수록 산이 과도하게 생성됨으로써 김치는 시어지고 맛이 떨어지게 된다(Kim M.H. et al., Korean J. Food Sci. Technol. 28(5):806-813. 1996). The lactic acid bacteria, which is the most important microorganism in kimchi fermentation, produces antimicrobial substances such as organic acid and bacteriocin to inhibit the proliferation of harmful bacteria during kimchi fermentation, produce various flavor components, and give a unique flavor to kimchi. (Kim MH et al., Korean J. Food Sci. Technol. 28 (5): 806-813, 1996).
사용되는 재료의 종류와 소금의 농도가 물론 중요하지만, 특히 김치 발효는 온도가 중요한 것으로 평가되고 있다. 단적인 예로서 저온 숙성시킨 김장 김치가 여름철 속성 발효된 김치에 비하여 맛이 좋으며, 초봄의 기온 상승으로 김장 김치의 맛이 급격히 나빠지게 되는 현상이다. The type of materials used and the concentration of salt are of course important, but in particular, kimchi fermentation is considered to be important for temperature. As a simple example, the low-temperature aged Kimchi Kimchi has a taste better than the fermented Kimchi in summer, and the taste of Kimchi Kimchi is rapidly deteriorated due to the rise of temperature in early spring.
뿐만 아니라, 저온 발효된 김치가 중온 또는 고온 발효된 김치보다 관능검사에서 더 높은 평가를 받은 연구 결과가 많이 보고된 바 있다(So M.H and Kim Y.B., Korean J. Soc. Food Sci. 27(4):495-505, 1995 등)In addition, there have been many reports of studies in which low-temperature fermented kimchi has been evaluated more highly in sensory tests than in medium- or high-temperature fermented kimchi (So MH and Kim YB, Korean J. Soc. Food Sci. : 495-505, 1995, etc.)
김치의 시원한 깊은 맛을 내는 주요인은 김치를 저온 숙성시 이상 젖산 발효균(heterofermentative lactic acid bacteria)인 류코노스톡 Leuconostoc mesenteroides가 우점적으로 증가하기 때문이다. The main reason for the cool deep flavor of Kimchi is that when fermenting Kimchi at low temperature, the fermenting lactic acid bacteria ( Leuconostoc mesenteroides ), which is a heterofermentative lactic acid bacteria, is increased.
상기 류코노스톡 메센테로이드 균주는 김치 발효 초기부터 생육이 시작되어 이산화탄소, 초산, 젖산, 에탄올 및 기타 휘발성 산물을 생산하여 김치 특유의 독특한 풍미와 향미를 가지도록 해준다. The Leuconostoc mesenteroide strain begins to grow from the early stage of fermentation of kimchi and produces carbon dioxide, acetic acid, lactic acid, ethanol and other volatile products, thereby providing a unique flavor and flavor unique to Kimchi.
또한, 탄산미를 부여하는 이산화탄소는 김치를 혐기적 조건으로 유지하여 호기성균의 번식을 억제시킴으로써, 김치 발효가 정상적으로 이루어 지도록 한다(So M.H. and Kim Y.B. Korean J. Food Sci. Technol. 27(4):506-515, 1995; So M.H. and Kim Y.B., Korean J. Food Sci. Technol. 27(4):495-505, 1995). In addition, the carbon dioxide imparting carbonic acid gives kimchi fermentation normally by suppressing the growth of aerobic bacteria by maintaining the kimchi in anaerobic conditions (So MH and Kim YB Korean J. Food Sci. Technol. 27 (4) : 506-515, 1995; So MH and Kim YB, J. Food Sci. Technol. 27 (4): 495-505, 1995).
또한, 김치 발효 중기에는 정상 젖산 발효균(homofermentative lactic acid bacteria)인 Lactobacillus plantarum 균주가 우세하게 증식하게 되는데, 이 균주는 젖산을 Leu. mesenteroides 젖산 생상량의 3~4배의 젖산을 생성하며, pH 3.0 이하로 떨어뜨리는 김치 신맛의 주요 원인균이다. Lactobacillus plantarum , a homofermentative lactic acid bacteria, predominantly proliferates during the middle of kimchi fermentation . Mesenteroides lactic acid is 3 ~ 4 times more lactic acid than lactic acid and it is the main cause of kimchi sour taste.
따라서, 락토바실러스 플란타리움이 우점하게 되면, pH가 낮아지면서 산에 약한 류코노스톡 메센테로이드는 사멸하게 되면서 김치가 시어지게 된다.Therefore, when Lactobacillus plantarum is dominant, the pH is lowered, and leuconostox mesenteroid weak in acid is killed and the kimchi is sown.
김치 발효는 관능검사(Mheen T.I. and Kwon T.W. Korean J. Food. Sci. Technol., 16:443. 1984; Rhie S.G. and Chun S.K. Korean J. Nutri. Food 11(3):63-66, 1982; Cho Y and Rhee H.S. Korean J. Soc. Food Sci. 7(1):15-25, 1991; ho Y and Rhee H.S. Korean J. Soc. Food Sci. 7(2):89-98, 1991), 풍미성분(Lim C.R. Park H.k. Han H.U. Korean J. Microbiol. 27(4):404-414, 1989; So M.H. and Kim Y.B., Korean J. Food Sci. Technol.) 분석결과를 통하여 저온 발효가 좋은 것으로 평가되었으며, 저온 발효 김치와 중온 발효 김치에 관여하는 유산균의 주요 균종이 서로 다르다는 결과가 보고되었고, 저온 발효 김치의 좋은 풍미는 저온성 균의 특성이 김치 발효에 반영된 결과로 볼 수 있다. Kimchi fermentation was examined by sensory evaluation (Mheen TI and Kwon TW Korean J. Food Sci. Technol., 16: 443 1984; Rhie SG and Chun SK Korean J. Nutri. Food 11 (3): 63-66, 1982; Cho Y and Rhee HS Korean J. Soc. Food Sci. 7 (2): 89-98, 1991), flavor components (Lim CR Park Hk Han HU Korean J. Microbiol 27 (4): 404-414, 1989; So MH and Kim YB, J. Food Sci. Technol. The results showed that the fermented kimchi fermented at low temperature and the fermented Kimchi were different from those of the fermented kimchi. The good flavor of low temperature fermented kimchi was the result of the characteristics of low temperature bacteria reflected in fermentation of kimchi.
