KR102677848B1 - Probiotics for fermenting kimchi and kimchi using the same - Google Patents
Probiotics for fermenting kimchi and kimchi using the same Download PDFInfo
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- KR102677848B1 KR102677848B1 KR1020210117038A KR20210117038A KR102677848B1 KR 102677848 B1 KR102677848 B1 KR 102677848B1 KR 1020210117038 A KR1020210117038 A KR 1020210117038A KR 20210117038 A KR20210117038 A KR 20210117038A KR 102677848 B1 KR102677848 B1 KR 102677848B1
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- South Korea
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- lactobacillus
- kimchi
- strain
- plantarum
- paracasei
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- 229940088594 vitamin Drugs 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/065—Microorganisms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/10—Preserving with acids; Acid fermentation
- A23B7/105—Leaf vegetables, e.g. sauerkraut
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
- A23L19/20—Products from fruits or vegetables; Preparation or treatment thereof by pickling, e.g. sauerkraut or pickles
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/143—Fermentum
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/165—Paracasei
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
Abstract
본 발명은 김치, 막걸리 등에서 내산성, 내담즙성, 항염증 우수 프로바이오틱스 후보 유산균을 선발하고, 상기 유산균을 적용한 김치를 제조하여, 품질이 균일하고 프로바이오틱스 특성이 우수한 김치를 제조할 수 있다.The present invention selects probiotic candidate lactic acid bacteria with excellent acid resistance, bile resistance, and anti-inflammatory properties from kimchi, makgeolli, etc., and manufactures kimchi using the lactic acid bacteria to produce kimchi of uniform quality and excellent probiotic properties.
Description
본 발명은 김치, 막걸리 등에서 내산성, 내담즙성, 항염증 우수 프로바이오틱스 후보 유산균을 선발하여, 상기 유산균을 적용한 김치를 제조하는 방법에 관한 것이다.The present invention relates to a method of selecting probiotic candidate lactic acid bacteria with excellent acid resistance, bile resistance, and anti-inflammatory properties from kimchi, makgeolli, etc., and manufacturing kimchi using the lactic acid bacteria.
프로바이오틱스는 인체에 이로운 역할을 하는 살아있는 균으로 락토바실러스 카세이(Lactobacillus casei)를 비롯한 락토바실러스(Lactobacillus) 속 11종, 비피도박테리움(Bifidobacterium) 속 4종, 및 스트렙토코커스(Streptococcus) 1종, 락토코커스(Lactococcus) 1종, 엔테로코커스(Enterococcus) 2종 총 19종이 식약처에 등록되어있으며 우리나라에서는 상기 19종만을 고시형으로 프로바이오틱스라 인정하고 있다. 하지만, 유산균 및 발효 복합물을 개발하여 현재까지 10종정도가 개별형으로 인증 받았으며 다양한 연구진이 다양한 유산균을 연구하고 있는 실정이다.Probiotics are live bacteria that play a beneficial role in the human body, including 11 species of the Lactobacillus genus including Lactobacillus casei , 4 species of the Bifidobacterium genus , 1 species of Streptococcus , and lactobacilli. A total of 19 species, including 1 species of Lactococcus and 2 species of Enterococcus , are registered with the Ministry of Food and Drug Safety, and in Korea, only the above 19 species are recognized as probiotics by notification. However, through the development of lactic acid bacteria and fermentation complexes, about 10 types have been certified as individual types to date, and various research teams are researching various lactic acid bacteria.
대부분의 프로바이오틱스는 동물에서 유래하여 그것을 사람을 포함한 동물인 숙주에게 공급하여 항암, 항염 등 다양한 기능성을 가지고 있지만, 동물에서 유래한 유산균과 비교하여 식물성에서 유래한 유산균은 많은 연구가 되어있지 않다.Most probiotics are derived from animals and are supplied to animal hosts, including humans, and have various functions such as anti-cancer and anti-inflammatory properties. However, compared to lactic acid bacteria derived from animals, not much research has been done on lactic acid bacteria derived from plants.
김치는 배추를 기존 재료로 하여 다양한 채소들이 유산균에 의해 발효된 세계적인 식물성 발효식품으로, 풍부한 미네랄, 비타민, 식이섬유 등 다양한 기능성이 보고되고 있다. Kimchi is a world-class vegetable fermented food made by fermenting various vegetables with lactic acid bacteria using cabbage as an existing ingredient, and has been reported to have various functionalities such as rich minerals, vitamins, and dietary fiber.
소비 트렌드와 김장 문화의 변화로 집에서 담궈먹는 김치보다는 상품화 김치 시장이 많이 확대되고 있다. 이를 위해 김치 품질의 균일화, 규격화를 위해 다양한 연구를 진행하고 있으나, 발효유제품처럼 아직까지 종균화가 이루어지지 못하였다는 점이 가장 큰 문제점이다. 초기 균수와 균종이 일정하지 않기 때문에 발효유제품과 같은 발효시스템을 사용할 수 없다. 김치의 상업화를 위해서는 우수한 유산균을 활용하여 종균화가 되어야한다. Due to changes in consumption trends and kimchi culture, the market for commercialized kimchi is expanding rather than kimchi made at home. To this end, various studies are being conducted to standardize and standardize the quality of kimchi, but the biggest problem is that spawning has not yet been achieved like in fermented milk products. Because the initial number and species of bacteria are not constant, fermentation systems such as fermented milk products cannot be used. In order to commercialize kimchi, it must be spawned using excellent lactic acid bacteria.
이에 김치에 적용 가능한 유산균을 종균화하여, 품질이 균일한 김치를 제조하는 방법이 요구되었다.Accordingly, there was a need for a method of producing kimchi of uniform quality by spawning lactic acid bacteria applicable to kimchi.
본 발명은 김치에서 잘 자라고, 내산성, 내담즙성을 가지며, 항염증, 항산화 효과가 있는 프로바이오틱스 균주를 선정하여 프로바이오틱스 제제를 제조하고, 프로바이오틱스 제제를 김치 제조 시 이용하여, 품질이 균일하고 프로바이오틱스 특성이 우수한 김치를 제조하는 것을 목적으로 한다.The present invention manufactures a probiotic preparation by selecting a probiotic strain that grows well in kimchi, has acid resistance, bile resistance, and has anti-inflammatory and antioxidant effects, and uses the probiotic preparation when making kimchi, so that the quality is uniform and the probiotic characteristics are maintained. The purpose is to produce excellent kimchi.
본 발명은, 락토바실러스 플란타럼 (L. plantarum) HJ-Ki(기탁번호 : KACC 92324P) 균주, 락토바실러스 퍼멘텀(L. fermentum) HJ-C1(기탁번호 : KACC 92322P) 균주, 및 락토바실러스 파라카세이(L. paracasei) MO-C1(기탁번호 : KACC 92325P) 균주 중 적어도 하나를 포함하여 제조되는 김치 발효용 프로바이오틱스 제제를 제공한다.The present invention relates to Lactobacillus plantarum ( L. plantarum ) HJ-Ki (accession number: KACC 92324P) strain, Lactobacillus fermentum ( L. fermentum ) HJ-C1 (accession number: KACC 92322P) strain, and Lactobacillus Paracasei ( L. paracasei ) MO-C1 (accession number: KACC 92325P) strain is provided to produce a probiotic preparation for kimchi fermentation.
본 발명의 일 실시예에 있어서, 상기 락토바실러스 플란타럼 (L. plantarum) HJ-Ki(기탁번호 : KACC 92324P) 균주는 서열번호 1로 표시되는 16s rRNA 염기서열을 포함하고, 상기 락토바실러스 퍼멘텀(L. fermentum) HJ-C1(기탁번호 : KACC 92322P) 균주는 서열번호 2로 표시되는 16s rRNA 염기서열을 포함하고, 상기 락토바실러스 파라카세이(L. paracasei) MO-C1(기탁번호 : KACC 92325P) 균주는 서열번호 3으로 표시되는 16s rRNA 염기서열을 포함할 수 있다.In one embodiment of the present invention, the Lactobacillus plantarum ( L. plantarum ) HJ-Ki (accession number: KACC 92324P) strain includes a 16s rRNA base sequence represented by SEQ ID NO: 1, and the Lactobacillus fur Mentum ( L. fermentum ) HJ-C1 (accession number: KACC 92322P) strain contains a 16s rRNA base sequence represented by SEQ ID NO: 2, and the Lactobacillus paracasei ( L. paracasei ) MO-C1 (accession number: KACC 92325P) strain may include the 16s rRNA base sequence represented by SEQ ID NO: 3.
본 발명은, 배추를 소금물에 절이는 단계, 양념을 준비하는 단계, 상기 양념에 락토바실러스 플란타럼 (L. plantarum) HJ-Ki(기탁번호 : KACC 92324P) 균주, 락토바실러스 퍼멘텀(L. fermentum) HJ-C1(기탁번호 : KACC 92322P) 균주, 및 락토바실러스 파라카세이(L. paracasei) MO-C1(기탁번호 : KACC 92325P) 균주 중 적어도 하나를 포함하는 프로바이오틱스 제제를 혼합하는 단계, 상기 소금물에 절여둔 상기 배추를 세척하고 물기를 제거하는 단계, 및 혼합된 상기 양념을 배추에 버무리는 단계를 포함하는 김치 제조방법을 제공한다.The present invention includes the step of pickling cabbage in salt water, the step of preparing seasoning, and the seasoning including Lactobacillus plantarum ( L. plantarum ) HJ-Ki (accession number: KACC 92324P) strain, Lactobacillus fermentum ( L. fermentum) ) mixing a probiotic preparation containing at least one of the HJ-C1 (accession number: KACC 92322P) strain and the Lactobacillus paracasei ( L. paracasei ) MO-C1 (accession number: KACC 92325P) strain, in the salt water It provides a kimchi manufacturing method including the steps of washing and removing moisture from the pickled cabbage, and mixing the mixed seasoning into the cabbage.
본 발명의 일 실시예에 있어서, 상기 김치 제조방법은 상기 양념에 버무린 상기 배추를 발효시키는 단계를 더 포함하며, 상기 발효는 김치 내 유산균을 제외한 일반 세균 대비 유산균 함량이 100배 이상이 되도록 6주 이내로 수행되는 것일 수 있다.In one embodiment of the present invention, the kimchi manufacturing method further includes the step of fermenting the cabbage mixed with the seasoning, and the fermentation is performed for 6 weeks so that the content of lactic acid bacteria in kimchi is 100 times or more compared to general bacteria excluding lactic acid bacteria. It may be carried out within a period of time.
본 발명의 일 실시예에 있어서, 상기 발효하는 단계는 1℃ 내지 10℃에서 수행될 수 있다.In one embodiment of the present invention, the fermentation step may be performed at 1°C to 10°C.
본 발명의 일 실시예에 따르면 상기 균주를 이용하여 일반적인 김치보다 산도가 높은 신김치를 제조할 수 있다. 본 발명의 일 실시예에 따른은 신김치 제조 방법은, 배추를 소금물에 절이는 단계, 양념을 준비하는 단계, 상기 양념에 락토바실러스 플란타럼 (L. plantarum) HJ-Ki(기탁번호 : KACC 92324P) 균주 또는 락토바실러스 파라카세이(L. paracasei) MO-C1(기탁번호 : KACC 92325P) 균주를 포함하는 프로바이오틱스 제제를 혼합하는 단계, 상기 소금물에 절여둔 상기 배추를 세척하고 물기를 제거하는 단계, 혼합된 상기 양념을 배추에 버무리는 단계, 및 상기 양념에 버무린 배추를 2주 내지 8주 동안 발효시키는 단계를 포함할 수 있다.According to one embodiment of the present invention, sour kimchi with higher acidity than general kimchi can be produced using the above strain. The method of producing sour kimchi according to an embodiment of the present invention includes the steps of pickling cabbage in salt water, preparing seasoning, and adding Lactobacillus plantarum ( L. plantarum ) HJ-Ki (Accession number: KACC 92324P) to the seasoning. mixing a probiotic preparation containing the strain or Lactobacillus paracasei ( L. paracasei ) MO-C1 (accession number: KACC 92325P) strain, washing the cabbage pickled in salt water and removing moisture, mixing It may include mixing the seasoning with cabbage, and fermenting the cabbage mixed with the seasoning for 2 to 8 weeks.
본 발명의 일 실시예에 있어서, 상기 발효시키는 단계는 4주 내지 8주 동안 수행되며, 상기 신김치의 총산도(%)는 0.7% 이상일 수 있다.In one embodiment of the present invention, the fermentation step is performed for 4 to 8 weeks, and the total acidity (%) of the sour kimchi may be 0.7% or more.
