KR101349692B1 - The Alcohol resistant strain of lactic acid bacteria, Pediococcus acidilactici and its use - Google Patents
The Alcohol resistant strain of lactic acid bacteria, Pediococcus acidilactici and its use Download PDFInfo
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- KR101349692B1 KR101349692B1 KR1020110129675A KR20110129675A KR101349692B1 KR 101349692 B1 KR101349692 B1 KR 101349692B1 KR 1020110129675 A KR1020110129675 A KR 1020110129675A KR 20110129675 A KR20110129675 A KR 20110129675A KR 101349692 B1 KR101349692 B1 KR 101349692B1
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- lactic acid
- alcohol
- pediococcus
- kctc
- acid bacteria
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- A23V2400/41—Pediococcus
- A23V2400/413—Acidilactici
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
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- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
본 발명은 신규 유산균 및 이의 응용에 관한 것으로서, 보다 상세하게는 막걸리로부터 분리된 유산균으로서 고농도의 알코올 존재 하에서도 생육이 가능하고, 알코올 존재 하의 저온에서는 성장하지 않고 장시간 생존하며, 중온에서는 성장하는 것을 특징으로 하는 페디오코커스 애시디락티시 AC-K3(KCTC 12039BP) 및 이를 포함하는 미생물제제의 제조에 관한 것이다. 본 발명의 유산균은 식품첨가제, 프로바이오틱스제, 정장제, 생균제, 사료첨가제, 의약품 및 기타 발효제품의 제조에 다양하게 이용될 수 있다. 특히 본 발명의 페디오코커스 애시디락티시 AC-K3(KCTC 12039BP)는 막걸리 제조의 유산균 후첨가제로서 유용하게 사용할 수 있다. The present invention relates to a novel lactic acid bacterium and its application, and more particularly, as a lactic acid bacterium separated from makgeolli, it is possible to grow even in the presence of a high concentration of alcohol, to survive for a long time without growing at low temperature in the presence of alcohol, and to grow at a medium temperature. Pediococcus is characterized in that the manufacturing of ACID-AC3 (KCTC 12039BP) and a microbial agent comprising the same. The lactic acid bacterium of the present invention can be variously used in the preparation of food additives, probiotics, suits, probiotics, feed additives, pharmaceuticals and other fermented products. In particular, pediococcus ashidilacti AC-K3 (KCTC 12039BP) of the present invention can be usefully used as a lactic acid bacterium post-additive of makgeolli.
Description
본 발명은 알코올 내성을 갖는 유산균 페디오코커스 애시디락티시 AC-K3(KCTC 12039BP) 및 이를 포함하는 미생물 제제에 관한 것으로, 더욱 상세하게는 막걸리로부터 분리된 유산균으로서 고농도의 알코올 존재 하에서도 생육이 가능하고, 알코올 존재 하의 저온에서는 성장하지 않고 장시간 생존하며, 중온에서는 성장하는 것을 특징으로 하는 페디오코커스 애시디락티시 AC-K3(KCTC 12039BP) 및 이를 포함하는 미생물제제에 관한 것이다.The present invention relates to a lactic acid bacterium pediococcus ACID-AC3 (KCTC 12039BP) having an alcohol resistance and a microbial agent comprising the same. The present invention relates to a pediococcus ashidilacti AC-K3 (KCTC 12039BP) and a microbial agent comprising the same, which are capable of being survived for a long time without growing at low temperature in the presence of alcohol.
우리 전통 음료 막걸리에 대한 관심과 소비가 늘어나면서 막걸리의 기능성에 관한 증폭하는 가운데 막걸리 속의 유산균의 기능성이 주목을 받고 있다. 과거 막걸리는 보존기간을 늘이기 위하여 유산균과 효모를 모두 사멸시킨 살균막걸리였으나 근년에는 생막걸리로 기호가 급격하게 이동하고 있다.
As the interest and consumption of Korean traditional rice wine increases, the functionalities of lactic acid bacteria in rice wine are attracting attention. In the past, Makgeolli was a sterilized Makgeolli, which killed both lactic acid bacteria and yeast in order to increase the preservation period.
유산균은 장에서 염증이나 암을 일으키는 유해세균의 증식을 억제하며, 배변을 편하게 해주고 면역력을 강화하는 등 프로바이오틱스 (probiotics)으로서의 역할을 하는 것으로 잘 알려져 있다.
Lactic acid bacteria are known to act as probiotics by inhibiting the proliferation of harmful bacteria causing inflammation and cancer in the intestine, facilitating defecation and strengthening immunity.
하지만 실제로 살아있는 유산균은 시판중인 ‘살균막걸리’ 속에는 전혀 없으며, 생막걸리도 많은 제품이 일본식의 입국을 통해 만들어지기 때문에 유산균의 수는 알려진 것보다 훨씬 적은 수가 들어 있다. 일부의 막걸리는 전통의 누룩을 이용해서 막걸리를 담그고 있지만 막걸리를 담그는 제법에 따라 막걸리의 유산균의 수는 큰 차이를 보이고 있다.
However, in reality, live lactic acid bacteria are not in the 'sterilized rice wine' on the market at all, and the number of live bacteria is much smaller than what is known because many products are made through Japanese entry. Although some of the rice wine is made using traditional yeast, the number of lactobacillus of rice wine varies greatly according to the manufacturing method of rice wine.
서미영 등은 막걸리 속의 유산균은 누룩 속에 존재하던 유산균이 초기에 번식하다가 효모에 의한 알코올 발효가 진행되면서 점차 생육이 영향을 받아 알코올 농도가 13% 이상이 되면 급격히 사멸한다고 한다 (한국식품과학회지, 2005, vol 37, No. 1, p61-66). 또한 배경화 등이 안동소주에서 분리한 페디오코커스 애시디락티시 (Pediococcus acidilactici)도 알코올 농도 12% 이상에서 전혀 성장하지 않는다고 했다 (Kor . J. Microbiol Biotechnol . 2007. Vol. 35, No. 4, p310-315). 따라서 12~13%의 알코올 농도에서도 생육할 수 있는 고농도 알코올 내성의 유산균은 막걸리 등 주류 발효에 유용하게 사용될 수 있을 것으로 기대된다.