저온성 균(Psychrotrophs)은 생육 최적온도와 무관하게 10℃ 이하의 냉장 온도에서 비교적 왕성하게 증식하는 미생물들을 지칭하며(Thomas S.B, J. Appl. Bactriol., 32:269, 1969), 저온 발효된 김치로부터 분리한 유산균들이 모두 저온성균임이 보고되었다(So M.H and Kim Y.B., Korean J. Soc. Food Sci. 27(4):495-505, 1995; So M.H and Kim Y.B., Korean J. Soc. Food Sci. 27(4):506-515, 1995). Psychrotrophs refer to microorganisms that grow relatively heavily at refrigeration temperatures below 10 ° C regardless of the optimum temperature for growth (Thomas SB, J. Appl. Bactriol., 32: 269, 1969) It has been reported that lactic acid bacteria isolated from kimchi are low temperature bacteria (So MH and Kim YB, Korean J. Soc. Food Sci. 27 (4): 495-505, 1995; So MH and Kim YB, Sci., 27 (4): 506-515, 1995).
또한, 저온 발효시에는 Lactobacillus plantarum 이 포함되지 않고, Lac . maltaromicus, Lac . sake, Lac . bavaricus, Lac . homohiochii 등이 포함된 것으로 보고된 바 있다(So M.H. and Kim Y.B. Korean J. Food Sci. Technol., 27(4):506-515, 1995). During low temperature fermentation, Lactobacillus plantarum And Lac . maltaromicus, Lac . sake , Lac . bavaricus , Lac . homohiochii, etc. (So MH and Kim YB Korean J. Food Sci. Technol., 27 (4): 506-515, 1995).
1990 년대까지 김치는 주로 겨울철 김장 김치를 제조하여 가정에서 자체적으로 만들어 먹은 식품이었으나, 1990 년대 이후부터 급속한 경제 성장과 국민소득의 증가, 생활 환경의 변화로 인하여 공장에서 본격적으로 상품화되기 시작하였고, 최근에는 상품 김치에 대한 인식 개선과 국내 수요량과 수출량이 꾸준히 증가하고 있는 실정이다. Until the 1990s, kimchi was mainly made from kimchi made in the winter and made itself at home. However, since the 1990s, it has started to be commercialized in the factory due to rapid economic growth, increase of the national income, The improvement of perception of commodity kimchi and domestic demand and export volume are steadily increasing.
김치가 외국의 사우크라우트나 피클처럼 공장에서 안정적인 방법으로 대량생산하기 위하여, 균주의 인위적인 조절, 즉 자연 균주가 아닌 스타터 균주를 이용한 김치가 생산되기 시작하였다. 최근에는 냉장유통 시스템을 도입하여 김치를 저온에서 발효 숙성시켜, 가식기간을 연장시키고 있다.In order to produce kimchi in large quantities in a stable manner at a factory like foreign sauces and pickles, kimchi using an artificial control of the strain, that is, a starter strain, which is not a natural strain, has begun to be produced. Recently, a refrigeration distribution system has been introduced to ferment and ferment kimchi at a low temperature, thereby extending the period of edible foods.
또한, 김치의 과숙 현상은 내산성 미생물에 의하여 pH가 낮아지면서 산패가 일어나 불쾌한 맛과 냄새가 발생하며, 배추의 조직연화 현상이 일어나게 되는 것이므로, 과숙 현상을 억제하기 위해서는 저온에서도 생육이 가능하고, 산성에 대한 내성이 높아 후기 발효에 관여하는 균주의 생육을 늦추어 줌으로써, 김치의 조직연화 현상의 발생을 지연시켜야 한다.In addition, the overcrowding phenomenon of Kimchi is caused by acid rancidity and low pH due to acid-resistant microorganisms, resulting in unpleasant taste and smell, resulting in tissue softening of Chinese cabbage. Therefore, in order to suppress overcrowding, And thus delayed the growth of strains involved in late fermentation, thereby delaying the occurrence of tissue softening phenomenon of kimchi.
이에 띠리, 내염성, 저온 생육 및 내산성이 우수한 균주를 개발하여 이를 스타터 균주로 사용한 김치의 개발이 절실히 요구되고 있는 실정이다.Therefore, there is an urgent need for the development of a kimchi which has excellent resistance to salt, salt tolerance, low temperature growth and acid resistance and which is used as a starter strain.
따라서, 본 발명의 목적은 낮은 pH와 높은 염도, 저온 숙성과 같은 김치 발효에 적합한 신규 유산균을 제공하는 것이다.Accordingly, an object of the present invention is to provide a novel lactic acid bacterium suitable for fermentation of kimchi such as low pH, high saltiness and low temperature aging.
본 발명의 다른 목적은 상기 신규 유산균을 포함하는 유산균 스타터를 제공하는 것이다.Another object of the present invention is to provide a lactic acid bacteria starter comprising the novel lactic acid bacterium.
본 발명의 또 다른 목적은 상기 신규 유산균을 포함하는 김치를 제공하는 것이다.It is still another object of the present invention to provide a kimchi containing the novel lactic acid bacteria.
본 발명의 또 다른 목적은 상기 신규 유산균을 포함하는 김치의 제조방법을 제공하는 것이다.It is still another object of the present invention to provide a process for producing a kimchi comprising the novel lactic acid bacterium.
본 발명은 상기와 같은 목적을 달성하기 위하여, 내염성 및 저온 생육이 우수한 류코노스톡 메센테로이드 AFY-3 (Leuconostoc mesenteroides AFY-3) KACC 92069P 균주를 제공한다.In order to achieve the above-mentioned object, the present invention provides a method for producing leuconostox mesenteroid AFY-3 ( Leuconostoc mesenteroides AFY-3) KACC 92069P strain.