본 발명의 일 실시예에 있어서, 상기 발효하는 단계는 1℃ 내지 10℃에서 수행될 수 있다.In one embodiment of the present invention, the fermentation step may be performed at 1°C to 10°C.
본 발명은, 상기 김치 제조방법으로 제조된 김치 또는 상기 신김치 제조방법으로 제조된 신김치를 제공한다.The present invention provides kimchi prepared by the kimchi manufacturing method or sour kimchi prepared by the sour kimchi manufacturing method.
본 발명의 일 실시예에 따르면, 품질이 균일하고 프로바이오틱스 특성이 우수한 김치를 제조할 수 있다. According to one embodiment of the present invention, kimchi of uniform quality and excellent probiotic properties can be produced.
도 1은 락토바실러스 플란타럼 (L. plantarum) HJ-Ki(기탁번호 : KACC 92324P) 균주의 계통도를 나타낸 것이다.
도 2는 락토바실러스 퍼멘텀(L. fermentum) HJ-C1(기탁번호 : KACC 92322P) 균주의 계통도를 나타낸 것이다.
도 3은 락토바실러스 파라카세이(L. paracasei) MO-C1(기탁번호 : KACC 92325P) 균주의 계통도를 나타낸 것이다.
도 4는 김치, 막걸리, 효소액 등을 0.85 중량% NaCl로 희석하고 선택배지에 접종하고 배양하여 단일 콜로니를 얻고, 시퀀싱하는 과정을 개략적으로 도시한 것이다.
도 5는 발효 기간에 따른 대조구와 10가지 균주의 흡광도(OD)값을 측정하여 나타낸 것이다.
도 6은 LPS(Lipopolysaccharide)의 처리에 따른, LPS(Lipopolysaccharide) 처리구, PC(Positive control), 및 7가지 균주의 NO 생성량을 나타낸 것이다.
도 7은 LPS(Lipopolysaccharide)의 처리에 따른, 대조구, LPS(Lipopolysaccharide) 처리구, 및 7가지 균주의 COX-2 발현 수준을 나타낸 것이다.
도 8은 PC(Positive control) 및 6가지 균주의 ABTS 라디칼 소거능 및 DPPH 라디칼 소거능을 나타낸 그래프이다.Figure 1 shows a family diagram of the Lactobacillus plantarum ( L. plantarum ) HJ-Ki (accession number: KACC 92324P) strain.
Figure 2 shows a systematic diagram of the Lactobacillus fermentum ( L. fermentum ) HJ-C1 (accession number: KACC 92322P) strain.
Figure 3 shows a systematic diagram of the Lactobacillus paracasei ( L. paracasei ) MO-C1 (accession number: KACC 92325P) strain.
Figure 4 schematically shows the process of diluting kimchi, makgeolli, enzyme solution, etc. with 0.85% by weight NaCl, inoculating and culturing a selective medium to obtain a single colony, and sequencing.
Figure 5 shows the measured absorbance (OD) values of the control group and 10 strains according to the fermentation period.
Figure 6 shows the NO production amount of LPS (Lipopolysaccharide) treatment group, PC (positive control), and 7 strains according to LPS (Lipopolysaccharide) treatment.
Figure 7 shows the COX-2 expression levels of the control group, LPS (Lipopolysaccharide) treated group, and 7 strains according to LPS (Lipopolysaccharide) treatment.
Figure 8 is a graph showing the ABTS radical scavenging ability and DPPH radical scavenging ability of PC (positive control) and six strains.
본 발명은 다양한 변경을 가할 수 있고 여러 가지 실시예를 가질 수 있는 바, 특정 실시예들을 도면에 예시하고 본문에서 상세하게 설명하고자 한다. 그러나, 이는 본 발명을 특정한 실시 형태에 대해 한정하려는 것이 아니며, 본 발명의 사상 및 기술 범위에 포함되는 모든 변경, 균등물 내지 대체물을 포함하는 것으로 이해되어야 한다. 본 발명을 설명함에 있어서 관련된 공지 기술에 대한 구체적인 설명이 본 발명의 요지를 흐릴 수 있다고 판단되는 경우 그 상세한 설명을 생략한다.Since the present invention can make various changes and have various embodiments, specific embodiments will be illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all changes, equivalents, and substitutes included in the spirit and technical scope of the present invention. In describing the present invention, if it is determined that a detailed description of related known technologies may obscure the gist of the present invention, the detailed description will be omitted.
이하, 본 명세서에 개시된 실시예들을 도면을 참조하여 상세하게 설명하고 자 한다. 상세한 설명, 도면들 및 청구항들에서 상술하는 예시적인 실시예들은 한정을 위한 것이 아니며, 기재된 실시예들과 다른 실시예들이 이용될 수 있으며, 여기서 개시되는 기술의 사상이나 범주를 벗어나지 않는 한 다른 변경들도 가능하다. Hereinafter, embodiments disclosed in this specification will be described in detail with reference to the drawings. The exemplary embodiments described above in the detailed description, drawings, and claims are not intended to be limiting, and embodiments different from the described embodiments may be used, and other modifications may be made without departing from the spirit or scope of the technology disclosed herein. It is also possible.
개시된 기술에 관한 설명은 구조적 내지 기능적 설명을 위한 실시예에 불과하므로, 개시된 기술의 권리범위는 본문에 설명된 실시예에 의하여 제한되는 것으로 해석되어서는 아니된다. 즉, 실시예는 다양한 변경이 가능하고 여러 가지 형태를 가질 수 있으므로 개시된 기술의 권리범위는 기술적 사상을 실현할 수 있는 균등물을 포함하는 것으로 이해되어야 한다.Since the description of the disclosed technology is only an example for structural or functional explanation, the scope of rights of the disclosed technology should not be construed as limited by the examples described in the text. In other words, since the embodiments can be modified in various ways and can take various forms, the scope of rights of the disclosed technology should be understood to include equivalents that can realize the technical idea.
여기서 사용된 모든 용어들은 다르게 정의되지 않는 한, 개시된 기술이 속하는 분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 것과 동일한 의미를 가진다. 일반적으로 사용되는 사전에 정의되어 있는 용어들은 관련기술의 문맥상 가지는 의미와 일치하는 것으로 해석되어야 하며, 본 출원에서 명백하게 정의하지 않는 한 이상적이거나 과도하게 형식적인 의미를 지니는 것으로 해석 될 수 없다.All terms used herein, unless otherwise defined, have the same meaning as commonly understood by a person of ordinary skill in the field to which the disclosed technology pertains. Terms defined in commonly used dictionaries should be interpreted as consistent with the meaning they have in the context of the related technology, and cannot be interpreted as having an ideal or excessively formal meaning unless clearly defined in the present application.
본 발명은 김치, 막걸리 등에서 내산성, 내담즙성, 항염증 우수 프로바이오틱스 후보 유산균을 선발하여, 상기 유산균을 적용한 김치를 제조하는 방법에 관한 것이다.The present invention relates to a method of selecting probiotic candidate lactic acid bacteria with excellent acid resistance, bile resistance, and anti-inflammatory properties from kimchi, makgeolli, etc., and manufacturing kimchi using the lactic acid bacteria.
본 발명의 일 실시예에 있어서, 상기 유산균은 본 발명은, 락토바실러스 플란타럼 (L. plantarum) HJ-Ki(기탁번호 : KACC 92324P) 균주, 락토바실러스 퍼멘텀(L. fermentum) HJ-C1(기탁번호 : KACC 92322P) 균주, 및 락토바실러스 파라카세이(L. paracasei) MO-C1(기탁번호 : KACC 92325P) 균주 중 적어도 하나를 포함할 수 있다. In one embodiment of the present invention, the lactic acid bacteria of the present invention are Lactobacillus plantarum ( L. plantarum ) HJ-Ki (accession number: KACC 92324P) strain, Lactobacillus fermentum ( L. fermentum ) HJ-C1 (Accession number: KACC 92322P) strain, and Lactobacillus paracasei ( L. paracasei ) MO-C1 (Accession number: KACC 92325P) strain.
그러나 상기 유산균은 이에 한정되는 것은 아니며, 락토바실러스 퍼멘텀(L. fermentum) C2, 락토바실러스 플란타룸(L. plantarum) MS-C1, 락토바실러스 플란타룸(L. plantarum) B3-I, 락토바실러스 플란타룸(L. plantarum) C6, 락토바실러스 플란타룸(L. plantarum) MCS-D, 락토바실러스 플란타룸(L. plantarum) ho, 락토바실러스 파라카세이(L. paracasei) MC-A1, 락토바실러스 파라카세이(L. paracasei) C3, 락토바실러스 파라카세이(L. paracasei) MJ-E1, 락토바실러스 파라카세이(L. paracasei) kg, 락토바실러스 파라카세이(L. paracasei) MCS-E, 락토바실러스 파라카세이(L. paracasei) MS-D1, 락토바실러스 람노서스(Lactobacillus rhamnosus) B1-A, 락토바실러스 람노서스(Lactobacillus rhamnosus) B1-D, 락토바실러스 람노서스(Lactobacillus rhamnosus) B4-M, 락토바실러스 람노서스(Lactobacillus rhamnosus) B4-L, 락토바실러스 브레비스(Lactobacillus brevis) MCS-F, 락토바실러스 브레비스(Lactobacillus brevis) Ha, 락토바실러스 브레비스(Lactobacillus brevis) hf, 락토바실러스 브레비스(Lactobacillus brevis) sf, 락토바실러스 브레비스(Lactobacillus brevis) De, 락토바실러스 사케이(Lactobacillus sakei) Kd, 락토바실러스 사케이(Lactobacillus sakei) Hd, 락토바실러스 사케이(Lactobacillus sakei) hc, 락토바실러스 사케이(Lactobacillus sakei) kc, 락토바실러스 사케이(Lactobacillus sakei) hi, 락토바실러스 파라부크네리(Lactobacillus parabuchneri) MS-C2, 락토바실러스 파라부크네리(Lactobacillus parabuchneri) ke, 락토바실러스 파라부크네리(Lactobacillus parabuchneri) kf, 락토바실러스 커베터스(Lactobacillus curvatus) Ya, 및 락토바실러스 커베터스(Lactobacillus curvatus) Yb 중 적어도 하나를 더 포함할 수 있다.However, the lactic acid bacteria are not limited to this, Lactobacillus fermentum ( L. fermentum) C2, Lactobacillus plantarum ( L. plantarum ) MS-C1, Lactobacillus plantarum ( L. plantarum ) B3-I, lactobacillus Bacillus plantarum ( L. plantarum ) C6, Lactobacillus plantarum ( L. plantarum ) MCS-D, Lactobacillus plantarum ( L. plantarum ) ho, Lactobacillus paracasei ( L. paracasei ) MC-A1, Lactobacillus paracasei ( L. paracasei ) C3, Lactobacillus paracasei ( L. paracasei ) MJ-E1, Lactobacillus paracasei ( L. paracasei ) kg, Lactobacillus paracasei ( L. paracasei ) MCS-E, Lactobacillus Paracasei ( L. paracasei ) MS-D1, Lactobacillus rhamnosus B1-A, Lactobacillus rhamnosus B1-D, Lactobacillus rhamnosus B4-M, Lactobacillus rhamnosus Lactobacillus rhamnosus B4-L, Lactobacillus brevis MCS-F, Lactobacillus brevis Ha, Lactobacillus brevis hf, Lactobacillus brevis sf, Lactobacillus brevis ( Lactobacillus brevis ) De, Lactobacillus sakei ( Lactobacillus sakei ) Kd, Lactobacillus sakei ( Lactobacillus sakei ) Hd, Lactobacillus sakei ( Lactobacillus sakei ) hc, Lactobacillus sakei ( Lactobacillus sakei ) kc, Lactobacillus sakei ( Lactobacillus sakei ) hi, Lactobacillus parabuchneri MS-C2, Lactobacillus parabuchneri ke, Lactobacillus parabuchneri kf, Lactobacillus curvatus It may further include at least one of Ya, and Lactobacillus curvatus Yb.
본 발명은, 락토바실러스 플란타럼 (L. plantarum) HJ-Ki(기탁번호 : KACC 92324P) 균주, 락토바실러스 퍼멘텀(L. fermentum) HJ-C1(기탁번호 : KACC 92322P) 균주, 및 락토바실러스 파라카세이(L. paracasei) MO-C1(기탁번호 : KACC 92325P) 균주 중 적어도 하나를 포함하여 제조되는 김치 발효용 프로바이오틱스 제제를 제공한다.The present invention relates to Lactobacillus plantarum ( L. plantarum ) HJ-Ki (accession number: KACC 92324P) strain, Lactobacillus fermentum ( L. fermentum ) HJ-C1 (accession number: KACC 92322P) strain, and Lactobacillus Paracasei ( L. paracasei ) MO-C1 (accession number: KACC 92325P) strain is provided to produce a probiotic preparation for kimchi fermentation.