Miyoung Seok et al. Reported that lactic acid bacteria in makgeolli are proliferated in the early stage of lactic acid bacteria, but are gradually affected by alcohol fermentation by yeast, and rapidly die when the alcohol concentration reaches 13% or higher (Korean Food Science Society, 2005). vol 37, No. 1, p61-66). Pediococcus Ashdi Lactici ( Pediococcus isolated from Andong Suzhou) acidilactici ) did not grow at all above 12% alcohol ( Kor . J. Microbiol) . Biotechnol . 2007. Vol. 35, No. 4, p310-315). Therefore, it is expected that lactic acid bacteria of high concentration alcohol resistance, which can be grown even at an alcohol concentration of 12-13%, can be usefully used for alcoholic fermentation such as makgeolli.
대한민국 특허 10-0424659에서는 프로바이오틱스로 시판되는 시판품보다 8~32%의 알코올 농도에 생존능이 우수한 락토바실러스 플란타럼 (Lactobacillus plantarum)을 분리하고 프로바이오틱스로 이용한 것이 있으나 알코올 내성의 특성을 부여했을 뿐, 그 특성을 특별히 응용하지는 않았다. 대한민국 공개특허 10-2006-0078894는 알코올 농도 1-13%(v/v)에서 내알코올성 및 pH 2-7에서 내산성을 가지고, 5-40℃에서 생육할 수 있는 저온성 락토바실러스 사케이(Lactobacillus sakei)를 분리하고 과실 주 제조 등에 응용하고 있으나 저온에 생육함으로서 막걸리 등의 저온으로 유통하는 주류에서도 유산균이 성장하여 주류의 품질을 떨어지게 하는 단점이 있을 수 있다.
Korean Patent 10-0424659 discloses Lactobacillus plantarum ( Lactobacillus) with superior viability at an alcohol concentration of 8-32% than commercially available products commercially available as probiotics. plantarum ) was isolated and used as a probiotic, but it gave the property of alcohol resistance, and did not apply the property in particular. Republic of Korea Patent Publication 10-2006-0078894 alcohol concentration is 1-13% (v / v) in the alcohol and with acid at pH 2-7, psychrotrophic Lactobacillus yarn K which can grow at 5-40 ℃ (Lactobacillus sakei ) is used to separate fruit liquor and produce fruit liquor, but lactic acid bacteria may grow in liquor distributed at low temperature, such as makgeolli, resulting in a poor quality of liquor.
본 발명과 관련된 선행문헌에 대해 개시하면 다음과 같다.Disclosed is a prior art related to the present invention.
(i) 특허문헌으로서 대한민국 특허 10-0424659 (백현동 등), 대한민국 공개특허 10-2006-0078894 (김일연 등)이 존재하고, (ii) 비특허문헌으로서 서미영 등의 “약,탁주 발효과정 중 미생물 균총의 변화(한국식품과학회지, 2005, Vol 37, No. 1 p61-66)" 및 배경화 등의 “안동소주 발효액으로부터 분리한 젖산 세균의 동정 및 발효 특성(Kor . J. Microbiol Biotechnol. 2007, Vol. 35, No. 4, p310-315)"이 존재하나, 본 발명의 기술적 특정과는 본질적으로 상이하다.
(i) Korean Patent No. 10-0424659 (Baek Hyun-dong, etc.) and Republic of Korea Patent Publication No. 10-2006-0078894 (Kim Il-yeon, etc.) exist as patent documents. changes in flora (Journal of the Korea Food, 2005, Vol 37, No. 1 p61-66) Identification and fermentation of lactic acid bacteria isolated from the fermentation broth Anton shochu ", and such background screen" (Kor. J. Microbiol Biotechnol . 2007, Vol. 35, No. 4, p310-315) ", but is essentially different from the technical specifics of the present invention.
본 발명은 알코올 내성 유산균을 분리하여 전통주 막걸리 및 여러 가지의 주류에 이용함으로서 막걸리를 비롯한 여러 가지 주류에 유산균의 기능성을 더하거나 높이는데 그 목적이 있다. 아울러 저온에서는 성장하지 않고 생존만함으로서, 저온에서 알코올 내성 유산균의 응용에 따른 주류의 품질변화를 최소화하는 것에도 목적이 있다. 나아가 주류 이외의 식품, 건강기능식품, 사료 및 의약품으로서의 용도로 적용하는데도 그 목적이 있다.
An object of the present invention is to add or increase the functionality of lactic acid bacteria to various alcoholic beverages including makgeolli by separating alcohol-resistant lactic acid bacteria and using traditional liquor makgeolli and various alcoholic beverages. In addition, by only survival without growing at low temperatures, the purpose of minimizing the change in the quality of alcohol according to the application of alcohol-resistant lactic acid bacteria at low temperatures. In addition, the purpose is to apply to the non-alcoholic foods, dietary supplements, feed and pharmaceuticals.
본 발명이 이루고자 하는 기술적 과제들은 이상에서 언급한 기술적 과제들로 제한되지 않으며, 언급되지 않은 또 다른 기술적 과제들은 본 발명의 기재로부터 당해 분야에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.The technical objects to be achieved by the present invention are not limited to the above-mentioned technical problems, and other technical subjects which are not mentioned can be clearly understood by those skilled in the art from the description of the present invention .
본 발명에서는 고농도 알콜에 내성을 가지는 페디오코커스 애시디락티시 AC-K3(KCTC 12039BP)를 제공한다.