또한, 본 발명은 상기 류코노스톡 메센테로이드 AFY-3 (Leuconostoc mesenteroides AFY-3) KACC 92069P 균주를 포함하는 유산균 스타터를 제공한다.The present invention also relates to the above-mentioned leuconostomesenteroid AFY-3 ( Leuconostoc mesenteroides AFY-3) KACC 92069P.
또한, 본 발명은 상기 류코노스톡 메센테로이드 AFY-3 (Leuconostoc mesenteroides AFY-3) KACC 92069P 균주를 포함하는 김치를 제공한다.The present invention also relates to the above-mentioned leuconostomesenteroid AFY-3 ( Leuconostoc mesenteroides AFY-3) KACC 92069P.
또한, 본 발명은 상기 류코노스톡 메센테로이드 AFY-3 (Leuconostoc mesenteroides AFY-3) KACC 92069P 균주를 발효시킨 김치에 접종하는 단계를 포함하는 김치의 제조방법을 제공한다.The present invention also relates to the above-mentioned leuconostomesenteroid AFY-3 ( Leuconostoc mesenteroides AFY-3) KACC 92069P strain in a fermented kimchi.
본 발명에 의한 류코노스톡 메센테로이드 AFY-3(Leuconostoc mesenteroides AFY-3) KACC 92069P 균주는 내염성, 저온 생육 및 내산성이 우수한 특징을 가지고 있기 때문에, 이를 유산균 스타터로 이용하여 제조된 김치는 풍미가 증가하고, 아삭한 식감이 향상되는 효과를 지니고 있다.The leuconostomercenteroid AFY-3 ( Leuconostoc mesenteroides AFY-3) KACC 92069P strain is characterized by salt tolerance, low temperature growth and acid resistance. Therefore, kimchi prepared by using this as a lactic acid bacteria starter has an effect of improving flavor and crispy texture.
도 1은 본 발명에 의한 류코노스톡 메센테로이드 AFY-3 균주의 접종 농도별 pH 변화를 나타낸 것이다.
도 2는 본 발명에 의한 류코노스톡 메센테로이드 AFY-3 균주의 접종 농도별 산도의 변화를 나타낸 것이다.
도 3은 본 발명에 의한 류코노스톡 메센테로이드 AFY-3 균주의 접종 농도별 일반 세균수의 변화를 나타낸 것이다.
도 4는 본 발명에 의한 류코노스톡 메센테로이드 AFY-3 균주의 접종 농도별 대장균군의 변화를 나타낸 것이다.
도 5는 본 발명에 의한 류코노스톡 메센테로이드 AFY-3 균주의 접종 농도별 대장균의 변화를 나타낸 것이다.
도 6은 본 발명에 의한 류코노스톡 메센테로이드 AFY-3 균주의 접종 농도별 경도의 변화를 나타낸 것이다.FIG. 1 shows the pH change of Inocystein methionide AFY-3 according to the inoculation concentration according to the present invention.
Fig. 2 shows changes in acidity of the fungus belonging to the present invention, according to the inoculation concentration of leuconovirus methensertoid AFY-3.
Fig. 3 shows the change in the number of general bacteria according to the inoculation concentration of the leukoconstrictor methionine AFY-3 strain according to the present invention.
Fig. 4 shows changes in coliform group according to the inoculation concentration of leuconovirus methensertoid AFY-3 strain according to the present invention.
FIG. 5 shows the change in E. coli according to the inoculation concentration of the yeast Leconovac methenseolide AFY-3 according to the present invention.
FIG. 6 shows the change in hardness of the leukoconstrictor methionine AFY-3 strain according to the present invention at each inoculation concentration.
본 발명은 내염성 및 저온 생육이 우수한 류코노스톡 메센테로이드 AFY-3 (Leuconostoc mesenteroides AFY-3) KACC 92069P 균주에 관한 것이다..The present invention relates to a method for producing Leuconostoc mesenteroides AFY-3, which is excellent in salt tolerance and low-temperature growth, AFY-3) KACC 92069P.
본 발명의 상기 균주는 서열번호 1의 16S rRNA 염기서열을 갖는데, 류코노스톡 메센테로이드의 표준 균주인 Leuconostoc mesenteroides LOCK 0986의 16S rRNA 염기서열과 99.0%의 상동성을 갖는다. The strain of the present invention has the 16S rRNA base sequence of SEQ ID NO: 1, which is a standard strain of Leuconostocercenteroid , Leuconostoc mesenteroides The 16S rRNA nucleotide sequence of LOCK 0986 and And has a homology of 99.0%.
본 발명의 상기 균주는 NaCl 5~10 %(w/v)가 첨가된 MRS(de Man, Rogosa and Sharpe) 액체배지에서 30℃, 72시간, 180 rpm의 조건에서 배양시, 생균수가 2.8x108 내지 2.9x108, 바람직하게는 2.86x108 CFU/mL인 내염성을 갖는다. When the strain of the present invention was cultured in a MRS (de Man, Rogosa and Sharpe) medium supplemented with 5 to 10% (w / v) NaCl at 30 ° C for 72 hours at 180 rpm, the number of viable cells was 2.8 x 10 8 To 2.9x10 < 8 >, preferably 2.86x10 < 8 > CFU / mL.
본 발명의 상기 균주는 pH 3의 MRS 액체배지에서 30℃의 온도 및 180 rpm의 회전속도로 48시간 동안 배양시, 생균수가 1.5x107 내지 2.5x107 CFU/mL, 바람직하게는 2.0x107 CFU/mL인 내산성을 갖는다.When the strain of the present invention is cultured in an MRS liquid medium at pH 3 for 48 hours at a temperature of 30 DEG C and a rotation speed of 180 rpm, the number of viable cells is 1.5 x 10 7 to 2.5 x 10 7 CFU / mL, preferably 2.0 x 10 7 CFU / mL. < / RTI >
본 발명의 상기 균주는 MRS 액체배지에서 7℃의 온도 및 120 rpm의 회전속도로 48시간 동안 배양시, 생균수가 2.8x108 내지 2.9x108 CFU/mL, 바람직하게는 2.85x108 CFU/mL인 저온생육 특성을 갖는다. The strain of the present invention is cultured in MRS liquid medium at a temperature of 7 캜 for 48 hours at a rotation speed of 120 rpm and the viable cell count is 2.8 x 10 8 to 2.9 x 10 8 CFU / mL, preferably 2.85 x 10 8 CFU / mL Temperature growth characteristics.