도 1은 락토바실러스 플란타럼 (L. plantarum) HJ-Ki(기탁번호 : KACC 92324P) 균주의 계통도를 나타낸 것이다.Figure 1 shows a family diagram of the Lactobacillus plantarum (L. plantarum ) HJ-Ki (accession number: KACC 92324P) strain.
도 2는 락토바실러스 퍼멘텀(L. fermentum) HJ-C1(기탁번호 : KACC 92322P) 균주의 계통도를 나타낸 것이다.Figure 2 shows a systematic diagram of the Lactobacillus fermentum (L. fermentum ) HJ-C1 (accession number: KACC 92322P) strain.
도 3은 락토바실러스 파라카세이(L. paracasei) MO-C1(기탁번호 : KACC 92325P) 균주의 계통도를 나타낸 것이다.Figure 3 shows a family diagram of the Lactobacillus paracasei (L. paracasei ) MO-C1 (accession number: KACC 92325P) strain.
본 발명의 일 실시예에 있어서, 상기 락토바실러스 플란타럼 (L. plantarum) HJ-Ki(기탁번호 : KACC 92324P) 균주는 서열번호 1로 표시되는 16s rRNA 염기서열을 포함하고, 상기 락토바실러스 퍼멘텀(L. fermentum) HJ-C1(기탁번호 : KACC 92322P) 균주는 서열번호 2로 표시되는 16s rRNA 염기서열을 포함하고, 상기 락토바실러스 파라카세이(L. paracasei) MO-C1(기탁번호 : KACC 92325P) 균주는 서열번호 3으로 표시되는 16s rRNA 염기서열을 포함할 수 있다.In one embodiment of the present invention, the Lactobacillus plantarum ( L. plantarum ) HJ-Ki (accession number: KACC 92324P) strain includes a 16s rRNA base sequence represented by SEQ ID NO: 1, and the Lactobacillus fur Mentum ( L. fermentum ) HJ-C1 (accession number: KACC 92322P) strain contains a 16s rRNA base sequence represented by SEQ ID NO: 2, and the Lactobacillus paracasei ( L. paracasei ) MO-C1 (accession number: KACC 92325P) strain may include the 16s rRNA base sequence represented by SEQ ID NO: 3.
상기 내산성과 관련하여, 본 발명에 따른 김치 발효용 프로바이오틱스 제제는 pH 3.0 내지 pH 3.3에서 1시간 경과 후, 79% 내지 80%의 생존률을 나타낼 수 있고, pH 3.0 내지 pH 3.3에서 2시간 경과 후, 79% 내지 86%의 생존률을 나타낼 수 있고, pH 3.0 내지 pH 3.3에서 3시간 경과 후, 79% 내지 85%의 생존률을 나타낼 수 있다.In relation to the acid resistance, the probiotic preparation for kimchi fermentation according to the present invention can exhibit a survival rate of 79% to 80% after 1 hour at pH 3.0 to pH 3.3, and after 2 hours at pH 3.0 to pH 3.3, It can show a survival rate of 79% to 86%, and after 3 hours at pH 3.0 to pH 3.3, it can show a survival rate of 79% to 85%.
본 발명은, 배추를 소금물에 절이는 단계, 양념을 준비하는 단계, 상기 양념에 락토바실러스 플란타럼 (L. plantarum) HJ-Ki(기탁번호 : KACC 92324P) 균주, 락토바실러스 퍼멘텀(L. fermentum) HJ-C1(기탁번호 : KACC 92322P) 균주, 및 락토바실러스 파라카세이(L. paracasei) MO-C1(기탁번호 : KACC 92325P) 균주 중 적어도 하나를 포함하는 프로바이오틱스 제제를 혼합하는 단계, 상기 소금물에 절여둔 상기 배추를 세척하고 물기를 제거하는 단계, 및 혼합된 상기 양념을 배추에 버무리는 단계를 포함하는 김치 제조방법을 제공한다.The present invention includes the step of pickling cabbage in salt water, the step of preparing seasoning, and the seasoning including Lactobacillus plantarum ( L. plantarum ) HJ-Ki (accession number: KACC 92324P) strain, Lactobacillus fermentum ( L. fermentum) ) mixing a probiotic preparation containing at least one of the HJ-C1 (accession number: KACC 92322P) strain and the Lactobacillus paracasei ( L. paracasei ) MO-C1 (accession number: KACC 92325P) strain, in the salt water It provides a kimchi manufacturing method including the steps of washing and removing moisture from the pickled cabbage, and mixing the mixed seasoning into the cabbage.
본 발명의 일 실시예에 있어서, 상기 양념은, 무 100 중량부 내지 150 중량부, 쪽파 50 중량부 내지 100 중량부를 준비하는 단계, 무 150 중량부 내지 200 중량부 및 양파 40 중량부 내지 60 중량부를 분쇄하여 준비하는 단계, 물과 찹쌀을 1:9(중량비) 내지 1:11(중량비)로 혼합하여 찹쌀풀을 준비하는 단계, 사과즙 30 중량부 내지 50 중량부에 고춧가루 80 중량부 내지 120 중량부, 까나리액젓 40 중량부 내지 50 중량부, 새우젓 30 중량부 내지 45 중량부, 매실액 20 중량부 내지 30 중량부, 다진 마늘 15 중량부 내지 25 중량부, 다진 생강 1.5 중량부 내지 2.5 중량부, 소금 5 중량부 내지 10 중량부, 건새우분말 20 중량부 내지 40 중량부, 멸치다시육수 140 중량부 내지 180 중량부, 및 상기 찹쌀풀 40 중량부 내지 60 중량부를 상기 무, 상기 쪽파, 분쇄한 상기 무, 분쇄한 상기 양파와 혼합하여 양념을 제조하는 단계, 및 상기 앙념을 20분 내지 40분 숙성시키는 단계를 포함할 수 있다.In one embodiment of the present invention, the seasoning includes preparing 100 to 150 parts by weight of radish, 50 to 100 parts by weight of chives, 150 to 200 parts by weight of radish, and 40 to 60 parts by weight of onion. Preparing by pulverizing the portion, preparing glutinous rice paste by mixing water and glutinous rice at a ratio of 1:9 (weight ratio) to 1:11 (weight ratio), 30 to 50 parts by weight of apple juice and 80 to 120 parts by weight of red pepper powder 40 to 50 parts by weight of canary fish sauce, 30 to 45 parts by weight of salted shrimp, 20 to 30 parts by weight of plum juice, 15 to 25 parts by weight of minced garlic, 1.5 to 2.5 parts by weight of minced ginger. , 5 to 10 parts by weight of salt, 20 to 40 parts by weight of dried shrimp powder, 140 to 180 parts by weight of anchovy broth, and 40 to 60 parts by weight of the glutinous rice paste, the radish, the chives, and the pulverized It may include preparing a seasoning by mixing the radish and the pulverized onion, and aging the seasoning for 20 to 40 minutes.
본 발명의 일 실시예에 있어서, 상기 김치 제조방법은 상기 양념에 버무린 상기 배추를 발효시키는 단계를 더 포함하며, 상기 발효는 김치 내 유산균을 제외한 일반 세균 대비 유산균 함량이 100배 이상이 되도록 6주 이내로 수행되는 것일 수 있다. 그러나 8주를 초과하지 않는 범위 내에서 발효를 더 수행할 수도 있다.In one embodiment of the present invention, the kimchi manufacturing method further includes the step of fermenting the cabbage mixed with the seasoning, and the fermentation is performed for 6 weeks so that the content of lactic acid bacteria in kimchi is 100 times or more compared to general bacteria excluding lactic acid bacteria. It may be carried out within a period of time. However, further fermentation may be carried out within a period not exceeding 8 weeks.
본 발명의 일 실시예에 있어서, 상기 발효하는 단계는 1℃ 내지 10℃에서 수행될 수 있다. 그러나 이에 한정되는 것은 아니며, 균주가 활성을 갖고 발효를 할 수 있는 온도 범위 내라면 1℃ 이하, 10℃ 이상에서 발효할 수도 있다.In one embodiment of the present invention, the fermentation step may be performed at 1°C to 10°C. However, it is not limited to this, and fermentation may be performed at 1℃ or lower and 10℃ or higher as long as the strain is active and within the temperature range at which fermentation can occur.
본 발명은, 상기 김치 제조방법으로 제조된 김치를 제공한다. The present invention provides kimchi prepared by the above kimchi manufacturing method.
이를 통해 락토바실러스 플란타럼 (L. plantarum) HJ-Ki(기탁번호 : KACC 92324P) 균주, 락토바실러스 퍼멘텀(L. fermentum) HJ-C1(기탁번호 : KACC 92322P) 균주, 및 락토바실러스 파라카세이(L. paracasei) MO-C1(기탁번호 : KACC 92325P) 균주 중 적어도 하나를 포함한 김치의 경우 상기 균주를 포함하지 않는 김치 대비 발효 8주차에 총 플라보노이드 함량이 우수한 김치를 제조할 수 있다. Through this, Lactobacillus plantarum ( L. plantarum ) HJ-Ki (accession number: KACC 92324P) strain, Lactobacillus fermentum (L. fermentum ) HJ-C1 (accession number: KACC 92322P) strain, and Lactobacillus paracasei ( L. paracasei ) In the case of kimchi containing at least one of the MO-C1 (accession number: KACC 92325P) strains, kimchi with excellent total flavonoid content can be produced at the 8th week of fermentation compared to kimchi that does not contain the above strain.
이에 더해, 락토바실러스 플란타럼 (L. plantarum) HJ-Ki(기탁번호 : KACC 92324P) 균주, 락토바실러스 퍼멘텀(L. fermentum) HJ-C1(기탁번호 : KACC 92322P) 균주, 및 락토바실러스 파라카세이(L. paracasei) MO-C1(기탁번호 : KACC 92325P) 균주 중 적어도 하나를 포함한 김치의 경우 상기 균주를 포함하지 않는 김치 대비 발효 6주차 내지 8주차에 DPPH 라디칼 소거능이 우수한 김치를 제조할 수 있다.In addition, Lactobacillus plantarum ( L. plantarum ) HJ-Ki (accession number: KACC 92324P) strain, Lactobacillus fermentum (L. fermentum ) HJ-C1 (accession number: KACC 92322P) strain, and Lactobacillus para. In the case of kimchi containing at least one of the L. paracasei MO-C1 (accession number: KACC 92325P) strains, kimchi with excellent DPPH radical scavenging ability can be produced in the 6th to 8th week of fermentation compared to kimchi not containing the above strain. there is.
본 발명은, 배추를 소금물에 절이는 단계, 양념을 준비하는 단계, 상기 양념에 락토바실러스 플란타럼 (L. plantarum) HJ-Ki(기탁번호 : KACC 92324P) 균주 및 락토바실러스 파라카세이(L. paracasei) MO-C1(기탁번호 : KACC 92325P) 균주 중 적어도 하나를 포함하는 프로바이오틱스 제제를 혼합하는 단계, 상기 소금물에 절여둔 상기 배추를 세척하고 물기를 제거하는 단계, 혼합된 상기 양념을 배추에 버무리는 단계, 및 상기 양념에 버무린 배추를 2주 내지 8주 동안 발효시키는 단계를 포함하는 신김치 제조방법을 제공한다.The present invention includes the steps of pickling cabbage in salt water, preparing a seasoning, and adding Lactobacillus plantarum ( L. plantarum ) HJ-Ki (accession number: KACC 92324P) strain and Lactobacillus paracasei ( L. paracasei) to the seasoning. ) MO-C1 (Accession number: KACC 92325P) mixing a probiotic preparation containing at least one of the strains, washing the cabbage pickled in salt water and removing moisture, mixing the mixed seasoning into the cabbage. It provides a method for producing sour kimchi, including the step of fermenting cabbage mixed with the seasoning for 2 to 8 weeks.
본 발명의 일 실시예에 있어서, 상기 발효시키는 단계는 4주 내지 8주 동안 수행되며, 상기 신김치의 총산도(%)는 0.7% 이상일 수 있다.In one embodiment of the present invention, the fermentation step is performed for 4 to 8 weeks, and the total acidity (%) of the sour kimchi may be 0.7% or more.
본 발명의 일 실시예에 있어서, 상기 발효하는 단계는 1℃ 내지 10℃에서 수행될 수 있다.In one embodiment of the present invention, the fermentation step may be performed at 1°C to 10°C.