The present invention provides pediococcus ashidilactici AC-K3 (KCTC 12039BP) resistant to high concentrations of alcohol.
상기 고농도 알콜은 6~13%(v/v)의 알콜인 것을 특징으로 할 수 있다.
The high concentration alcohol may be characterized in that the alcohol of 6 to 13% (v / v).
상기 페디오코커스 애시디락티시는 저온에서 성장하지 않고, 생존만 하는 것을 특징으로 할 수 있으며, 상기 저온은 1~10℃인 것을 특징으로 할 수 있다.
The pediococcus ashidilacthy may be characterized in that it does not grow at low temperature, but only survives, and the low temperature may be 1 to 10 ° C.
또한 본 발명은 (i) 상기 페디오코커스 애시디락티시 AC-K3(KCTC 12039BP)을 함유하는 (ii) 정장용 조성물, 생균제 조성물, 사료용조성물, 식품첨가용 조성물 및 프로바이오틱스 조성물로 이루어진 군에서 선택된 어느 하나의 조성물을 제공한다.
In another aspect, the present invention (i) any one selected from the group consisting of (ii) a formal composition, a probiotic composition, a feed composition, a food additive composition and a probiotic composition containing (p) the Pediococcus ashidilacti AC-K3 (KCTC 12039BP). One composition is provided.
또한 본 발명은 (i) 상기 페디오코커스 애시디락티시 AC-K3(KCTC 12039BP)을 함유하는 (ii) 막걸리, 포도주, 된장, 고추장, 청국장, 간장 및 식초로 이루어진 군에서 선택된 어느 하나의 발효제품을 제공한다.
In addition, the present invention (i) fermentation of any one selected from the group consisting of (ii) makgeolli, wine, miso, red pepper paste, cheonggukjang, soy sauce and vinegar containing (Piecococcus) adi lacticitic AC-K3 (KCTC 12039BP) Provide the product.
또한 본 발명은 상기 페디오코커스 애시디락티시 AC-K3(KCTC 12039BP)를 막걸리의 후첨가제로 이용하여 막걸리를 제조하는 방법을 제공한다.
In another aspect, the present invention provides a method for producing makgeolli using the pediococcus ashidilacti AC-K3 (KCTC 12039BP) as a post-additive of makgeolli.
본 발명의 다른 일면으로 상기 페디오코커스 애시디락티시 AC-K3(KCTC 12039BP)를 탄소원, 질소원, 유기산 및 미네랄로 구성된 배지에서 20~40℃로 8~48시간 동안 배양하는 것을 특징으로 하는 페디오코커스 애시디락티시 AC-K3(KCTC 12039BP)의 배양방법을 제공한다.
In another aspect of the present invention, the pedicoccus ashidilacti AC-K3 (KCTC 12039BP) in a medium consisting of a carbon source, a nitrogen source, an organic acid and minerals incubated for 8 to 48 hours at 20 ~ 40 ℃ Provided is a method for culturing Okoccus ashidilacti AC-K3 (KCTC 12039BP).
나아가 본 발명은 상기 배양방법으로 수득한 페디오코커스 애시디락티시 AC-K3(KCTC 12039BP)의 배양액으로부터 (i) 균체를 회수한 후, (ii) 단백질보호제, 당류계 보호제 및 pH조절제로 이루어진 군에서 선택된 어느 하나 이상을 혼합하고, (iii) 동결건조한 동결건조품의 제조방법을 제공한다.
Furthermore, the present invention recovers (i) the cells from the culture solution of pediococcus ashidilacti AC-K3 (KCTC 12039BP) obtained by the above culture method, and then comprises (ii) a protein protecting agent, a saccharide protecting agent and a pH adjusting agent. Mixing any one or more selected from the group, and (iii) provides a method for producing a lyophilized lyophilized product.
본 발명은 알코올 내성이 강한 유산균을 분리하는 단계, 알코올 내성 유산균이 저온에서 성장 혹은 생존능을 확인하는 단계 및 알코올 내성 유산균의 알코올 내성 유산균을 대량 배양하여 제품화하는 단계로 이루어진 것에 특징이 있다고 할 수 있다.The present invention may be characterized in that it comprises the step of separating the alcohol resistant lactic acid bacteria, the alcohol resistant lactic acid bacteria to confirm the growth or viability at low temperature, and the step of culturing the alcohol-resistant lactic acid bacteria of alcohol resistant lactic acid bacteria in mass production. .
본 발명은 알코올 13%이상에서도 사멸하지 않고 성장하는 강한 내성을 가지는 유산균을 분리함으로서 막걸리와 같은 저도주에서 성장할 수 있을 뿐만 아니라 또한 냉장 및 저온 유통온도인 4~10℃에 성장하지 않고 생존만하는 유산균을 분리함으로서 막걸리 같은 유산균 함유 주류에 첨가하여 품질변화 없이 유산균의 기능성을 부여할 수 있다.The present invention not only grows in lactic acid bacteria that have strong resistance to growth but also dies at more than 13% of alcohol, and can not only grow in low wines such as makgeolli, but also survive only without growing at 4-10 ° C., which is cold and cold temperature. By separating the lactic acid bacteria can be added to liquor containing liquor, such as makgeolli can give the functionality of the lactic acid bacteria without any quality change.
도 1은 1차 선별한 알코올 내성 유산균의 알코올 13% 함유 MRS broth 배지에서 37℃로 정치배양 시의 성장곡선이다.
도 2는 선별한 AC-S1과 AC-K3(KCTC 12039BP)의 16S rRNA 유전자 염기서열 배열이다. AC-S1과 AC-K3(KCTC 12039BP)는 Pediococcud acidilactici로 동정되었다. 도에서 DSM20336은 Pediococcus pentosaceus의 기준균주(type srtrain)인 DSM 20336의 16S rRNA complete sequence (NCBI ACCESSION NR_042058)이고, DSM20284는 Pediococcus acidilactici의 기준균주(type strain)인 DSM 20284의 16S rRNA complete sequence (NCBI ACCESSION NR_042057)를 나타낸다. K3는 AC-K3(KCTC 12039BP) 균주를, S1은 AC-S1 균주를 나타낸다.