또한, 본 발명은 상기 류코노스톡 메센테로이드 AFY-3 (Leuconostoc mesenteroides AFY-3) KACC 92069P 균주를 포함하는 유산균 스타터에 관한 것이다.The present invention also relates to the above-mentioned leuconostomesenteroid AFY-3 ( Leuconostoc mesenteroides AFY-3) KACC 92069P.
본 발명의 상기 유산균 스타터는 유산균 스타터의 전체 중량 대비, 상기 류코노스톡 메센테로이드 AFY-3 (Leuconostoc mesenteroides AFY-3) KACC 92069P 균주를 0.1~5.0 중량%, 바람직하게는 0.5~2.0 중량% 포함할 수 있다.The lactic acid bacteria starter according to the present invention is characterized in that the ratio of the leucono stokescenteide AFY-3 ( Leuconostoc mesenteroides AFY-3) KACC 92069P strain 0.1 to 5.0% by weight, preferably 0.5 to 2.0% by weight.
본 발명의 상기 유산균 스타터는 통상의 방법에 따라 제조하는 것으로 족하므로, 상기 유산균 스타터의 제조방법에 대한 상세한 내용은 생략한다.Since the lactic acid bacteria starter of the present invention may be produced by a conventional method, details of the production method of the lactic acid bacteria starter will be omitted.
또한, 본 발명은 상기 류코노스톡 메센테로이드 AFY-3 (Leuconostoc mesenteroides AFY-3) KACC 92069P 균주를 포함하는 김치에 관한 것이다.The present invention also relates to the above-mentioned leuconostomesenteroid AFY-3 ( Leuconostoc mesenteroides AFY-3) KACC 92069P.
본 발명의 상기 김치는 상기 균주를 김치 전체 중량 대비 0.5∼2 중량％, 바람직하게는 1~1.5 중량% 포함할 수 있다. The Kimchi of the present invention may contain 0.5 to 2% by weight, preferably 1 to 1.5% by weight, of the above strain relative to the total weight of the kimchi.
또한, 본 발명은 상기 류코노스톡 메센테로이드 AFY-3 (Leuconostoc mesenteroides AFY-3) KACC 92069P 균주를 발효시킨 김치에 접종하는 단계를 포함하는 김치의 제조방법에 관한 것이다.The present invention also relates to the above-mentioned leuconostomesenteroid AFY-3 ( Leuconostoc mesenteroides AFY-3) KACC 92069P fermented with a strain of Kimchi.
본 발명에서, 상기 김치는 배추 김치, 백김치, 깍두기, 열무 김치, 총각 김치, 갓김치로 이루어진 군에서 선택되는 어느 하나일 수 있다. In the present invention, the kimchi may be any one selected from the group consisting of Chinese cabbage kimchi, white kimchi, kakdugi, radish kimchi, bonggak kimchi, mustard kimchi.
본 발명의 상기 김치의 제조방법은 통상의 방법으로 제조한 김치에 적정량의 상기 균주를 접종하는 단계를 포함하는 것으로 족하므로, 상기 김치의 제조방법에 대한 상세한 설명은 생략한다.The method of manufacturing the kimchi according to the present invention may include a step of inoculating an appropriate amount of the strain into a kimchi prepared by a conventional method, and thus a detailed description of the method for producing the kimchi will be omitted.
이하, 본 발명의 내용을 실시예 및 실험예를 통하여 구체적으로 설명한다. 그러나, 이들은 본 발명을 보다 상세하게 설명하기 위한 것으로 본 발명의 권리범위가 이들에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples. However, these are for the purpose of illustrating the present invention in more detail, and the scope of the present invention is not limited thereto.
<< 실시예Example 1> 내염성이1> Salt resistance 우수한 유산균의 분리 Isolation of excellent lactic acid bacteria
김치의 염도가 일반적으로 5~8 %(w/v)임을 감안하여 강원 지역에서 수집한 김치 및 젓갈에서 선택배지인 MRS 배지를 이용하여 분리한 균주들을 대상으로 고염도에서 생육이 우수한 균주를 1차 선발하였다. Considering that the salinity of kimchi is generally 5 ~ 8% (w / v), the strains isolated from kimchi and salted seafood collected from Gangwon area using MRS medium were selected as 1 I was selected.
내염성 측정은 MRS 배지 제조시 NaCl을 이용하여 염도가 5~10 %(w/v)가 되도록 제조하고, 스트리킹을 통해 생육 여부를 1차 스크리닝한 후, MRS 액체배지 (de Man, Rogosa and Sharpe; Difco, USA)에서 30℃, 72시간, 180 rpm의 조건에서 배양한 다음, OD600 nm에서 측정하여 생균수를 계수하였다. The salt tolerance was measured by using NaCl to produce a salt concentration of 5-10% (w / v) in the preparation of MRS medium. The MRS broth was firstly screened for staining and then grown in MRS liquid medium (de Man, Rogosa and Sharpe; Difco, USA) at 30 ° C for 72 hours, 180 rpm, and then counted at OD 600 nm.
일반적으로 106 CFU/mL 이상의 생육을 보여 줄 경우, 생육이 양호한 것으로 평가하는데, 1차 선발한 주요 균주의 생균수를 나타내는 하기 표 1에서 보는 바와 같이, 108 CFU/mL 이상의 생균수를 나타내었다. Typically, 10 6 when to present a more CFU / mL growth, for evaluation as having good growth, as shown to represent the number of viable cells of the major strains selected first in Table 1, indicate the number of viable cells at least 10 8 CFU / mL .
대상 균주는 MRS 선택배지에서 분리된 753점을 대상으로 NaCl을 이용하여 염도를 5, 7, 10 %(w/v)로 첨가한 MRS 고체배지를 이용하여 선발한 결과, 5 %(w/v)에서는 466점, 7 %(w/v)에서는 133점, 10 %(w/v)에서 15점의 균주의 생육을 확인하였다. The target strains were selected by using MRS solid medium supplemented with 5%, 7%, and 10% (w / v) salinity using NaCl at 753 points isolated from MRS selection medium. ) At 466 points, 133% at 7% (w / v) and 15 at 10% (w / v).