본 발명은, 상기 신김치 제조방법으로 제조된 신김치를 제공한다.The present invention provides sour kimchi produced by the above sour kimchi production method.
이를 통해 락토바실러스 플란타럼 (L. plantarum) HJ-Ki(기탁번호 : KACC 92324P) 균주 또는 락토바실러스 파라카세이(L. paracasei) MO-C1(기탁번호 : KACC 92325P) 균주를 포함한 김치의 경우 상기 균주를 포함하지 않는 김치 대비 산도가 현저히 높으며, 이에 따른 신김치의 제조가 가능하다.Through this, in the case of kimchi containing Lactobacillus plantarum ( L. plantarum ) HJ-Ki (accession number: KACC 92324P) strain or Lactobacillus paracasei ( L. paracasei ) MO-C1 (accession number: KACC 92325P) strain, the above The acidity is significantly higher than that of kimchi that does not contain strains, making it possible to produce sour kimchi.
<실험예 1. 발효식품에서 락토바실러스 프로바이오틱스 선발><Experimental Example 1. Selection of Lactobacillus probiotics from fermented foods>
<실험예 1-1. 락토바실러스 예상 균주 스크리닝><Experimental Example 1-1. Screening of expected Lactobacillus strains>
시판되는 김치와 충북농업기술원 식품개발팀 연구원 가정에서 제조한 김장김치와 시판 살균하지 않는 막걸리 등 발효식품에서 유산균을 선발하고자 하였다. 현재 식약처에 고시되어 있는 프로바이오틱스는 대부분 락토바실러스이므로 락토바실러스 선택배지를 사용하여 1차적으로 자연발효조건에 다양한 균주에서 락토바실러스만 선발하였다. We attempted to select lactic acid bacteria from fermented foods such as commercially available kimchi, kimchi made at home by researchers at the Food Development Team of the Chungbuk Agricultural Research and Extension Services, and commercially available unsterilized makgeolli. Since most of the probiotics currently notified by the Ministry of Food and Drug Safety are Lactobacillus, only Lactobacillus was initially selected from various strains under natural fermentation conditions using Lactobacillus selection medium.
도 4는 김치, 막걸리, 효소액 등을 0.85 중량% NaCl로 희석하고 선택배지에 접종하고 배양하여 단일 콜로니를 얻고, 시퀀싱하는 과정을 개략적으로 도시한 것이다.Figure 4 schematically shows the process of diluting kimchi, makgeolli, enzyme solution, etc. with 0.85% by weight NaCl, inoculating and culturing a selective medium to obtain a single colony, and sequencing.
도 4를 참조하면, 시료(김치, 막걸리, 효소액 등)를 스토마커로 분쇄하여 배수별로 희석(0.85 중량% NaCl)하였다. 이후 락토바실러스 선택 배지(LBS-media; Lactobacillus selection media)와 MRS 배지(media)에 아세트산(acetic acid)을 첨가하여 배지를 준비하였다. 첫 배양에는 락토바실러스 선택 배지를 사용하였으며, 계대배양에는 MRS 배지(media)를 사용하였다. 배양은 37℃에서 24시간 동안 배양하고 단일 콜로니를 얻을 때까지 계대배양하였다. 순수한 단일 콜로니를 얻은 후 그람염색 한 후 현미경 통해서 그람양성, 단간균 또는 간균인 콜로니들만 수집하고 아닌 경우 리스트에서 제거하였다. 상기 조건에 따라 수집된 균주는 16S rRNA 시퀀싱을 수행하였다.Referring to Figure 4, samples (kimchi, makgeolli, enzyme solution, etc.) were ground with a stem marker and diluted by multiple (0.85% by weight NaCl). Afterwards, the medium was prepared by adding acetic acid to Lactobacillus selection media (LBS-media) and MRS media. Lactobacillus selection medium was used for the first culture, and MRS medium was used for subculture. Cultures were incubated at 37°C for 24 hours and subcultured until single colonies were obtained. After obtaining a pure single colony, Gram staining was performed. Only colonies that were Gram-positive, monobacilli, or bacilli were collected through a microscope, and if not, they were removed from the list. 16S rRNA sequencing was performed on the strains collected according to the above conditions.
<실험예 1-2. 락토바실러스 예상 균주 동정><Experimental Example 1-2. Identification of expected Lactobacillus strains>
실험예 1-1에서 분리한 락토바실러스 예상 균주를 OD(optical density)값이 빠르고 현미경으로 본 세포의 모양 등을 통해 선발한 결과, 34가지 균주가 선발되었다. 선발된 분리균은 16S rRNA의 염기서열의 분석을 통해 동정하였다. 프라이머는 유니버설 PCR 프라이머(Universal PCR primer)인 27F (5-’AGAGTTTGATCMTGGCTCAG-3’) 1492R (5’-TACGGYTACCTTGTTACGACTT-3’)을 사용하였고, PCR은 초기 변성(initial denaturation) 5분, 94℃에서 45초간 변성 (denaturation), 55℃에서 60초간 풀림(annealing) 및 72℃에서 60초간 확장 사이클(extension cycle)을 35회 수행하였다. PCR산물의 염기서열은 ㈜솔젠트 (Daejeon, Korea)에 의뢰하여 분석하였다. 계통도(Phylogenetic tree) 작성은 BLAST 네트워크 서비스(network service)와 미국 국립생물정보센터(National Center for Biotechnology Information)에서 얻은 다른 균종의 16s rRNA 유전자 염기서열을 이용하여 RDP(Ribosomal Database Project)의 시퀀스 매치 프로그램(seqMatch program)에서 서열 일치도가 높은 표준 균주들의 16S rRNA 유전자 염기서열들을 얻었다. 그리고 DDBJ(DNA Data Bank of Japan)를 통해 계통분류학적 유연관계를 분석한 후, 트리뷰(treeview)를 이용하여 이웃-결합 방법(neighbour-joining method)과 거리 매트릭스 데이터(distance matrix data)를 사용하여 확인하였다. As a result of selecting the expected Lactobacillus strains isolated in Experimental Example 1-1 based on the fast OD (optical density) value and the shape of the cells seen under a microscope, 34 strains were selected. The selected isolates were identified through analysis of the 16S rRNA base sequence. Primers used were the universal PCR primers 27F (5-'AGAGTTTGATCMTGGCTCAG-3') 1492R (5'-TACGGYTACCTTGTTACGACTT-3'), and PCR was performed with initial denaturation for 5 minutes at 94°C for 45 minutes. Denaturation for 60 seconds, annealing at 55°C for 60 seconds, and extension cycles at 72°C for 60 seconds were performed 35 times. The base sequence of the PCR product was analyzed by Solgent Co., Ltd. (Daejeon, Korea). Phylogenetic tree was created using the sequence match program of the RDP (Ribosomal Database Project) using 16s rRNA gene sequences of other fungal species obtained from the BLAST network service and the U.S. National Center for Biotechnology Information. The 16S rRNA gene sequences of standard strains with high sequence identity were obtained from (seqMatch program). After analyzing the phylogenetic relationships through DDBJ (DNA Data Bank of Japan), the neighbor-joining method and distance matrix data were used using treeview. It was confirmed.
표 1은 선발된 34가지 균주의 시퀀싱 결과를 나타낸 표이다.Table 1 is a table showing the sequencing results of the 34 selected strains.
표 1을 참조하면, 모든 균주가 락토바실러스(Lactobacillus) 속에 포함되어 있었으며 퍼멘텀(fermentum) 2종, 플란타럼(plantarum) 7종, 파라카세이(paracasei) 6종, 람노서스(rhamnosus) 4종, 브레비스(brevis) 5종, 사케이(sakei) 5종, 파라부쉬네리(parabuchneri) 3종, 컬바투스(curvatus) 2종 이었다.Referring to Table 1, all strains were included in the genus Lactobacillus , including 2 types of fermentum , 7 types of plantarum , 6 types of paracasei , and 4 types of rhamnosus . , 5 species of brevis , 5 species of sakei , 3 species of parabuchneri , and 2 species of curvatus .
<실험예 1-3. 내산성 측정><Experimental Example 1-3. Acid resistance measurement>
발효식품에서 분리한 유산균이 프로바이오틱스로서의 기능을 가지기 위해서는 소화관을 통과해서 장까지 생존해야 한다. 선택배지와 그람양성 등 현미경 관찰에서 수집한 균주를 소화관 중 위와 같은 환경에서 장까지 살아갈 수 있는 능력을 알아보기 위해 위산과 유사한 pH의 조건에서 내산성 확인을 하였다. 이를 위해 MRS 배지(broth)에 접종하여 37℃에서 3시간 배양방법으로 테스트하였다. 생존률 확인은 균주를 MRS 배지(broth) 20ml에 37℃ 24hr 배양 후 스프레딩(spreading) 하여 균주 수를 측정하는데, 염산(HCl)을 이용하여 pH 3.0로 보정하여 만든 MRS 배지(broth)에 1 중량% 접종하여 37℃의 온도 조건에서 1시간, 2시간, 3시간 배양 후 각각 스프레딩(spreading) 하여 균주 수를 측정하였다. In order for lactic acid bacteria isolated from fermented foods to function as probiotics, they must survive through the digestive tract and into the intestines. The acid resistance of strains collected through microscopic observation, including selection medium and Gram-positive strains, was checked under pH conditions similar to stomach acid to determine their ability to survive in the stomach-like environment in the digestive tract and into the intestines. For this purpose, it was inoculated into MRS broth and tested by culturing at 37°C for 3 hours. To check the survival rate, the number of strains is measured by culturing the strains in 20 ml of MRS broth at 37°C for 24 hours and then spreading them. 1 weight of the strain is added to MRS broth adjusted to pH 3.0 using hydrochloric acid (HCl). After inoculation and culturing for 1 hour, 2 hours, and 3 hours at a temperature of 37°C, the number of strains was measured by spreading.
생존률(%) = pH 3.0 조건에서 1시간 배양한 균주/ 균주 수 * 100 Survival rate (%) = strain cultured for 1 hour under pH 3.0 conditions/number of strains * 100
표 2는 표 1에서 선발한 34가지 균주의 내산성을 시간에 따라 측정하여 표로 나타낸 것이다.Table 2 shows the acid resistance of the 34 strains selected in Table 1 measured over time.
<실험예 1-4. 내담즙성 측정><Experimental Example 1-4. Measurement of bile tolerance>
표 3은 황소의 담즙이 포함된 배지에 균주를 배양하여 내담즙성 생존률(%)을 측정하여 표로 나타낸 것이다.Table 3 shows the biliary resistance survival rate (%) measured by culturing the strain in a medium containing bull bile.
표 3에서 내산성 실험 3시간 뒤에 70% 이상 생육을 보이는 균주 중 락토바실러스(Lactobacillus) 속의 종류와 내산성을 고려하여 19 균주를 선정하여 내담즙성 실험을 수행하였다. 구강을 통해 섭취된 유산균은 위액(내산성 실험)을 통과한 뒤 담즙이 존재하는 십이지장을 거쳐 최종 목적 부위는 장에 도달해야 그 기능적인 효과를 가지게 되므로 내담즙성 테스트는 매우 중요하다. 0.3 중량% 황소의 담즙(Difco사, oxgall)이 포함되어 있는 MRS 배지(broth)에 균주를 1 중량%(OD 값 일정) 접종하여 37℃의 온도조건에서 6시간 배양한 후 스프레딩(spreading) 하여 균수를 측정하였다.In Table 3, among the strains that showed growth of more than 70% after 3 hours of the acid resistance test, 19 strains were selected considering the type of Lactobacillus genus and acid resistance, and a bile resistance test was performed. The bile resistance test is very important because lactic acid bacteria ingested through the mouth must pass through gastric juice (acid resistance test), then pass through the duodenum where bile exists, and reach the final destination in the intestines to have a functional effect. 1% by weight (constant OD value) of the strain was inoculated into MRS broth containing 0.3% by weight bull's bile (Difco, oxgall), cultured for 6 hours at 37°C, and then spread. The number of bacteria was measured.