도 3은 시판 막걸리의 알코올 농도인 6%, 막걸리 발효 종료 시의 알코올 농도인 12% 및 13%로 MRS broth배지에 알코올을 첨가하고 나서, 선별한 페디오코커스 애시디락티시 (Pediococcus acidilactici) AC-S1과 AC-K3(KCTC 12039BP)를 접종배양했을 때의 성장곡선을 대조균으로 페디오코커스 애시디락티시 (Pediococcus acidilactici) 기준균주 (type strain)인 KCTC 15064균주와 대비하여 나타내었다.
도 4는 선별한 페디오코커스 애시디락티시 (Pediococcus acidilactici) AC-S1과 AC-K3(KCTC 12039BP)를 시판중인 살균 막걸리 (알코올 함량 6%)에 접종하고 4℃, 10℃ 및 25℃에서 방치할 경우의 생균수 변화도이다.FIG. 1 is a growth curve during static culturing at 37 ° C. in MRS broth medium containing 13% alcohol of alcohol-resistant lactic acid bacteria selected first.
Figure 2 is a 16S rRNA gene sequence sequence of the selected AC-S1 and AC-K3 (KCTC 12039BP). AC-S1 and AC-K3 (KCTC 12039BP) are Pediococcud acidilactici was identified. DSM20336 in the Pediococcus 16S rRNA complete sequence (NCBI ACCESSION NR_042058) of DSM 20336, a type srtrain of pentosaceus , and DSM20284 is Pediococcus It represents the 16S rRNA complete sequence (NCBI ACCESSION NR_042057 ) of the reference strain DSM 20284 in acidilactici (type strain). K3 represents AC-K3 (KCTC 12039BP) strain and S1 represents AC-S1 strain.
FIG. 3 shows the selection of Pediococcus acidilactici AC after adding alcohol to MRS broth medium at 6% alcohol concentration of 12% and 13% alcohol concentration at the end of fermentation. Phedi the growth curve of the culture when inoculated -S1 and AC-K3 (KCTC 12039BP) to control bacteria Lactococcus o her CD rakti when (Pediococcus acidilactici ) compared with the KCTC 15064 strain, a type strain.
4 shows selected Pediococcus ashidilactici ( Pediococcus) acidilactici ) Variation in viable cell count when inoculated with commercial sterilized makgeolli (6% alcohol content) and AC-S1 and AC-K3 (KCTC 12039BP) at 4 ℃, 10 ℃ and 25 ℃.
본 발명은 고농도 알콜에 내성을 가지는 페디오코커스 애시디락티시 AC-K3(KCTC 12039BP)에 관한 것이다.
The present invention relates to pediococcus ashidilactici AC-K3 (KCTC 12039BP) resistant to high alcohol concentrations.
상기 고농도 알콜은 6~13%(v/v)의 알콜인 것을 특징으로 할 수 있다.
The high concentration alcohol may be characterized in that the alcohol of 6 to 13% (v / v).
상기 페디오코커스 애시디락티시는 저온에서 성장하지 않고, 생존만 하는 것을 특징으로 할 수 있으며, 상기 저온은 1~10℃인 것을 특징으로 할 수 있다.
The pediococcus ashidilacthy may be characterized in that it does not grow at low temperature, but only survives, and the low temperature may be 1 to 10 ° C.
또한 본 발명은 (i) 상기 페디오코커스 애시디락티시 AC-K3(KCTC 12039BP)을 함유하는 (ii) 정장용 조성물, 생균제 조성물, 사료용조성물, 식품첨가용 조성물 및 프로바이오틱스 조성물로 이루어진 군에서 선택된 어느 하나의 조성물에 관한 것이다.
In another aspect, the present invention (i) any one selected from the group consisting of (ii) a formal composition, a probiotic composition, a feed composition, a food additive composition and a probiotic composition containing (p) the Pediococcus ashidilacti AC-K3 (KCTC 12039BP). It relates to one composition.
또한 본 발명은 (i) 상기 페디오코커스 애시디락티시 AC-K3(KCTC 12039BP)을 함유하는 (ii) 막걸리, 포도주, 된장, 고추장, 청국장, 간장 및 식초로 이루어진 군에서 선택된 어느 하나의 발효제품에 관한 것이다.
In addition, the present invention (i) fermentation of any one selected from the group consisting of (ii) makgeolli, wine, miso, red pepper paste, cheonggukjang, soy sauce and vinegar containing (Piecococcus) adi lacticitic AC-K3 (KCTC 12039BP) It is about a product.
또한 본 발명은 상기 페디오코커스 애시디락티시 AC-K3(KCTC 12039BP)를 막걸리의 제조방법상 발효과정 후의 단계에 첨가하는 것으로 당업계에서 공연히 알려진 개념인 후첨가제로 이용하여 막걸리를 제조하는 방법에 관한 것이다.
In another aspect, the present invention is a method for producing makgeolli by using the pediococcus ashidilacti AC-K3 (KCTC 12039BP) as a post-additive which is known in the art to add to the step after the fermentation process in the manufacturing method of makgeolli. It is about.
본 발명의 다른 일면은 상기 페디오코커스 애시디락티시 AC-K3(KCTC 12039BP)를 탄소원, 질소원, 유기산 및 미네랄로 구성된 배지에서 20~40℃로 8~48시간 동안 배양하는 것을 특징으로 하는 페디오코커스 애시디락티시 AC-K3(KCTC 12039BP)의 배양방법에 관한 것이다.