7 %(w/v) 이상에서 생육이 우수한 균주와 5 %(w/v)에서 생육이 월등히 우수한 균주 400점을 대상으로 저온 생육을 검토하였다.Low temperature growth was investigated in 400 strains that were superior to 7% (w / v) and 5% (w / v).
<< 실시예Example 2> 저온2> Low temperature 생육이 우수한 유산균의 분리 Isolation of lactic acid bacteria with excellent growth
내염성이 우수했던 400점을 대상으로 김치 저장 온도인 7℃에서 균주의 생육을 검정하기 위하여, MRS 액체배지에 3 mL이 들어 있는 라운드 튜브에 배양할 균주 20㎕ 씩 접종하여 쉐이킹 인큐베이터(제이오텍 , MI-400)의 온도를 7℃로 세팅한 후, 2시간 후부터 48시간 동안 120 rpm으로 배양하였다. In order to test the growth of the strain at the storage temperature of 7 ° C, 400 μl of which was excellent in salt tolerance, 20 μl of a strain to be cultured in a round tube containing 3 ml of MRS liquid medium was inoculated into a shaking incubator MI-400) was set at 7 DEG C, followed by incubation at 120 rpm for 48 hours from 2 hours.
상기 배양을 한 후에 육안으로 생육이 확인된 샘플을 OD600nm에서 흡광도를 측정하여 생균수를 측정한 결과를 하기의 표 2(저온 생육 우수 주요 균주)에 나타내었다.After the cultivation, the sample in which the growth was visually confirmed was measured for absorbance at OD 600 nm , and the viable cell count was measured. The results are shown in Table 2 (main strain excellent in low temperature growth).
<< 실시예Example 3> 내산성이3> Acid resistance 우수한 유산균의 분리 Isolation of excellent lactic acid bacteria
주요 균주의 내산성 특성을 나타내는 하기의 표 3은 김치의 중기 발효 주요 유산균인 Lactobacillus sp.의 젖산 분비에 대한 내산성을 검정하기 위하여, pH 별 균주의 생균수를 나타낸 결과이다. Table 3, which shows the acid resistance characteristics of the major strains, shows the number of viable cells of each strain of pH in order to test the acid resistance of Lactobacillus sp.
MRS 액체배지에 HCl을 적정하여 pH를 맞춘 후 미리 접종했던 균주를 20㎕ 씩 분주하여 접종하였다. The pH was adjusted by titrating HCl in the MRS liquid medium and inoculated with 20 쨉 l of the strain previously inoculated.
쉐이킹 인큐베이터(제이오텍 , MI-400)에서 30℃, 48시간, 180 rpm의 조건으로 배양한 후, 육안으로 1차 스크리닝한 다음, 생육이 확인된 균주를 OD600nm에서 측정하여 생균수를 측정하였다. The cells were cultured in a shaking incubator (J-Otec, MI-400) at 30 DEG C for 48 hours under the condition of 180 rpm, followed by primary screening, and the number of live cells was measured by measuring the strain at OD 600 nm .
측정 결과, 하기 표 3에서 보는 바와 같이, AFY-3 등 유산균의 특성에 맞게 pH 3.0에서도 107 CFU/mL 이상의 생균수를 보여 주었고, 중성인 pH 7.0 에서 왕성한 생육을 나타내었다.As shown in the following Table 3, in accordance with the characteristics of lactic acid bacteria such as AFY-3, 10 7 CFU / mL And showed vigorous growth at neutral pH 7.0.
<< 실시예Example 4> 내염성4> Salt resistance , 저온 생육도 및 , Low temperature growth rate and 내산성이Acid resistance 우수한 유산 균주의 동정 Identification of excellent lactic acid strains
상기 실시예 1 내지 3에서와 같이, 내염성, 내산성 및 저온 적응성을 검토한 후, 3개 부분에서 우수한 생육을 보여 주는 균주인 AFY-3 균주를 선정하였다. As in Examples 1 to 3, AFY-3 was selected as a strain showing excellent growth in three parts after examination of salt tolerance, acid resistance and low temperature adaptability.
16S rRNA 유전자의 염기서열에 의한 균주 동정을 위하여, 유니버설 프라이머(universal primer)로서 27F(5'-AGAGTTTGATCATGGCTCAG-3'; 서열번호 2)과 1492R(5'-TACGGATACCTTGTTACGACTT-3'; 서열번호 3) 이용하여 16S rRNA 유전자를 PCR로 증폭한 후, 전체 약 1463bp 염기서열을 마크로젠(Macrogen Inc., Korea)에 의뢰하여 시퀀싱한 결과는 서열번호 1과 같다. (5'-AGAGTTTGATCATGGCTCAG-3 '; SEQ ID NO: 2) and 1492R (5'-TACGGATACCTTGTTACGACTT-3'; SEQ ID NO: 3) were used as universal primers for isolating strains by the base sequence of 16S rRNA gene The 16S rRNA gene was amplified by PCR, and the entire sequence of about 1463 bp was sequenced with Macrogen Inc., Korea. The result is shown in SEQ ID NO: 1.
상기 16S rRNA 유전자의 염기서열은 NCBI 데이타베이스로부터 BLAST N 프로그램을 사용하여 표준 균주의 16S rRNA 유전자 염기서열들과 상동성을 비교한 결과는 하기의 표 4와 같다.The nucleotide sequence of the 16S rRNA gene was compared with the 16S rRNA gene sequences of the standard strain using the BLAST N program from the NCBI database. The results are shown in Table 4 below.