생존률(%) = 0.3 중량% 황소의 담즙(oxgall) 조건에서 6시간 배양한 균주/ 균주 수 * 100Survival rate (%) = 0.3% by weight strain cultured for 6 hours in oxgall conditions/number of strains * 100
표 3을 참조하면, 담즙 조건하에서 대부분의 균주가 생존율이 높은 경향성을 보였다. 프로바이오틱스 대조구로는 전세계에서 가장 많이 쓰이고 있는 락토바실러스 람노서스(Lactobacillus rhamnosus) LGG 균주를 사용하였다. 락토바실러스 람노서스(Lactobacillus rhamnosus) LGG 균주는 내담즙 생존률이 91%로 나타났고, 분리균주 중에서는 락토바실러스 플란타럼(L. plantarum) HJ-Ki의 내담즙 생존률이 96%로 가장 높은 값을 나타내었고, 대부분 80% 이상의 값을 나타내었다.Referring to Table 3, most strains tended to have high survival rates under bile conditions. As a probiotic control, the Lactobacillus rhamnosus LGG strain, which is most commonly used in the world, was used. Lactobacillus rhamnosus LGG strain showed an intrabiliary survival rate of 91%, and among the isolates, Lactobacillus plantarum ( L. plantarum ) The biliary survival rate of HJ-Ki was the highest at 96%, and most were over 80%.
<실험예 1-5. 김치모사배지에서 생육관찰><Experimental Example 1-5. Observation of growth on kimchi simulation medium>
내담즙성을 확인한 19가지의 분리균주 중 10개의 프로바이오틱스 균주만을 김치 종균으로 사용하기 위하여 다음 실험으로 김치와 같은 유사한 환경에서 배양하여 생장을 확인하였다. In order to use only 10 probiotic strains among the 19 isolates with confirmed bile resistance as kimchi starters, growth was confirmed by culturing them in a similar environment as kimchi in the following experiment.
본 실험에서 사용한 김치모사배지를 제조하기 위하여 배추 700g, 무 200g, 부추 50 g, 생강 10g, 마늘 20g, 굵은 소금 60g, 피쉬 펩톤(Fish peptone) 5g 멸균 증류수를 준비하였다. 각각의 채소는 세척하고 적당한 사이즈로 절단하여 준비한 주재료와 굵은 소금을 믹서기에 넣고 5분간 분쇄하였다. 분쇄된 분쇄물은 깔때기를 이용하여 1L 시약병에 채우고 냉장고에서 24시간 동안 절여서 영양성분을 식물조직에서 추출하였다. 상기 추출액에 피쉬 펩톤(Fish peptone) 5g을 첨가한 뒤 70℃의 진탕 항온 수조(shaking water bath)에서 30분간 저온 살균한 뒤 7000rpm, 10분 조건에서 원심분리하고 상층액을 분리하였다. 분리된 상층액은 멸균수로 2배 희석하여 적절한 크기의 멸균 용기에 옮겨 보관하면서 실험하였다.To prepare the kimchi simulation medium used in this experiment, 700 g of cabbage, 200 g of radish, 50 g of chives, 10 g of ginger, 20 g of garlic, 60 g of coarse salt, 5 g of fish peptone, and sterilized distilled water were prepared. Each vegetable was washed and cut into appropriate sizes. The prepared main ingredients and coarse salt were placed in a blender and ground for 5 minutes. The pulverized material was filled into a 1L reagent bottle using a funnel and pickled in the refrigerator for 24 hours to extract nutrients from plant tissue. 5 g of fish peptone was added to the extract, sterilized at 70°C in a shaking water bath for 30 minutes, centrifuged at 7000 rpm for 10 minutes, and the supernatant was separated. The separated supernatant was diluted two-fold with sterilized water, transferred to a sterile container of an appropriate size, and stored for testing.
MRS 배지에서 전배양한 대조구(류코노스톡 메센테로이데스(Leuconostoc mesenteroides) ATCC8293)와 앞서 분리한 10개 균주는 김치모사배지에 접종하여 8℃인큐베이터에서 5일간 배양하였고 24시간마다 흡광도를 측정하였다.The control group ( Leuconostoc mesenteroides ATCC8293) pre-cultured in MRS medium and the 10 previously isolated strains were inoculated into kimchi simulation medium and cultured in an 8°C incubator for 5 days, and the absorbance was measured every 24 hours.
도 5는 발효 기간에 따른 대조구와 10가지 균주의 흡광도(OD)값을 측정하여 나타낸 것이다.Figure 5 shows the measured absorbance (OD) values of the control and 10 strains according to the fermentation period.
도 5를 참조하면 흡광도 측정 결과 대조구는 김치와 유사한 환경에서 급속하게 생육하여 발효 4일차까지 흡광도가 증가하여 가장 높은 값을 나타내었다가 발효 5일차에는 급격하게 감소하였다. 분리균주는 중 락토바실러스 플란타룸(L. plantarum) HJ-Ki 균주가 김치모사배지에서 가장 우수한 생육을 보여 김치원재료 및 낮은 온도에서 종균으로 잘 사용이 가능할 것으로 판단되었다. 다음으로 락토바실러스 플란타룸(L. plantarum) Ho 및 락토바실러스 퍼멘텀(L. fermentum) HJ-C1 균주가 김치와 유사한 조건에서 잘 생육하였으며, 분리균주 중에는 락토바실러스 파라카세이(L. paracasei) MO-C1 균주의 생육은 발효 4일차 까지는 높은 활성을 유지하다가 발효 5일차에는 대조구 및 실험구 중 가장 낮은 값을 나타내었다. Referring to Figure 5, the absorbance measurement results showed that the control group grew rapidly in an environment similar to kimchi, and the absorbance increased until the 4th day of fermentation, reaching the highest value, and then sharply decreased on the 5th day of fermentation. Among the isolated strains, the Lactobacillus plantarum ( L. plantarum ) HJ-Ki strain showed the best growth on kimchi simulation medium and was judged to be suitable for use as a kimchi raw material and starter at low temperatures. Next, Lactobacillus plantarum ( L. plantarum ) Ho and Lactobacillus fermentum ( L. fermentum ) HJ-C1 strains grew well under conditions similar to kimchi, and among the isolates, Lactobacillus paracasei ( L. paracasei ) MO The growth of the -C1 strain maintained high activity until the 4th day of fermentation, but showed the lowest value among the control and experimental groups on the 5th day of fermentation.
따라서 내산성, 내담즙성 및 김치모사배지에서 가장 우수한 균주로는 락토바실러스 퍼멘텀(L. fermentum) HJ-C1, 락토바실러스 플란타룸(L. plantarum) HJ-Ki, 락토바실러스 플란타룸(L. plantarum) Ho, 락토바실러스 파라카세이(L. paracasei) MO-C1, 락토바실러스 람노서스(L. rhamnosus) B1-A 다섯균주를 최종 선정하였다.Therefore, the strains that have the best acid resistance, bile resistance, and kimchi simulation medium include Lactobacillus fermentum ( L. fermentum ) HJ-C1, Lactobacillus plantarum ( L. plantarum ) HJ-Ki, and Lactobacillus plantarum ( L . plantarum ) Ho, Lactobacillus paracasei ( L. paracasei ) MO-C1, and five strains of Lactobacillus rhamnosus ( L. rhamnosus ) B1-A were finally selected.
<실험예 1-6. 세포주 및 배양조건><Experimental Example 1-6. Cell lines and culture conditions>
쥐 유래 대식세포주인 RAW 264.7은 한국세포주은행(KCLB; Korean Cell Line Bank, Seoul, Korea)에서 분양받았으며, 세포주는 10 중량% FBS(fatal bovine serum, Gibco, Waltham, Massachusetts, USA)과 1 중량% 페니실린-스트렙토마이신(P/S; penicillin-streptomycin, GenDEPOT, Katy, TX, USA)이 첨가된 DMEM(Dulbecco's Modified Eagle Medium, HyClone, San Angelo, TX, USA) 배지에 넣고, 세포주는 37℃, 5% CO2 조건이 유지되는 배양기(Sanyo, Sakata, Japan)에서 배양하였다. 계대 배양은 세포가 플레이트에 80~90 중량% 정도 밀집(confluency)되었을 때 실시하였고 3~4번 계대 배양 후 실험에 이용하였다.RAW 264.7, a mouse-derived macrophage cell line, was purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea), and the cell line contained 10% by weight FBS (fatal bovine serum, Gibco, Waltham, Massachusetts, USA) and 1% by weight. The cell lines were placed in DMEM (Dulbecco's Modified Eagle Medium, HyClone, San Angelo, TX, USA) medium supplemented with penicillin-streptomycin (P/S; penicillin-streptomycin, GenDEPOT, Katy, TX, USA), and the cell lines were incubated at 37°C for 5 days. Cultured in an incubator (Sanyo, Sakata, Japan) maintained at % CO 2 conditions. Subculture was performed when the cells reached a confluency of 80 to 90% by weight on the plate, and were used for experiments after subculture 3 to 4 times.
<실험예 1-7. 항염증 활성평가><Experimental Example 1-7. Anti-inflammatory activity evaluation>
균주의 항염증 활성 평가는 LPS로 자극된 RAW 264.7 세포에서 가열 사멸된 균주의 효과를 측정하는 방식으로 수행하였다. RAW 264.7 세포(5×105cfu )를 96 웰 플레이트(well plate)에 접종하고 20시간 동안 배양하였다. 이후, 상등액을 모두 제거하고 FBS(fatal bovine serum)가 함유되지 않은 세럼-프리(serum-free) DMEM 160 μL와 시료 100 μL를 첨가한 이후 염증 유발 물질로 1 μg/mL의 대장균(Escherichia coli)으로부터 분리된 LPS(lipopolysaccharide from Escherichia coli, Sigma-Aldrich) 20 μL를 처리하고 24시간 동안 재배양하였다. 배양 후, 각 웰(well)의 배양 상층액을 동량의 그리스(Griess) 시약과 혼합하고 실온에서 15분 동안 암실에서 반응시킨 뒤 540nm에서 측정하였다. 아질산염 농도는 아질산나트륨의 희석액을 표준으로 하여 계산하였다.Evaluation of the anti-inflammatory activity of the strain was performed by measuring the effect of the heat-killed strain in RAW 264.7 cells stimulated with LPS. RAW 264.7 cells (5×10 5 cfu ) was inoculated into a 96 well plate and cultured for 20 hours. Afterwards, all supernatants were removed and 160 μL of serum-free DMEM without FBS (fatal bovine serum) and 100 μL of sample were added, followed by 1 μg/mL of Escherichia coli as an inflammatory substance. 20 μL of LPS (lipopolysaccharide from Escherichia coli , Sigma-Aldrich) isolated from was treated and re-cultured for 24 hours. After incubation, the culture supernatant from each well was mixed with an equal amount of Griess reagent, reacted in the dark at room temperature for 15 minutes, and measured at 540 nm. Nitrite concentration was calculated using diluted sodium nitrite as a standard.
도 6은 LPS(Lipopolysaccharide)의 처리에 따른, LPS(Lipopolysaccharide) 처리구, PC(Positive control), 및 7가지 균주의 NO 생성량을 나타낸 것이다.Figure 6 shows the NO production amount of LPS (Lipopolysaccharide) treatment group, PC (positive control), and 7 strains according to LPS (Lipopolysaccharide) treatment.
도 6을 참조하면, LPS 처리는 LPS를 처리하지 않은 대조군에 비해 NO 생성을 현저히 증가시켰다. 그러나 정조절(PC; positive control)로 NO 합성효소 억제제인 메틸 아르기닌(methyl arginine)을 처리한 경우, LPS군에 비해 NO생성을 용량 의존적으로 억제하였다. 또한 B1-A 균주를 제외한 실험된 모든 균주들은 LPS 그룹에 비해 NO생성에 대한 유의한 억제 활성을 나타내었다. 특히 H0 균주는 LGG 균주와 비슷한 활성을 나타내었고, HJ-C1 균주, HJ-Ki 균주, MO-Ci 균주는 WCFS1 균주 보다 더 유의한 억제 활성을 보였다.Referring to Figure 6, LPS treatment significantly increased NO production compared to the control group not treated with LPS. However, when treated with methyl arginine, an NO synthase inhibitor, as a positive control (PC), NO production was suppressed in a dose-dependent manner compared to the LPS group. Additionally, all strains tested except the B1-A strain showed significant inhibitory activity on NO production compared to the LPS group. In particular, the H0 strain showed similar activity to the LGG strain, and the HJ-C1 strain, HJ-Ki strain, and MO-Ci strain showed more significant inhibitory activity than the WCFS1 strain.