In another aspect of the present invention, the pedicoccus ashidilacti AC-K3 (KCTC 12039BP) is a pedi, characterized in that for 8 to 48 hours incubation at 20-40 ℃ in a medium consisting of carbon source, nitrogen source, organic acid and minerals The present invention relates to a method of culturing Okoccus ashidilactici AC-K3 (KCTC 12039BP).
나아가 본 발명은 상기 배양방법으로 수득한 페디오코커스 애시디락티시 AC-K3(KCTC 12039BP)의 배양액으로부터 (i) 균체를 회수한 후, (ii) 단백질보호제, 당류계 보호제 및 pH조절제로 이루어진 군에서 선택된 어느 하나 이상을 혼합하고, (iii) 동결건조한 동결건조품의 제조방법을 제공한다.
Furthermore, the present invention recovers (i) the cells from the culture solution of pediococcus ashidilacti AC-K3 (KCTC 12039BP) obtained by the above culture method, and then comprises (ii) a protein protecting agent, a saccharide protecting agent and a pH adjusting agent. Mixing any one or more selected from the group, and (iii) provides a method for producing a lyophilized lyophilized product.
이하 본 발명인 페디오코커스 애시디락티시 (Pediococcus acidilactici) AC-KC (KCTC 12039BP)에 대해 보다 상세하게 개시한다. 애시디락티시는 생막걸리, 누룩, 김치 등의 발효 식품, 유산균 발효유등의 유제품 및 어류, 동물, 인체 등의 장관으로부터 분리할 수 있으나 보다 분리 확률을 높이기 위해서는 알코올이 함유된 발효식품으로부터 분리하는 것이 바람직하다. 본 발명의 분리배지로는 식품공전상의 BCP 베지 등 유산균 구별배지 (differentiation medium) 또는 선택배지 (selective medium), MRS 배지 등 유산균용 배양배지 등 유산균관련 배지를 이용할 수 있을 뿐만 아니라 유산균이 생육할 수 있는 일반 미생물배지도 이용할 수 있으나 유산균관련 배지 나아가 유산균 선택배지 (selective medium) 또는 구별배지 (differentiation medium) 를 이용하는 것이 본 발명의 균주를 획득하는데 유리하다.
Hereinafter, the present inventors Pediococcus ashidilactici ( Pediococcus acidilactici ) AC-KC (KCTC 12039BP) is disclosed in more detail. Ashdilacthyse can be separated from fermented foods such as fresh makgeolli, yeast and kimchi, dairy products such as lactic acid bacteria fermented milk, and fish, animal and human intestines. It is preferable. As the separation medium of the present invention, lactic acid bacteria-related mediums, such as lactic acid bacteria differentiation medium (differentiation medium) such as BCP vegetation in food industry or selective medium, culture medium for lactic acid bacteria such as MRS medium, as well as lactic acid bacteria can be grown. General microorganisms can also be used, but it is advantageous to obtain a strain of the present invention by using a lactic acid bacteria-related medium and also a lactic acid bacteria selective medium or a differentiation medium.
분리한 균주의 저온에서의 성장 및 생존능을 확인하기 위해서는 MRS 등의 배지류와 막걸리 등의 분리균주를 응용할 주류 및 식품 등을 베이스로 하여 1~40℃의 구간에서 배양하여 측정할 수 있다. 바람직하게는 응용할 주료 및 식품등의 대상물을 이용하는 것이 실제적이다.
In order to confirm the growth and viability at low temperature of the isolated strain can be measured by culturing in the section of 1 ~ 40 ℃ based on the mainstream and food to be applied to the isolated strain such as MRS and makgeolli. Preferably, it is practical to use objects such as alcohol and food to be applied.
분리된 균주의 대량배양을 위해서는 탄소원으로서 포도당, 과당, 만노즈, 갈락토오즈 등의 단당류와 말토즈 등의 이당류 및 이들로 구성된 다당류의 산 또는 효소분해물을 단독 혹은 혼합하여 이용할 수 있다. 질소원 및 영양요구물질의 목적으로 효모추출물, 각종 펩톤류 및 쇠고기 추출물 등의 동물단백질 추출물을 단독 혹은 혼합하여 대량배양에 사용할 수 있다. 인을 공급하기 위해서 인산칼륨류, 인산나트륨류를 보통 사용하며, 기타 인산염도 사용할 수 있다. 무기이온의 공급 목적으로서는 마그네숨염, 망간염, 철화합물 및 칼슘염 등을 사용할 수 있다. 또한 영양요구물질 또는 생육촉진물질로서 초산염, 구연산염 등의 유기산 염을 첨가하거나 올레인산, 폴리솔베이트 (polysorbate)같은 지방산류 및 비타민류도 첨가할 수 있다.이 밖에 필요에 따라서 상기 이외의 필요한 영양분을 더 첨가할 수도 있다.
For mass culture of the isolated strain, monosaccharides such as glucose, fructose, mannose and galactose, disaccharides such as maltose, and polysaccharides or acids or enzymatic products of these may be used alone or as a carbon source. For the purpose of nitrogen source and nutritional requirements, animal protein extracts such as yeast extract, various peptones and beef extracts can be used alone or in combination for mass culture. Potassium phosphates and sodium phosphates are commonly used to supply phosphorus, and other phosphates may also be used. Magnesium salt, manganese salt, iron compound, calcium salt, etc. can be used for the purpose of supplying an inorganic ion. In addition, organic acid salts such as acetate and citrate may be added as nutritional requirements or growth promoting substances, or fatty acids and vitamins such as oleic acid and polysorbate may be added. It may also be added.
배양된 균체의 동결건조 제품화를 위해서 탈지분유 (skim milk)같은 단백질보호제와 트레할로스, 설탕, 덱스트린, 전분 같은 당류계 보호제 및 인산염 같은 pH조절제를 단독 혹은 혼합하여 여러 가지 방식으로 사용할 수 있다.