상기 표 4에서 보는 바와 같이, 본 발명에 의한 상기 AFY-3 균주는 표준 균주인 Leuconostoc mesenteroides LOCK 0986와 99.0%의 상동성을 보이고 있음을 알 수 있고, 생화학 분석과 계통학적 분류를 고려하여 상기 AFY-3 균주를 "류코노스톡 메센테로이드(Leuconostoc mesenteroides) AFY-3"로 동정하고, 이를 2015년 5월 8일에 농업유전자원정보센터에 기탁번호 KACC92069P로 기탁하였다.As shown in Table 4, the AFY-3 strain according to the present invention is a strain of Leuconostoc mesenteroides The AFY-3 strain was identified as " Leuconostoc mesenteroides AFY-3" in consideration of biochemical analysis and phylogenetic classification, and the AFY- And deposited on May 8, 2015 with the accession number KACC92069P to the Information Center for Agricultural Genetic Resources.
<< 실시예Example 5> 류코노스톡5> Ryukono Stock 메센테로이드Mesenteroid AFYAFY -3 균주 배양액의 제조-3 Production of Culture Medium
상기 실시예 4에서 동정한 류코노스톡 메센테로이드 AFY-3 균주를 MRS 액체배지(Difco사, USA) 10 mL에 0.1 %(w/v) 접종한 후, 30℃에서 24시간 동안 배양하여 상기 류코노스톡 메센테로이드 AFY-3 균주의 배양액을 제조하였다. 0.1% (w / v) of Inocuum Stokescens AFY-3 strain identified in Example 4 was inoculated in 10 mL of MRS liquid medium (Difco, USA), and cultured at 30 DEG C for 24 hours, A culture solution of a strain of Kono Stokescecenoid AFY-3 was prepared.
< 실시예 6 내지 실시예 8> 류코노스톡 메센테로이드 AFY -3 균주를 첨가한 김치의 제조 <Examples 6 to Example 8> Ryukono Stock Mesenteroid Preparation of Kimchi with AFY -3 strain
하기 표 5의 조성과 같이, 소금으로 절인 배추를 염도가 2.0~2.5 %(w/v)가 되도록 한 후 절여진 배추(72.5 중량%)에 무채 (10 중량 %), 고춧가루, 마늘 등 양념(17.5 중량%)을 첨가하여 혼합하였다. (10% by weight), red pepper powder, garlic (such as garlic), and the like were added to salted Chinese cabbage (72.5% by weight) after the salted Chinese cabbage was made to have a salinity of 2.0 to 2.5% 17.5 wt%) was added and mixed.
이와 같이 제조된 김치에 상기 실시예 5에서 제조한 류코노스톡 메센테로이드 AFY-3 균주 배양액을 전체 김치 중량에 대해 0.5 중량%(실시예 6), 1.0 중량%(실시예 7), 2.0 중량%(실시예 8) 첨가하여 각각 김치를 제조하였다.(Example 6), 1.0% by weight (Example 7), 2.0% by weight, and the like were added to the thus prepared kimchi in an amount of 0.5% by weight based on the total weight of the kimchi, (Example 8) to prepare a kimchi, respectively.
<< 비교예Comparative Example 1> 유산균1> lactic acid bacteria 스타터를 첨가하지 않은 김치의 제조 Manufacture of Kimchi without starter
상기 류코노스톡 메센테로이드 AFY-3 균주를 접종한 것을 제외하고는, 상기 실시예 6과 동일한 방법으로 실시하여 김치를 제조하였다.A kimchi was prepared in the same manner as in Example 6 except that the above-mentioned Leuconostoc mesenteroid AFY-3 strain was inoculated.
<< 실시예Example 7> 김치의7> Kimchi's 이화학적 및 미생물학적 특성 Physicochemical and microbiological characteristics
상기 실시예 8 및 비교예 1에서 제시된 김치의 pH, 총산, 경도, 일반 세균수, 대장균군, 유산균수를 측정하였다. The pH, total acid, hardness, general bacterial count, coliform group and lactic acid bacterial counts of kimchi presented in Example 8 and Comparative Example 1 were measured.
이때, AFY-3 균주의 pH는 pH meter(Starter3100, Korea)로 측정하였고, 총산은 0.1N NaOH 용액으로 적정하고, 대표산을 젖산으로 하여 계산하였다. At this time, the pH of the AFY-3 strain was measured with a pH meter (Starter 3100, Korea), the total acid was titrated with 0.1 N NaOH solution, and the representative acid was calculated using lactic acid.
경도는 경도계(Brookfield CT3-10K, USA)를 이용하여 배추 잎 중 외각 줄기 가운데 부위를 선별하여 총 5반복으로 측정하였다. The hardness was measured by using a hardness tester (Brookfield CT3-10K, USA).
일반 세균수는 Aerobic count petriflim(3M, USA)을 1 mL 분주하여 35℃에서 24-48시간 배양하여 붉은색 집락만을 계수하였다. The number of normal bacteria was counted in red color by culturing at 35 ℃ for 24-48 hours with 1 mL of aerobic count petriflim (3M, USA).
대장균과 대장균군은 Colifrom count petrifilim(3M, USA)를 이용하여 1 mL을 분주하여 35℃에서 24시간 배양한 후, CO2가스가 발생한 붉은색 집락만을 계수하였다. Escherichia coli and E. coli were cultured at 35 ° C for 24 hours using 1 ml of colifrom count petrifilim (3M, USA), and only red colonies of CO 2 gas were counted.
그 결과, 모든 접종 농도에서 pH는 8일 이후 급격하게 감소한 후, 8-18일은 서서히 감소하는 경향을 보였고, 18일 이후 크게 변화하지 않았다 (도 1). As a result, pH at all inoculation concentrations decreased sharply after 8 days, then gradually decreased from 8-18 days, and did not change significantly after 18 days (Fig. 1).
총산(젖산으로서)의 결과를 정리해 보면 0~8일은 산도 0.5% 이하로 미숙기, 8~18일은 적숙기, 18일 이후에는 산도가 1%를 초과하여 김치 발효가 과숙기에 접어든 것을 확인 할 수 있었다 (도 2). 이는 적숙기부터 또 다른 젖산균들이 활발히 생육했기 때문인 것으로 생각된다. The results of total acid (as lactic acid) are summarized as follows: the acidity is less than 0.5% in 0 ~ 8 days, the acidity is more than 1% in 8 ~ 18 days and 18 ~ (Fig. 2). This is thought to be due to the active growth of other lactic acid bacteria from the early maturity.