RAW 247.7 세포에서 염증을 유발하는 전구염증매개 효소인 COX-2(cyclooxygenase-2)의 발현양을 확인하기 위해 세포를 6개의 웰 플레이트(well plate)에 접종하고 24시간 배양하였다. 시료를 처리한 이후 LPS(1 μg/mL)를 처리하여 24시간 배양한 뒤, 배지를 제거하고 PBS로 세척한 후, 트리졸 RNA 분리 시약(Trizol RNA isolation reagent)(Invitrogen, Carlsbad, USA)으로 RNA를 추출하였다. 리젠 익스프레스 제1 스트랜드 cDNA 합성 키트(LeGene Express 1st Strand cDNA Synthesis System Kit)(LeGene Biosciences, San Diego, CA, USA)를 사용하여 적절한 양의 RNA를 cDNA로 역전사 시켰다. COX-2의 발현 정도는 합성된 cDNA, 10pmol의 특정 프라이머 및 SYBR 그린 믹스(SYBR Green Mix)를 포함하는 PCR 마스터 믹스(PCR Master Mix)를 CFX96 실시간 PCR(CFX96 real-time PCR)(Bio-Rad, CA, USA)를 이용하여 실시간 유전자 정량 분석하였다. To check the expression level of COX-2 (cyclooxygenase-2), a pro-inflammatory mediating enzyme that causes inflammation, in RAW 247.7 cells, cells were inoculated into 6 well plates and cultured for 24 hours. After processing the sample, it was treated with LPS (1 μg/mL) and cultured for 24 hours, the medium was removed, washed with PBS, and then isolated with Trizol RNA isolation reagent (Invitrogen, Carlsbad, USA). RNA was extracted. An appropriate amount of RNA was reverse transcribed into cDNA using the LeGene Express 1st Strand cDNA Synthesis System Kit (LeGene Biosciences, San Diego, CA, USA). The expression level of COX-2 was measured using CFX96 real-time PCR (Bio-Rad) using PCR Master Mix containing synthesized cDNA, 10 pmol of specific primers, and SYBR Green Mix. , CA, USA) was used for real-time quantitative gene analysis.
도 7은 LPS(Lipopolysaccharide)의 처리에 따른, 대조구, LPS(Lipopolysaccharide) 처리구, 및 7가지 균주의 COX-2 발현 수준을 나타낸 것이다.Figure 7 shows the COX-2 expression levels of the control group, LPS (Lipopolysaccharide) treated group, and 7 strains according to LPS (Lipopolysaccharide) treatment.
상기 도 7은 GAPDH(Glyceraldehyde-3-phosphate dehydrogenase)를 참조 유전자로 하여 표준화(normalization)한 후 결과를 분석하여 그래프로 나타내었다. 표적 유전자의 상대적 발현 수준은 델타-델타 Ct(ΔΔCt) 방법으로 계산하였다. LPS 처리는 LPS 처리를 하지 않은 대조군에 비해 COX-2의 mRNA 발현 수준을 극적으로 증가시켰다. 실험된 모든 균주는 LPS 그룹에 비해 COX-2의 발현 수준을 유의하게 억제하였다. 이러한 결과는 균주의 처리가 상업적인 프로바이오틱스인 WCFS1 및 LGG와 유사하게 COX-2의 발현을 억제하는 활성을 가지고 있기 때문에 PGE2의 생성을 억제할 수 있음을 나타낸다.FIG. 7 shows a graph showing the analysis of the results after normalization using GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) as a reference gene. The relative expression levels of target genes were calculated using the delta-delta Ct (ΔΔCt) method. LPS treatment dramatically increased the mRNA expression level of COX-2 compared to the control group without LPS treatment. All tested strains significantly suppressed the expression level of COX-2 compared to the LPS group. These results indicate that treatment of the strain can inhibit the production of PGE2 because it has the activity of inhibiting the expression of COX-2, similar to commercial probiotics WCFS1 and LGG.
<실험예 1-8. 항산화 활성 측정><Experimental Example 1-8. Measurement of antioxidant activity>
앞서 실험한 5가지 분리균주의 프로바이오틱스로의 기능을 알아보기 위하여 다음 실험으로 항산화 활성 실험을 수행하였다. MRS 배지에서 키운 유산균들은 600nm 흡광도에서 배양액의 OD값이 1이 되도록 설정한 후 상기 배양액을 원심분리하여 상등액을 사용하였다. In order to investigate the function of the five isolated strains tested previously as probiotics, an antioxidant activity test was performed in the following experiment. For lactic acid bacteria grown in MRS medium, the OD value of the culture medium was set to 1 at an absorbance of 600 nm, and then the culture medium was centrifuged and the supernatant was used.
DPPH 활성은 0.4mM DPPH 용액을 흡광도 값이 1.3 내지 1.4가 되도록 배양액(10배로 희석)을 0.2mL에 DPPH(Sigma-Aldrich, USA) 용액 0.8mL를 가한 후 실온에서 30분간 방치 후, 분광광도계(Cary UV- Vis spectrophotometer, Agilent Technologies, Santa Clara, CA, USA)를 사용하여 525nm에서 흡광도를 측정하였다. 흡광도를 측정할 때 셀에 분주되는 각 시료에 의한 흡광도의 차이는 증류수만의 흡광도를 측정하여 보정해 주었고, 이때 전자공여능은 시료 첨가구와 비첨가구의 흡광도 차이를 백분율(%)로 구하였다.DPPH activity was determined by adding 0.8 mL of DPPH (Sigma-Aldrich, USA) solution to 0.2 mL of culture medium (diluted 10 times) so that the absorbance value of 0.4mM DPPH solution was 1.3 to 1.4, leaving it at room temperature for 30 minutes, and measuring it with a spectrophotometer ( Absorbance was measured at 525 nm using a Cary UV-Vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). When measuring absorbance, the difference in absorbance due to each sample dispensed into the cell was corrected by measuring the absorbance of distilled water alone, and the electron donating ability was calculated as a percentage (%) of the difference in absorbance between the sample added and the sample not added.
또한, ABTS 양이온 탈색 분석(ABTS＋cation decolorization assay) 방법에 의하여 측정하였다. 7.4mM의 2,2’-아지노-비스-(3-에틸벤조티아졸린-6-설포닉산)(2,2'-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid))(ABTS, Sigma-Aldrich, USA) 와 2.6mM의 포타슘 퍼설페이트(potassium persulfate)(Sigma-Aldrich, USA)를 하루 동안 암소에 방치하여 ABTS 양이온을 형성시켰다. 이 용액을 735nm에서 흡광도 값이 1.3 내지 1.4가 되도록 몰 흡광계수(ε＝3.6×104M－1cm－1)를 이용하여 증류수로 희석하였다. 희석된 ABTS용액 1mL에 10배로 희석한 배양액 상등액 50㎕를 가하여 735nm 흡광도에서의 변화를 정확히 30분 후에 측정하였으며, ABTS 라디칼의 소거활성은 시료 첨가구와 시료를 첨가하지 않은 경우의 흡광도를 백분율로 나타내었다.Additionally, it was measured using the ABTS+cation decolorization assay. 7.4mM of 2,2'-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, Sigma) -Aldrich, USA) and 2.6mM potassium persulfate (Sigma-Aldrich, USA) were left in the dark for one day to form ABTS cations. This solution was diluted with distilled water using a molar extinction coefficient (ε = 3.6×10 4 M −1 cm −1 ) so that the absorbance value was 1.3 to 1.4 at 735 nm. 50 ㎕ of culture supernatant diluted 10 times was added to 1 mL of diluted ABTS solution, and the change in absorbance at 735 nm was measured exactly 30 minutes later. The scavenging activity of ABTS radicals was expressed as a percentage of the absorbance when the sample was added and when no sample was added. It was.
도 8은 PC(Positive control) 및 6가지 균주의 ABTS 라디칼 소거능 및 DPPH 라디칼 소거능을 나타낸 그래프이다.Figure 8 is a graph showing the ABTS radical scavenging ability and DPPH radical scavenging ability of PC (positive control) and six strains.
도 8에서 PC(Positive control)로는 1 중량% 비타민C를 사용하였고, 또 다른 대조구로는 김치 스타터로 사용할 수 있는 류코노스톡 메센테로이데스(Leuconostoc mesenteroides) 균주를 사용하였을 때, ABTS 라디컬 소거능의 경우는 대조구 류코노스톡(Leuconostoc) 속에 비해 락토바실러스 퍼멘텀(L. fermentum) HJ-C1과 락토바실러스 플란타럼(L. plantarum) HJ-Ki 두 균주가 더 높은 값을 나타나내었고, DPPH 라디컬 소거능은 류코노스톡(Leuconostoc) 균주보다 모든 분리균주가 높은 값을 나타내 항산화능이 우수한 것으로 나타났다.In Figure 8, 1% by weight vitamin C was used as PC (positive control), and when Leuconostoc mesenteroides strain, which can be used as a kimchi starter, was used as another control, the ABTS radical scavenging ability In this case, compared to the control Leuconostoc genus, the two strains Lactobacillus fermentum ( L. fermentum ) HJ-C1 and Lactobacillus plantarum ( L. plantarum ) HJ-Ki showed higher values, and DPPH radii The curl-scavenging ability of all isolates was higher than that of the Leuconostoc strain, showing excellent antioxidant ability.
<실험예 2. 프로바이오틱스를 종균으로 첨가한 김치의 품질조사><Experimental Example 2. Quality investigation of kimchi with probiotics added as a starter>
실험예 1에서 최종적으로 우수한 락토바실러스 퍼멘텀(Lactobacillus fermentum) HJ-C1, 락토바실러스 플란타럼(L. plantarum) HJ-Ki, 락토바실러스 파라카세이(L. paracasei) MO-C1 균주를 김치에 첨가하여 발효과정에 따라 김치의 품질을 조사하였다. In Experimental Example 1, excellent Lactobacillus fermentum ( Lactobacillus fermentum ) HJ-C1, Lactobacillus plantarum ( L. plantarum ) HJ-Ki, and Lactobacillus paracasei ( L. paracasei ) MO-C1 strains were finally added to kimchi. The quality of kimchi was investigated according to the fermentation process.
<실험예 2-1. 김치 제조><Experimental Example 2-1. Kimchi manufacturing>
최종 우수 유산균 3종(락토바실러스 퍼멘텀(Lactobacillus fermentum) HJ-C1, 락토바실러스 플란타럼(L. plantarum) HJ-Ki, 락토바실러스 파라카세이(L. paracasei) MO-C1 균주)과 유산균을 종균으로 첨가하지 않는 일반 김치는 동일한 방법으로 준비하였다. 김치용 배추(1,200g)는 적당한 크기로 썰어 소금물(75g/500mL)에 1시간 절이고, 양념재료에 들어갈 찹쌀풀(찹쌀1: 물10)을 쑤어 식혀두었다. 무(138g)는 채를 썰고 쪽파(75g)는 3cm 길이로 썰어 준비하였다. 양념을 만들기 위하여 무(173g), 양파(47g)는 갈고, 사과는 즙(39g)에 고춧가루(100g), 까나리액젓(45g), 새우젓(38g), 매실액(25g), 다진마늘(20g), 다진생강(2g), 소금(8g), 건새우분말(30g), 식혀둔 찹쌀풀(50g), 멸치다시육수(160g)를 섞어 양념을 30분가량 숙성시켰다. 절여둔 배추를 3번 씻어 30분 동안 물을 뺀 배추에 채썬 무, 쪽파, 양념을 넣어 버무렸다.The final three types of excellent lactic acid bacteria (Lactobacillus fermentum ( Lactobacillus fermentum ) HJ-C1, Lactobacillus plantarum ( L. plantarum ) HJ-Ki, Lactobacillus paracasei ( L. paracasei ) MO-C1 strain) and lactic acid bacteria were sown. Regular kimchi without any additives was prepared in the same way. Cabbage for kimchi (1,200g) was cut into appropriate sizes and pickled in salt water (75g/500mL) for 1 hour. Then, the glutinous rice paste (1 part glutinous rice to 10 parts water) to be added to the seasoning was added and left to cool. Radish (138g) was sliced and chives (75g) were prepared by cutting them into 3cm lengths. To make the seasoning, grind radish (173g) and onion (47g), juice of apple (39g), red pepper powder (100g), canary fish sauce (45g), salted shrimp (38g), plum juice (25g), and minced garlic (20g). , minced ginger (2g), salt (8g), dried shrimp powder (30g), cooled glutinous rice paste (50g), and anchovy broth (160g) were mixed and the seasoning was aged for about 30 minutes. Wash the pickled cabbage three times, drain the water, and mix with shredded radish, chives, and seasoning for 30 minutes.