For lyophilization of cultured cells, protein protecting agents such as skim milk, saccharide-based protecting agents such as trehalose, sugar, dextrin, starch, and pH adjusting agents such as phosphate may be used alone or in various ways.
이하 도면을 참조한 실시예에 의거하여 본 발명에 대해 구체적으로 설명한다.
Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.
[실시예 1] 알코올 내성 균주의 분리 및 선별Example 1 Isolation and Screening of Alcohol Resistant Strains
알코올 내성 유산균을 탐색하기 위하여 시판중인 생막걸리, 누룩, 김치 및 유산균 발효유에서 샘플을 1g 정도를 취하여 생리식염수에 현탁액을 만든 다음에 serial dilution법으로 희석하여 식품공전상의 유산균 분리배지인 BCP 한천배지 [효모추출물(yeast extract) 2.5g/l, 펩톤(peptone) 5g/l, 포도당(dextrose) 1g/l, 트윈 80 (Tween 80) 1g/l, L-시스테인(L-cysteine) 0.1g/l, 한천(agar) 15g/l, pH6.8~7.0, 브로모크레졸 퍼플 (bromocresol purple 0.004~0.006%, 121℃x15분 멸균]의 평판에 도말하였다. 37℃에서 24~48시간 배양한 후에 집락(colony) 주위가 노란색을 띄는 집락(colony)를 순수분리하여 MRS agar 배지 [미국 Difco 제품: 프로테오즈 펩톤 No. 3(proteose peptone No. 3) 10g/l. 소고기 추출물(beef extract) 10g/l, 효모 추출물(yeast extract) 5g/l, 포도당(dextrose) 20g/l, 폴리솔베이트 80(polysorbate 80) 1g/l, 구연산암모늄(ammonium citrate) 2g/l, 초산나트륨(sodium acetate) 5g/l, 황산마그네슘(magnesium sulfate) 0.1g/l, 황산망간(manganese sulfate) 0.05g/l, 제2인산칼륨(dipotassium phosphate) 2g/l, 한천(agar) 15g/l, 121℃x15분 멸균]의 평판에 이식시키고 37℃에서 24시간 이상 배양하여 알코올 내성 테스트에 사용하였다.
To detect alcohol-resistant lactic acid bacteria, take about 1 g of sample from commercial Makgeolli, Nuruk, Kimchi, and lactic acid bacteria fermented milk, make a suspension in physiological saline, and dilute by serial dilution method. Yeast extract 2.5g / l, peptone 5g / l, glucose 1g / l,
상기와 같이 분리한 유산균주 1 백금이를 알코올이 0%(v/v), 10%(v/v), 15%(v/v)가 함유된 MRS broth 배지 (미국 Difco제품, 상기 MRS agar에서 한천을 제외한 배지) 10ml에 접종하고 37℃에 24시간 배양하였다. Spectrophotometer를 이용하여 배양액의 OD600을 측정하여 알코올 10%(v/v) 및 15%(v/v)가 함유된 MRS broth 배지에서 OD가 증가한 균주를 1차로 선별하였다. 1차 선별한 균주를 다시 0%(v/v), 10%(v/v), 13%(v/v)의 알코올이 함유된 MRS broth 배지에 1백금이 접종하여 48시간에 OD600을 측정한 결과 13% 알코올이 함유된 배지에서 가장 높은 성장을 보인 2균주, AC-K3(KCTC 12039BP)와 AC-S1을 선별하였다 (도 1).
[실시예 2] 알코올 내성 선별균주의 동정 Example 2 Identification of Alcohol Resistant Screening Strains
선별한 2종의 유산균 AC-K3(KCTC 12039BP)와 AC-S1을 MRS broth배지에 액체 배양한 후 genomic DNA를 분리하여 16S rRNA 유전자의 염기서열분석을 통하여 계통분류학적 동정을 시도하였다. 세균의 27F 프라이머와 1492R 프라이머를 이용하여 16S rRNA 유전자를 PCR하여 염기서열 분석을 한 결과, 두 종의 유산균은 모두 Pediococcus acidilactici와 가장 가까운 것으로 확인되었다 (도 2).
Two kinds of lactic acid bacteria AC-K3 (KCTC 12039BP) and AC-S1 were cultured in MRS broth medium, and then genomic DNA was isolated and phylogenetic identification was performed through sequencing of 16S rRNA gene. As a result of sequencing of 16S rRNA gene using bacterial 27F primer and 1492R primer, both strains of lactic acid bacteria were Pediococcus. It was found to be closest to acidilactici (FIG. 2).
[실시예 3] 분리 알코올 내성 유산균의 알코올 내성 확인Example 3 Confirmation of Alcohol Resistance of Isolated Alcohol-Resistant Lactic Acid Bacteria
선별한 2종의 유산균, 페디오코커스 애시디락티시 (Pediococcus acidilactici) AC-K3(KCTC 12039BP)와 페디오코커스 애시디락티시 (Pediococcus acidilactici) AC-S1을 알코올 0%(v/v), 6%(v/v), 12%(v/v) 및 13%(v/v) 첨가한 MRS broth 배지 10ml에 MRS agar에서 37℃, 24 시간 배양한 균체 1백금이를 접종하고, 37℃에서 144시간 정치배양하면서 도중에 배양액 샘플을 취하여 10배 희석한 다음에 OD660을 측정함으로서 성장정도를 측정하였다 (도 3). 대조균주로서 페디오코커스 애시디락티시 (pediococcus acidilactici) 기준주 (type strain) KCTC 15064를 동일하게 배양하고 비교했다. 알코올의 첨가농도는 시판 막걸리의 알코올 농도인 6%(v/v), 막걸리 발효 종료 시의 알코올 농도인 12%(v/v) 및 13%(v/v)를 참고로 하여 정했다. 그 결과 분리한 두 알코올 내성주는 대조균주가 성장하지 않는 알코올 12% 및 13%에서 성장함으로서 알코올 내성주임을 확인할 수 있었다.