일반 세균수를 보면, 김치 발효가 증가됨에 따라 초기에는 큰 폭으로 증가하다 산도가 급격히 증가하는 적숙기에 접어들면서 증가폭이 감소하였고 (도 3), As for the number of general bacteria, the increase in kimchi fermentation was markedly increased at the initial stage, and the increase rate was decreased as the fermentation period, in which the acidity increased sharply (Fig. 3)
대장균군은 발효 8일 이후 pH가 5.0 이하로 크게 감소하면서 모두 사멸함을 확인하였다 (도 4). The E. coli group showed a significant decrease in pH after the 8th day of fermentation to below 5.0 (Fig. 4).
또한, 대장균은 측정 초기부터 검출되지 않았다 (도 5). In addition, E. coli was not detected from the beginning of the measurement (FIG. 5).
경도는 시간이 경과 할수록 대조구에 비해, AFY-3을 2.0 %(w/v) 접종한 김치의 경도가 높음을 확인하였고, 농도별로는 유의성이 없었다 (표 5 및 도 6). 하기 표 6에서, 단위는 kg이다.The hardness of kimchi inoculated with AFY-3 2.0% (w / v) was higher than that of the control, and the hardness was not significantly different according to the concentration (Table 5 and FIG. 6). In the following Table 6, the unit is kg.
상술한 바와 같이, 본 발명의 바람직한 실시예를 참조하여 설명하였지만, 해당 기술 분야의 숙련된 당업자라면 하기의 청구범위에 기재된 본 발명의 사상 및 영역으로부터 벗어나지 않는 범위 내에서 본 발명을 다양하게 수정 및 변경시킬 수 있음을 이해할 수 있을 것이다. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined in the following claims. It will be understood that the present invention can be changed.
<110> Kangwon-Do <120> Leuconostoc mesenteroides with an excellent salt tolerance and low temperatured growth, and Kimchi comprising the same <130> 10181 <160> 3 <170> KopatentIn 2.0 <210> 1 <211> 1463 <212> DNA <213> 16S rRNA of Leuconostoc mesenteroides <400> 1 ggggtatgcc cgctagacgt ttgatcgtga gctcagaacg caacacggaa ggcgcatgga 60 tcttatcagt gagtggcgaa cgggtgagta acacatagga caacctgcct caaggctggg 120 gataacattt ggaaacagat gctaataccg aataaaactt agtgtcgcat gacacaaagt 180 taaaaggcgc ttcggcgtca cctagagatg gatccgcggt gcattagtta gttggtgggg 240 taaaggccta ccaagacaat gatgcatagc cgagttgaga gactgatcgg ccacattggg 300 actgagacac ggcccaaact cctacgggag gctgcagtag ggaatcttcc acaatgggcg 360 aaagcctgat ggagcaacgc cgcgtgtgtg atgaaggctt tcgggtcgta aagcactgtt 420 gtatgggaag aacagctaga ataggaaatg attttagttt gacggtacca taccagaaag 480 ggacggctaa atacgtgcca gcagccgcgg taatacgtat gtcccgagcg ttatccggat 540 ttattgggcg taaagcgagc gcagacggtt tattaagtct gatgtgaaag cccggagctc 600 aactccggaa tggcattgga aactggttaa cttgagtgca gtagaggtaa gtggaactcc 660 atgtgtagcg gtggaatgcg tagatatatg gaagaacacc agtggcgaag gcggcttact 720 ggactgcaac tgacgttgag gctcgaaagt gtgggtagca aacaggatta gataccctgg 780 tagtccacac cgtaaacgat gaacactagg tgttaggagg tttccgcctc ttagtgccga 840 agctaacgca ttaagtgttc cgcctgggga gtacgaccgc aaggttgaaa ctcaaaggaa 900 ttgacgggga cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac 960 cttaccaggt cttgacatcc tttgaagctt ttagagatag aagtgttctc ttcggagaca 1020 aagtgacagg tggtgcatgg tcgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc 1080 gcaacgagcg caacccttat tgttagttgc cagcattcag atgggcactc tagcgagact 1140 gccggtgaca aaccggagga aggcggggac gacgtcagat catcatgccc cttatgacct 1200 gggctacaca cgtgctacaa tggcgtatac aacgagttgc caacccgcga gggtgagcta 1260 atctcttaaa gtacgtctca gttcggattg tagtctgcaa ctcgactaca tgaagtcgga 1320 atcgctagta atcgcggatc atcacgccgc ggcgaatacg ttcccgggtc ttgtacacat 1380 cgcccgtcac cccatggcag tttgtactgc ccaacgctca gagtccgatc ctgctcgatc 1440 gaaccaatga gtccagaagc ttt 1463 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 27F of forward primer <400> 2 agagtttgat catggctcag 20 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 1492R of reverse primer <400> 3 tacggatacc ttgttacgac tt 22 <110> Kangwon-Do <120> Leuconostoc mesenteroides with an excellent salt tolerance and low temperature growth, and Kimchi comprising the same <130> 10181 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 1463 <212> DNA <213> 16S rRNA of Leuconostoc mesenteroides <400> 1 ggggtatgcc cgctagacgt ttgatcgtga gctcagaacg caacacggaa ggcgcatgga 60 tcttatcagt gagtggcgaa cgggtgagta acacatagga caacctgcct caaggctggg 120 gataacattt ggaaacagat gctaataccg aataaaactt agtgtcgcat gacacaaagt 180 taaaaggcgc ttcggcgtca cctagagatg gatccgcggt gcattagtta gttggtgggg 240 taaaggccta ccaagacaat gatgcatagc cgagttgaga gactgatcgg ccacattggg 300 actgagacac ggcccaaact cctacgggag gctgcagtag ggaatcttcc acaatgggcg 360 aaagcctgat ggagcaacgc cgcgtgtgtg atgaaggctt tcgggtcgta aagcactgtt 420 gtatgggaag aacagctaga ataggaaatg attttagttt gacggtacca taccagaaag 480 ggacggctaa atacgtgcca gcagccgcgg taatacgtat gtcccgagcg ttatccggat 540 ttattgggcg taaagcgagc