제조된 김치를 대조구, 실험구로 나누었다. 대조구에는 유산균을 첨가하지 않고, 나머지 실험구에는 락토바실러스 퍼멘텀(Lactobacillus fermentum) HJ-C1, 락토바실러스 플란타럼(L. plantarum) HJ-Ki, 락토바실러스 파라카세이(L. paracasei) MO-C1 균주를 각각 첨가하였다. 유산균은 김치모사배지에서 배양한 후 600nm 흡광도에서 1에 맞춰서 준비하고 배추 전체 무게 대비 5% 중량비로 준비하였다. 각각의 유산균을 첨가한 뒤 4℃에서 발효하면서 2주 간격으로 실험분석하였다.The manufactured kimchi was divided into control group and experimental group. No lactic acid bacteria were added to the control group, and the remaining experimental groups included Lactobacillus fermentum HJ-C1, Lactobacillus plantarum ( L. plantarum ) HJ-Ki, and Lactobacillus paracasei ( L. paracasei ) MO-C1. Each strain was added. Lactic acid bacteria were cultured in kimchi-mimicking medium, then prepared at an absorbance of 1 at 600 nm, and prepared at a weight ratio of 5% compared to the total weight of the cabbage. After adding each lactic acid bacteria, the samples were fermented at 4°C and analyzed at two-week intervals.
<실험예 2-2. pH 및 산도 측정><Experimental Example 2-2. pH and acidity measurement>
김치의 pH 측정은 시료 20g에 증류수 180mL를 넣고, 교반기를 사용하여 1시간 균질화 시키고 현탁액을 감압여과하여 얻은 여액을 pH 미터(pH meter)(Sartorius AG, Gottingen, Germany)를 사용하여 측정하였다. 산도는 AOAC(1995) 방법에 따라 추출액 10 mL에 1 중량% 페놀프탈레인(phenolphthalein)을 지시약으로 하여 0.1 N NaOH용액으로 중화 적정하여 젖산(lactic acid)으로 나타내었다. The pH of kimchi was measured by adding 180 mL of distilled water to 20 g of the sample, homogenizing it for 1 hour using a stirrer, filtering the suspension under reduced pressure, and measuring the filtrate using a pH meter (Sartorius AG, Gottingen, Germany). Acidity was expressed as lactic acid by neutralizing and titrating 10 mL of the extract with 0.1 N NaOH solution using 1% by weight phenolphthalein as an indicator according to the method of AOAC (1995).
표 4는 제조한 4가지 김치의 pH를 측정하여 표로 나타낸 것이다.Table 4 shows the measured pH of the four types of kimchi produced.
표 4를 참조하면, A 내지 D 김치의 0주차 pH는 서로 유사하였으나, 발효시간이 경과할수록 B 및 D 김치는 pH가 급격하게 낮아져 초기 pH 6.45에서 pH 4.12까지 줄어들었고, pH 6.46에서 pH 4.03까지 낮아졌으나 8주가 지나도 대조구와 C 실험구는 pH 5 이상을 유지하였다.표 5는 제조한 4가지 김치의 총산도(%)를 측정하여 표로 나타낸 것이다.Referring to Table 4, the pH at week 0 of A to D kimchi was similar to each other, but as fermentation time elapsed, the pH of B and D kimchi decreased rapidly, from the initial pH of 6.45 to pH 4.12, and from pH 6.46 to pH 4.03. It decreased, but even after 8 weeks, the control group and experimental group C maintained pH above 5. Table 5 shows the measured total acidity (%) of the four types of kimchi produced.
표 5를 참조하면, A 내지 D 김치의 0주차 산도는 0.14% 내지 0.17%로 비슷한 수준이었으나, 2주차 이후, B 및 D 김치의 산도는 대조구(A) 대비 현저하게 높은 산도를 유지하였다. 특히, 발효 8주차의 B 및 D 김치는 각각 0.88% 및 1.0%의 총산을 나타냈는 바, 대조구인 A 김치의 총산인 0.59%보다 현저하게 높은 총산을 나타내었다.Referring to Table 5, the acidity of the A to D kimchi at week 0 was at a similar level of 0.14% to 0.17%, but after the second week, the acidity of the B and D kimchi maintained a significantly higher acidity compared to the control (A). In particular, B and D kimchi in the 8th week of fermentation showed total acids of 0.88% and 1.0%, respectively, which were significantly higher than the total acid of 0.59% in control A kimchi.
<실험예 2-3. 미생물 조사><Experimental Example 2-3. Microbial Investigation>
유산균을 첨가한 김치의 미생물학적 품질평가를 위해 일반 세균 수와 유산균 수를 분석하였다. 미생물 검사를 위해 김치 시료 1 g을 0.85 중량% 멸균 생리식염수에 단계적으로 희석하여 제조하였으며, 총 균수는 PCA(plate count agar)(Difco, Sparks, MD, USA)를 사용하고, 유산균 수는 MRS 배지(MRS agar)(Difco, Detroit, MI, USA)를 사용하여 37℃에서 24시간 배양한 후, 생성된 집락수를 계산하고 그 평균 집락수에 희석배수를 곱하여 균수를 산출하였다.To evaluate the microbiological quality of kimchi containing lactic acid bacteria, the number of general bacteria and lactic acid bacteria were analyzed. For microbial testing, 1 g of kimchi sample was prepared by stepwise dilution in 0.85% by weight sterilized saline solution. The total number of bacteria was determined using PCA (plate count agar) (Difco, Sparks, MD, USA), and the number of lactic acid bacteria was determined using MRS medium. After culturing at 37°C for 24 hours using (MRS agar) (Difco, Detroit, MI, USA), the number of colonies produced was calculated and the average number of colonies was multiplied by the dilution factor to calculate the number of bacteria.
표 6은 발효 기간에 따른 A 내지 D 김치의 세균의 총 수(log CFU/g)와 유산균의 수(log CFU/g)를 나타낸 표이다.Table 6 is a table showing the total number of bacteria (log CFU/g) and the number of lactic acid bacteria (log CFU/g) of A to D kimchi according to the fermentation period.
cell
countTotal
cell
count
acid
bacteriaLactic
acid
bacteria
표 6을 참조하면, A 내지 D 김치의 일반세균과 유산균 수를 측정한 결과 일반세균은 대조구의 경우 증가하다가 감소하였지만, 나머지 유산균 첨가 김치는 발효기간별 천천히 감소하였으며, 발효 8주차에는 대조구 및 유산균 첨가구 유사한 값을 나타냈다. 하지만 유산균 수의 차이는 달랐는데, 첫날에는 대조구의 경우 5.45 log CFU/g 였으나 유산균 첨가 실험구는 7.59 log CFU/g 내지 7.82 log CFU/g로 약 2 log배(100배) 차이가 났으나, 발효가 진행되면서 대조구의 유산균 수도 증가하여 발효 8주차에는 유사한 값을 나타내었다. 대조구의 경우 6주간은 거의 변화가 없었다가 8주차에 급격히 유산균이 증가한 특징이 있다. 따라서 초기 0주 내지 6주간은 종균으로 첨가한 김치의 경우 100배 이상의 유산균이 더 많이 존재하였다.Referring to Table 6, as a result of measuring the number of general bacteria and lactic acid bacteria in A to D kimchi, the general bacteria increased and then decreased in the control, but the remaining kimchi with lactic acid bacteria decreased slowly according to the fermentation period, and in the 8th week of fermentation, the number of general bacteria in the control and lactic acid bacteria added decreased slowly. Households showed similar values. However, the difference in the number of lactic acid bacteria was different. On the first day, the control group was 5.45 log CFU/g, but the experimental group with lactic acid bacteria added was 7.59 log CFU/g to 7.82 log CFU/g, a difference of about 2 log (100 times). As the process progressed, the number of lactic acid bacteria in the control group increased, reaching similar values in the 8th week of fermentation. In the case of the control group, there was little change for 6 weeks, but the number of lactic acid bacteria increased sharply in the 8th week. Therefore, for the first 0 to 6 weeks, more than 100 times more lactic acid bacteria were present in the kimchi added as a starter.
<실험예 2-4. 생리활성 조사><Experimental Example 2-4. Physiological activity investigation>
김치시료 10g과 탈이온수(DW) 90ml을 샘플백에 넣어 30분간 스토마커로 추출하였으며, 추출물 50 ㎕에 2 중량% Na2CO3 1 mL를 혼합하여 3분 방치하여 50 중량% 폴린-시오칼투(Folin-Ciocalteu’s) 페놀 시약(Sigma-Aldrich, St. Louis, MO, USA) 50 ㎕를 혼합하여 1시간 반응시킨 후, 750 nm에서 흡광도 값을 측정하였다. 표준물질로 갈산(gallic acid, Sigma-Aldrich, USA)을 사용하여 검량선을 작성하였고, 추출물 중의 mg 갈산 등가물(gallic acid equivalent)(GAE, dry basis)로 나타내었다. 10 g of kimchi sample and 90 ml of deionized water (DW) were placed in a sample bag and extracted with a stem marker for 30 minutes. 50 ㎕ of the extract was mixed with 1 mL of 2% by weight Na 2 CO 3 and left for 3 minutes to obtain 50% by weight of Pauline-Ciocaltu. (Folin-Ciocalteu's) 50 ㎕ of phenol reagent (Sigma-Aldrich, St. Louis, MO, USA) was mixed and reacted for 1 hour, and then the absorbance value was measured at 750 nm. A calibration curve was prepared using gallic acid (Sigma-Aldrich, USA) as a standard material, and expressed as mg gallic acid equivalent (GAE, dry basis) in the extract.
표 7은 발효 기간에 따른 A 내지 D 김치의 총 폴리페놀 함량, 총 플라보노이드 함량, 및 DPPH 라디칼 소거능을 나타낸 표이다.Table 7 is a table showing the total polyphenol content, total flavonoid content, and DPPH radical scavenging ability of kimchi A to D according to the fermentation period.
polyphenol
(mg%)Total
polyphenols
(mg%)
(㎍/㎕)Total flavonoids
(㎍/㎕)
표 7을 참조하면, 대조구에 비해 유산균을 첨가한 김치의 총 폴리페놀 함량이 다소 높게 나타났으며, 특히 락토바실러스 파라카세이(L. paracasei) MO-C1(D) 첨가 김치는 발효기간이 경과할수록 총 폴리페놀 함량의 값이 높아졌다.Referring to Table 7, the total polyphenol content of kimchi added with lactic acid bacteria was slightly higher than that of the control, and in particular, the kimchi added with Lactobacillus paracasei ( L. paracasei ) MO-C1(D) increased as the fermentation period elapsed. The value of total polyphenol content increased.
총 플라보노이드 함량은 1:1로 희석한 김치시료 200 μL에 1 N NaOH 600 μL와 디에틸렌글리콜(diethylene glycol) 4 mL를 가하여 37℃에서 1시간 반응시킨 후 반응액의 흡광도 값을 420 nm에서 측정하였고, 표준물질로는 루틴(rutin)(Sigma-Aldrich Co.)을 이용하여 검량선을 작성하여 총 플라보노이드 함량을 구하였다. 총 플라보노이드의 함량은 총 폴리페놀 함량과 같은 경향성을 보였는데 특히 모든 샘플에서 발효가 경과될수록 총 플라보노이드 함량의 값이 증가하였고 락토바실러스 파라카세이(L. paracasei) MO-C1(D)>락토바실러스 플란타럼(L. plantarum) HJ-Ki(B)>락토바실러스 퍼멘텀(L. fermentum) HJ-C1(C)>대조구 순으로 나타났다.The total flavonoid content was determined by adding 600 μL of 1 N NaOH and 4 mL of diethylene glycol to 200 μL of the kimchi sample diluted 1:1, reacting at 37°C for 1 hour, and measuring the absorbance value of the reaction solution at 420 nm. A calibration curve was prepared using rutin (Sigma-Aldrich Co.) as a standard material to determine the total flavonoid content. The total flavonoid content showed the same trend as the total polyphenol content. In particular, the value of total flavonoid content increased as fermentation progressed in all samples, and Lactobacillus paracasei ( L. paracasei ) MO-C1(D)> Lactobacillus p. It appeared in the following order: L. plantarum HJ-Ki (B) > Lactobacillus fermentum ( L. fermentum ) HJ-C1 (C) > control.
8주차에서 총 플라보노이드 함량은 락토바실러스 플란타럼(L. plantarum) HJ-Ki(B) 균주를 첨가한 김치에서 750.78(㎍/㎕), 락토바실러스 퍼멘텀(L. fermentum) HJ-C1(C) 균주를 첨가한 김치에서 712.33(㎍/㎕), 락토바실러스 파라카세이(L. paracasei) MO-C1(D) 균주를 첨가한 김치에서 775.78(㎍/㎕)을 나타내어 679.44(㎍/㎕)의 플라보노이드 함량을 나타낸 대조구 대비 높은 수치를 나타내었다.At week 8, the total flavonoid content was 750.78 (㎍/㎕) in kimchi containing Lactobacillus plantarum ( L. plantarum ) HJ-Ki (B) strain, Lactobacillus fermentum ( L. fermentum ) Kimchi containing the HJ-C1 (C) strain showed 712.33 (㎍/㎕), and Kimchi containing the Lactobacillus paracasei ( L. paracasei ) MO-C1 (D) strain showed 775.78 (㎍/㎕), showing 679.44 ( The flavonoid content was higher than that of the control group (㎍/㎕).