Selected 2 kinds of lactic acid bacteria, Pediococcus ashidilactici ( Pediococcus acidilactici ) AC-K3 (KCTC 12039BP) and Pediococcus ashidilactici ( Pediococcus acidilactici ) 10 ml of MRS broth medium containing 0% (v / v), 6% (v / v), 12% (v / v) and 13% (v / v) of alcohol at 37 ° C. in MRS agar , Inoculated with
[실시예 4] 막걸리에서의 안정성 Example 4 Stability in Rice Wine
시판 중인 살균 막걸리를 무균적으로 멸균된 시험관에 분주하고 선별한 2종의 유산균, 페디오코커스 애시디락티시 (Pediococcus acidilactici) AC-K3(KCTC 12039BP)와 페디오코커스 애시디락티시 (Pediococcus acidilactici) AC-S1을 MRS agar에서 37℃, 24 시간 배양한 균체 1백금이를 생균수가 106~107이 되도록 접종하고 4℃, 10℃ 및 25℃에 방치하면서 생균수의 변화를 측정하였다. 생균수는 건강기능식품공전에 나와 있는 BCP agar 배지를 이용한 pour plate법으로 측정하였다. 그 결과, 4~10℃에서 성장하지는 않고, 생존하는 것으로 나타났으며, 반면에 25℃에서는 성장하는 것으로 나타났다.
Two kinds of lactic acid bacteria, Pediococcus ashidilactici, which were obtained by dispensing commercially available sterile rice wine in aseptically sterilized test tubes acidilactici ) AC-K3 (KCTC 12039BP) and Pediococcus acidilactici AC-S1 were inoculated to live
[실시예 5] 분리균주의 대량배양Example 5 Mass Culture of Separated Strains
페디오코커스 애시디락티시 (Pediococcus acidilactici) AC-K3(KCTC 12039BP)를 MRSm agar에 도말하고 37℃, 48hrs 증식(amplification)시킨 다음에, 배양균체 1 loop를 취하여 MRSm broth 10 ml이 들어있는 30 ml 시험관에 접종하여 37℃에서 12 hrs 시간 배양하였다. 그 배양액 0.5 ml을 멸균된 5%(w/v) skim milk, 50%(v/v) glycerol 수용현탁액 0.5ml과 잘 혼합하여 -70℃의 deep freezer에 동결함으로서 종균을 보관한다. MRSm agar 및 broth의 배지조성은 카제인 펩톤 1%(w/v), 효모추출물 0.4%(w/v), 쇠고기추출물 0.8%(w/v), 제2인산칼륨 0.2%(w/v), 초산나트륨 0.5%(w/v), 구연산암모늄 0.2%(w/v), 황산마그네슘 0.02%(w/v), 황산망간 0.01%(w/v), 폴리솔베이트 80 (트윈 80) 0.1%(w/v), 한천(agar) 첨가시는 1.5%(w/v)이며 121℃에서 15분 멸균하여 사용한다. Pediococcus acidilactici ) AC-K3 (KCTC 12039BP) was smeared on MRSm agar and amplified at 37 ° C for 48hrs.Then, 1 loop of culture was taken and inoculated into a 30 ml test tube containing 10 ml of MRSm broth. Incubated for hrs time. 0.5 ml of the culture is mixed with sterile 5% (w / v) skim milk and 0.5 ml of 50% (v / v) glycerol aqueous suspension, and the seed is stored by freezing in a deep freezer at -70 ° C. The medium composition of MRSm agar and broth was
30ml 시험관에 준비한 멸균 MRSm broth배지에 상기의 -70℃에 보관한 종균 1 ml을 해동하여 접종하고 37℃로 12 시간, 정치하여 활성화 배양 (activation culture)했다. 활성화된 배양액 1ml을 50ml의 serum bottle에 준비된 35ml의멸균 MRSm broth 배양액에 멸균 주사기를 이용하여 접종하고 37℃로 12 시간, 정치하여 종배양(seed culture)한다.
1 ml of the spawn stored at -70 ° C was thawed and inoculated into a sterile MRSm broth medium prepared in a 30 ml test tube, and was incubated at 37 ° C for 12 hours to activate culture. 1 ml of the activated culture was inoculated into a 35 ml sterile MRSm broth culture prepared in a 50 ml serum bottle using a sterile syringe and left incubated at 37 ° C. for 12 hours for seed culture.
그리고 5L 발효조에 본배양 배지 (포도당 4%(w/v), 소이펩톤 1%(w/v), 효모추출물 1%(w/v), 제2인산칼륨 0.1%(w/v), 구연산 칼륨 0.1%(w/v), 황산마그네슘 0.01%(w/v), 황산망간 0.005%(w/v))를 3.5L 사입하고 121℃에서 15분 살균하였다. 살균 후에 seed 배양액 35 ml을 접종하여 접종량이 1%(v/v)되게 하였다. 배양조건은 37℃, pH 5.5로 배양하고 pH 조절제로서는 0.5M NaOH를 사용했으며, 균체의 침강을 방지하기 위하여 100 rpm으로 낮게 교반하였다. 배지멸균 후 배양완료 시까지 혐기성을 유지하기 위하여 N2 gas를 충진하고, 약 0.2 kg/cm2의 내압을 유지하여 오염의 위험 가능성을 낮추었으며 내압이 떨어지면 그때그때 N2 gas를 보충하였다. 12시간 배양 종료액의 OD660은 10배 희석한 값으로 0.727이었으며 생균수는 5.7 x 109 cfu/ml이었다.