gcagacggtt tattaagtct gatgtgaaag cccggagctc 600 aactccggaa tggcattgga aactggttaa cttgagtgca gtagaggtaa gtggaactcc 660 atgtgtagcg gtggaatgcg tagatatatg gaagaacacc agtggcgaag gcggcttact 720 ggactgcaac tgacgttgag gctcgaaagt gtgggtagca aacaggatta gataccctgg 780 tagtccacac cgtaaacgat gaacactagg tgttaggagg tttccgcctc ttagtgccga 840 agctaacgca ttaagtgttc cgcctgggga gtacgaccgc aaggttgaaa ctcaaaggaa 900 ttgacgggga cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac 960 cttaccaggt cttgacatcc tttgaagctt ttagagatag aagtgttctc ttcggagaca 1020 aagtgacagg tggtgcatgg tcgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc 1080 gcaacgagcg caacccttat tgttagttgc cagcattcag atgggcactc tagcgagact 1140 gccggtgaca aaccggagga aggcggggac gacgtcagat catcatgccc cttatgacct 1200 gggctacaca cgtgctacaa tggcgtatac aacgagttgc caacccgcga gggtgagcta 1260 atctcttaaa gtacgtctca gttcggattg tagtctgcaa ctcgactaca tgaagtcgga 1320 atcgctagta atcgcggatc atcacgccgc ggcgaatacg ttcccgggtc ttgtacacat 1380 cgcccgtcac cccatggcag tttgtactgc ccaacgctca gagtccgatc ctgctcgatc 1440 gaaccaatga gtccagaagc ttt 1463 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 27F of forward primer <400> 2 agagtttgat catggctcag 20 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 1492R of reverse primer <400> 3 tacggatacc ttgttacgac tt 22
Claims (11)
상기 균주는 서열번호 1의 16S rRNA 염기서열을 갖는 것을 특징으로 하는 류코노스톡 메센테로이드 AFY-3 (Leuconostoc mesenteroides AFY-3) KACC 92069P 균주.The method according to claim 1,
Wherein said strain has the 16S rRNA base sequence of SEQ ID NO: 1, leuconostomercenteroid AFY-3 ( Leuconostoc mesenteroides AFY-3) KACC 92069P strain.
상기 균주는 NaCl 5~10 %(w/v)가 첨가된 MRS(de Man, Rogosa and Sharpe) 액체배지에서 30℃, 72시간, 180 rpm 조건에서 배양시, 생균수가 2.8x108 내지 2.9x108인 내염성을 갖는 것을 특징으로 하는 류코노스톡 메센테로이드 AFY-3 (Leuconostoc mesenteroides AFY-3) KACC 92069P 균주.The method according to claim 1,
The strain was cultured in a MRS (de Man, Rogosa and Sharpe) medium supplemented with 5 to 10% (w / v) NaCl at 30 DEG C for 72 hours at 180 rpm When the number of culture, live cells flow, characterized in that with 2.8x10 8 to 2.9x10 8 in salt tolerance Pocono stock mesen steroid-3 AFY (mesenteroides Leuconostoc AFY-3) KACC 92069P strain.
상기 균주는 pH 3의 MRS 액체배지에서 30℃의 온도 및 180 rpm의 회전속도로 48시간 동안 배양시, 생균수가 1.5x107 내지 2.5x107 CFU/mL인 내산성을 갖는 것을 특징으로 하는 류코노스톡 메센테로이드 AFY-3 (Leuconostoc mesenteroides AFY-3) KACC 92069P 균주.The method according to claim 1,
This strain is the Pocono flow, characterized in that when the number of cultured for 48 hours at a rotating speed of 180 rpm and a temperature of 30 ℃ in MRS broth of pH 3, live cells with 1.5x10 7 to 2.5x10 7 CFU / mL of the acid resistance Stock Methensertoid AFY-3 ( Leuconostoc mesenteroides AFY-3) KACC 92069P strain.
상기 균주는 MRS 액체배지에서 7℃의 온도 및 120 rpm의 회전속도로 48시간 동안 배양시, 생균수가 2.8x108 내지 2.9x108 CFU/mL인 저온생육 특성을 갖는 것을 특징으로 하는 류코노스톡 메센테로이드 AFY-3 (Leuconostoc mesenteroides AFY-3) KACC 92069P 균주.The method according to claim 1,
Wherein said strain has low-temperature growth characteristics in which the number of viable cells is 2.8 x 10 8 to 2.9 x 10 8 CFU / mL when cultured in an MRS liquid medium at a temperature of 7 ° C and a rotation speed of 120 rpm for 48 hours. Teroid AFY-3 ( Leuconostoc mesenteroides AFY-3) KACC 92069P strain.
상기 균주를 김치 전체 중량 대비 0.5∼2 중량％ 포함하는 것을 특징으로 하는 김치.8. The method of claim 7,
Wherein the strain comprises 0.5 to 2 wt% of the total weight of the kimchi.
상기 김치는 배추 김치, 백김치, 깍두기, 열무 김치, 총각 김치, 갓김치로 이루어진 군에서 선택되는 어느 하나인 것을 특징으로 하는 김치. 8. The method of claim 7,
Wherein the kimchi is any one selected from the group consisting of cabbage kimchi, white kimchi, kakdugi, yamu kimchi, bamboo kimchi, and mustard kimchi.
상기 김치는 배추 김치, 백김치, 깍두기, 열무 김치, 총각 김치, 갓김치로 이루어진 군에서 선택되는 어느 하나인 것을 특징으로 하는 김치의 제조방법.11. The method of claim 10,
Wherein the kimchi is any one selected from the group consisting of cabbage kimchi, white kimchi, kakdugi, yamu kimchi, bamboo kimchi, and mustard kimchi.
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| KR20230019483A (en) * | 2020-07-15 | 2023-02-08 | 주식회사 풀무원 | Leuconostoc mesenteroides PBio03 and methods for preparing kimchi using the same |
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| KR20230019483A (en) * | 2020-07-15 | 2023-02-08 | 주식회사 풀무원 | Leuconostoc mesenteroides PBio03 and methods for preparing kimchi using the same |
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