In a 5L fermenter, the main culture medium (4% (w / v) glucose, 1% (w / v) soy peptone, 1% (w / v) yeast extract, 0.1% (w / v) potassium diphosphate, citric acid Potassium 0.1% (w / v), magnesium sulfate 0.01% (w / v), manganese sulfate 0.005% (w / v)) were added 3.5L and sterilized for 15 minutes at 121 ℃. After sterilization, 35 ml of seed culture was inoculated to inoculate 1% (v / v). Culture conditions were incubated at 37 ℃, pH 5.5, 0.5M NaOH was used as a pH regulator, and stirred at a low 100 rpm to prevent the settling of the cells. In order to maintain anaerobic until the completion of culture after sterilization of the medium, N 2 gas was charged, and the internal pressure of about 0.2 kg / cm 2 was lowered to reduce the risk of contamination, and when internal pressure dropped, N 2 gas was supplemented at that time. The OD 660 of the 12-hour culture was diluted 10-fold to 0.727 and the viable cell count was 5.7 x 10 9 cfu / ml.
[실시예 6] 대량배양액의 균체수확 및 동결건조 Example 6 Cell Harvesting and Freeze-Drying of Bulk Culture Solution
5L 배양액을 냉장원심분리기(refigerated centrifuge)을 이용하여 4℃, 6,000rpm으로 원심분리한 결과 습윤균체 (wet cell) 32g을 얻었다. 습윤균체 32g에 동결보호제 용액 (트레할로스 20%(w/v), 말토덱스트린 10%(w/v), 탈지분유 1%(w/v)) 31g을 혼합하여 동결건조기에서 96시간 건조한 결과, 동결건조된 원말 16g을 얻었으며 동결건조 원말의 생균수는 건강기능식품공전에 나와 있는 BCP agar 배지를 이용한 pour plate법에 준하여 측정한 결과, 4.0 x 1011 cfu/g으로 측정되었다.
The 5L culture was centrifuged at 6,000 rpm at 4 ° C. using a refrigerated centrifuge to obtain 32 g of wet cells. After drying for 32 hours in a freeze dryer, 31 g of a freeze protection agent solution (
이상 본 발명의 구체적 실시형태와 관련하여 본 발명을 설명하였으나 이는 예시에 불과하며 본 발명은 이에 제한되지 않는다. 당업자는 본 발명의 범위를 벗어나지 않고 설명된 실시형태를 변경 또는 변형할 수 있으며, 이러한 변경 또는 변형도 본 발명의 범위에 속한다. 또한, 본 명세서에서 설명한 각 구성요소의 물질은 당업자가 공지된 다양한 물질로부터 용이하게 선택하여 대체할 수 있다. 또한 당업자는 본 명세서에서 설명된 구성요소 중 일부를 성능의 열화 없이 생략하거나 성능을 개선하기 위해 구성요소를 추가할 수 있다. 뿐만 아니라, 당업자는 공정 환경이나 장비에 따라 본 명세서에서 설명한 방법 단계의 순서를 변경할 수도 있다. 따라서 본 발명의 범위는 설명된 실시형태가 아니라 특허청구범위 및 그 균등물에 의해 결정되어야 한다.The present invention has been described above in connection with specific embodiments of the present invention, but this is only an example and the present invention is not limited thereto. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. In addition, the materials of each component described herein can be readily selected and substituted for various materials known to those skilled in the art. Those skilled in the art will also appreciate that some of the components described herein can be omitted without degrading performance or adding components to improve performance. In addition, those skilled in the art may change the order of the method steps described herein depending on the process environment or equipment. Therefore, the scope of the present invention should be determined by the appended claims and equivalents thereof, not by the embodiments described.
본 발명은 막걸리에서 유래하는 페디오코커스 애시락티시 AC-K3(KCTC 12039BP) 및 이를 포함하는 미생물제제를 제공하여 주류 등의 식품산업에 이용될 수 있다. 특히 알코올 내성과 더불어 저온에 성장하지 않는 특성을 이용하면 저온으로 유통되는 막걸리 발효의 유산균 후첨가제로서 특히 유용하다. 이외에도 본발명의 유산균은 식품첨가제, 프로바이오틱스제, 정장제, 생균제, 사료첨가제, 의약품 및 기타 발효제품의 제조에 다양하게 이용될 수 있다.
The present invention can be used in the food industry, such as liquor by providing a microorganism including the Pediococcus acelacthysis AC-K3 (KCTC 12039BP) derived from makgeolli and the same. In particular, it is particularly useful as a lactic acid bacterium post-additive of makgeolli fermentation distributed at low temperature by using a property that does not grow at low temperature in addition to alcohol resistance. In addition, the lactic acid bacteria of the present invention can be used in various preparations for food additives, probiotics, suits, probiotics, feed additives, pharmaceuticals and other fermented products.
Claims (10)
Pediococcus ashidilacti AC-K3 (KCTC 12039BP), which does not grow at a low temperature of 1 to 10 ° C., but survives, and is resistant to alcohol of 6 to 13% (v / v).
The pediococcus ashidilacti AC-K3 (KCTC 12039BP) according to claim 1, wherein the pediococcus ashidilacthysis is resistant to 12-13% (v / v) alcohol.
The pediococcus ashidilacti AC-K3 (KCTC 12039BP) according to claim 1, wherein the pediococcus ashidilacthysis is resistant to alcohol of 13% (v / v).
(i) any fermentation product selected from the group consisting of (ii) makgeolli, wine, soybean paste, red pepper paste, cheonggukjang, soy sauce and vinegar, containing (1) pediococcus ashidilacti AC-K3 (KCTC 12039BP).
Method for producing makgeolli using pediococcus ashidilacti AC-K3 (KCTC 12039BP) of claim 1 as a post-additive of makgeolli.
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| US20100040730A1 (en) * | 2003-06-12 | 2010-02-18 | Lallemand Sas | Alcohol-tolerant malolactic strains for the maturation of wines with average or high ph |
| KR20090077993A (en) * | 2008-01-14 | 2009-07-17 | 김현풍 | Process for preparing makgeolli(korean rice wine) |
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