DPPH 라디칼 소거활성은 0.4 mM DPPH 용액을 흡광도 값이 1.3 내지 1.4가 되도록 제조한 김치시료 추출액(20배로 희석)을 0.2 mL에 DPPH(Sigma-Aldrich, USA) 용액 0.8 mL를 가한 후 실온에서 30분간 방치 후, 분광광도계(Cary UV- Vis spectrophotometer, Agilent Technologies, Santa Clara, CA, USA)를 사용하여 525 nm에서 흡광도를 측정하였다. 흡광도를 측정할 때 셀에 분주되는 각 시료에 의한 흡광도의 차이는 증류수만의 흡광도를 측정하여 보정해 주었고, 이때 전자공여능은 시료 첨가구와 비첨가구의 흡광도 차이를 백분율(%)로 구하였다.DPPH radical scavenging activity was obtained by adding 0.8 mL of DPPH (Sigma-Aldrich, USA) solution to 0.2 mL of kimchi sample extract (diluted 20-fold) prepared so that the absorbance value of 0.4 mM DPPH solution was 1.3 to 1.4, and then incubated at room temperature for 30 minutes. After standing, the absorbance was measured at 525 nm using a spectrophotometer (Cary UV-Vis spectrophotometer, Agilent Technologies, Santa Clara, CA, USA). When measuring absorbance, the difference in absorbance due to each sample dispensed into the cell was corrected by measuring the absorbance of distilled water alone, and the electron donating ability was calculated as a percentage (%) of the difference in absorbance between the sample added and the sample not added.
그 결과를 표 7에 나타내었다. 표 7을 참조하면, 총 폴리페놀, 총 플라보이드 함량에 이어 DPPH 라디컬 소거능도 락토바실러스 파라카세이(L. paracasei) MO-C1(D)>락토바실러스 플란타럼(L. plantarum) HJ-Ki(B)>락토바실러스 퍼멘텀(L. fermentum) HJ-C1(C)>대조구 순으로 나타났다. The results are shown in Table 7. Referring to Table 7, following the total polyphenol and total flavonoid content, the DPPH radical scavenging activity is Lactobacillus paracasei ( L. paracasei ) MO-C1(D)>Lactobacillus plantarum ( L. plantarum ) HJ-Ki (B)>Lactobacillus fermentum ( L. fermentum ) Appeared in the order HJ-C1(C)>Control.
4주차 까지는 락토바실러스 플란타럼(L. plantarum) HJ-Ki(B), 락토바실러스 퍼멘텀(L. fermentum) HJ-C1(C), 락토바실러스 파라카세이(L. paracasei) MO-C1(D) 균주 첨가구의 DPPH 라디칼 소거능과 대조구의 DPPH 라디칼 소거능의 차이가 미미하였으나, 발효 6주차 내지 8주차에는 대조구 대비 높은 DPPH 라디칼 소거능을 나타내었다.Until the 4th week, Lactobacillus plantarum ( L. plantarum ) HJ-Ki (B), Lactobacillus fermentum ( L. fermentum ) The difference between the DPPH radical scavenging ability of HJ-C1(C) and Lactobacillus paracasei ( L. paracasei ) MO-C1(D) strains and the control group was slight, but in the 6th to 8th week of fermentation, the DPPH radical scavenging ability was higher than that of the control group. DPPH radical scavenging ability was shown.
<실험예 2-5. 김치 기호도 평가><Experimental Example 2-5. Kimchi preference evaluation>
제조한 A 내지 D 김치의 기호도 평가를 진행하였다. 색, 향, 신맛, 짠맛, 매운맛, 식감 및 전반적인 기호도를 9점 척도법으로 분석하였다.The preference of the manufactured A to D kimchi was evaluated. Color, aroma, sourness, saltiness, spiciness, texture, and overall preference were analyzed using a 9-point scale.
표 8은 A 내지 D 김치의 기호도 평가를 진행한 결과를 나타낸 표이다.Table 8 is a table showing the results of evaluating the preference of kimchi A to D.
표 8을 참조하면, 기호도 평가에서 향과 전반적인 기호도에서 락토바실러스 퍼멘텀(L. fermentum) HJ-C1을 첨가한 C 김치가 가장 좋은 평가를 받았는데, 이는 낮은 산도와 종균이 이형 젖산발효 균주로서 당을 발효하여 젖산뿐만 아니라 초산, 탄산 및 다양한 대사산물을 내어 기호도가 좋았을 것으로 판단된다.Referring to Table 8, in the preference evaluation, C kimchi with Lactobacillus fermentum ( L. fermentum ) HJ-C1 received the best evaluation in terms of flavor and overall preference, as it has low acidity and the starter is a heterozygous lactic acid fermentation strain. It is believed that fermentation produced not only lactic acid but also acetic acid, carbonic acid, and various metabolites, making it highly palatable.
이상의 설명은 본 발명의 기술 사상을 예시적으로 설명한 것에 불과한 것으로서, 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자라면 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 다양한 수정 및 변형이 가능할 것이다. 따라서, 본 발명에 개시된 실시예들은 본 발명의 기술 사상을 한정하기 위한 것이 아니라 설명하기 위한 것이고, 이러한 실시예에 의하여 본 발명의 기술 사상의 범위가 한정되는 것은 아니다. 본 발명의 보호 범위는 아래의 청구범위에 의하여 해석되어야 하며, 그와 동등한 범위 내에 있는 모든 기술 사상은 본 발명의 권리범위에 포함되는 것으로 해석되어야 할 것이다.The above description is merely an illustrative explanation of the technical idea of the present invention, and various modifications and variations will be possible to those skilled in the art without departing from the essential characteristics of the present invention. Accordingly, the embodiments disclosed in the present invention are not intended to limit the technical idea of the present invention, but are for illustrative purposes, and the scope of the technical idea of the present invention is not limited by these embodiments. The scope of protection of the present invention should be interpreted in accordance with the claims below, and all technical ideas within the equivalent scope should be construed as being included in the scope of rights of the present invention.
<110> REPUBLIC OF KOREA <120> Probiotics for fermenting kimchi and kimchi using the same <130> FP-2107046-KR <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 1413 <212> RNA <213> Lactobacillus plantarum <400> 1 gcagtcgaac gaactctggt aatgattggt gcttgcatca tgaattacat ttgagtgagt 60 ggcgaactgg tgagtaacac gtgggaaacc tgcccagaag cgggggataa cacctggaaa 120 cagatgctaa taccgcataa caacttggac cgcatggtcc gagtttgaaa gatggcttcg 180 gctatcactt ttggatggtc ccgcggcgta ttagctagat ggtgaggtaa cggctcacca 240 tggcaatgat acgtagccga cctgagaggg taatcggcca cattgggact gagacacggc 300 ccaaactcct acgggaggca gcagtaggga atcttccaca atggacgaaa gtctgatgga 360 gcaacgccgc gtgagtgaag aagggtttcg gctcgtaaaa ctctgttgtt aaagaagaac 420 atatctgaga gtaactgttc aggtattgac ggtatttaac cagaaagcca cggctaacta 480 cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg tccggattta ttgggcgtaa 540 agcgagcgca ggcggttttt taagtctgat gtgaaagctt tcggctcaac cgaagaagtg 600 catcggaaac tgggaaactt gagtgcagaa gaggacagtg gaactccatg tgtagcggtg 660 aaatgcgtag atatatggaa gaacaccagt ggcgaaggcg gctgtctggt ctgtaactga 720 cgctgaggct cgaaagtatg ggtagcaaac aggattagat accctggtag tccataccgt 780 aaacgatgaa tgctaagtgt tggagggttt ccgcccttca gtgctgcagc taacgcatta 840 agcattccgc ctggggagta cggccgcaag gctgaaactc aaaggaattg acgggggccc 900 gcacaagcgg tggagcatgt ggtttaattc gaagctacgc gaagaacctt accaggtctt 960 gacatactat gcaaatctaa gagattagac gttcccttcg gggacatgga tacaggtggt 1020 gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac 1080 ccttattatc agttgccagc attaagttgg gcactctggt gagactgccg gtgacaaacc 1140 ggaggaaggt ggggatgacg tcaaatcatc atgcccctta tgacctgggc tacacacgtg 1200 ctacaatgga tggtacaacg agttgcgaac tcgcgagagt aagctaatct cttaaagcca 1260 ttctcagttc ggattgtagg ctgcaactcg cctacatgaa gtcggaatcg ctagtaatcg 1320 cggatcagca tgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca 1380 tgagagtttg taacacccaa agtcggtggg gta 1413 <210> 2 <211> 1466 <212> RNA <213> Lactobacillus fermentum <400> 2 gtgctataca tgcagtcgaa cgcgttggcc caattgattg atggtgcttg cacctgattg 60 attttggtcg ccaacgagtg gcggacgggt gagtaacacg taggtaacct gcccagaagc 120 gggggacaac atttggaaac agatgctaat accgcataac aacgttgttc gcatgaacaa 180 cgcttaaaag atggcttctc gctatcactt ctggatggac ctgcggtgca ttagcttgtt 240 ggtggggtaa tggcctacca aggcgatgat gcatagccga gttgagagac tgatcggcca 300 caatgggact gagacacggc ccatactcct acgggaggca gcagtaggga atcttccaca 360 atgggcgcaa gcctgatgga gcaacaccgc gtgagtgaag aagggtttcg gctcgtaaag 420 ctctgttgtt aaagaagaac acgtatgaga gtaactgttc atacgttgac ggtatttaac 480 cagaaagtca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgtta 540 tccggattta ttgggcgtaa agagagtgca ggcggttttc taagtctgat gtgaaagcct 600 tcggcttaac cggagaagtg catcggaaac tggataactt gagtgcagaa gagggtagtg 660 gaactccatg tgtagcggtg gaatgcgtag atatatggaa gaacaccagt ggcgaaggcg 720 gctacctggt ctgcaactga cgctgagact cgaaagcatg ggtagcgaac aggattagat 780 accctggtag tccatgccgt aaacgatgag tgctaggtgt tggagggttt ccgcccttca 840 gtgccggagc taacgcatta agcactccgc ctggggagta cgaccgcaag gttgaaactc 900 aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc gaagctacgc 960 gaagaacctt accaggtctt gacatcttgc gccaacccta gagatagggc gtttccttcg 1020 ggaacgcaat gacaggtggt gcatggtcgt cgtcagctcg tgtcgtgaga tgttgggtta 1080 agtcccgcaa cgagcgcaac ccttgttact agttgccagc attaagttgg gcactctagt 1140 gagactgccg gtgacaaacc ggaggaaggt ggggacgacg tcagatcatc atgcccctta 1200 tgacctgggc tacacacgtg ctacaatgga cggtacaacg agtcgcgaac tcgcgagggc 1260 aagcaaatct cttaaaaccg ttctcagttc ggactgcagg ctgcaactcg cctgcacgaa 1320 gtcggaatcg ctagtaatcg cggatcagca tgccgcggtg aatacgttcc cgggccttgt 1380 acacaccgcc cgtcacacca tgagagtttg taacacccaa agtcggtggg gtaacctttt 1440 aggagccagc cgcctaaggt gacaga 1466 <210> 3 <211> 1453 <212> RNA <213> Lactobacillus paracasei <400> 3 atgcaagtcg acgagttctc gttgatgatc ggtgcttgca ccgagattca acatggaacg 60 agtggcggac gggtgagtaa cacgtgggta acctgccctt aagtggggga taacatttgg 120 aaacagatgc taataccgca tagatccaag aaccgcatgg ttcttggctg aaagatggcg 180 taagctatcg cttttggatg gacccgcggc gtattagcta gttggtgagg taatggctca 240 ccaaggcgat gatacgtagc cgaactgaga ggttgatcgg ccacattggg actgagacac 300 ggcccaaact cctacgggag gcagcagtag ggaatcttcc acaatggacg caagtctgat 360 ggagcaacgc cgcgtgagtg aagaaggctt tcgggtcgta aaactctgtt gttggagaag 420 aatggtcggc agagtaactg ttgtcggcgt gacggtatcc aaccagaaag ccacggctaa 480 ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttatccggat ttattgggcg 540 taaagcgagc gcaggcggtt ttttaagtct gatgtgaaag ccctcggctt aaccgaggaa 600 gcgcatcgga aactgggaaa cttgagtgca gaagaggaca gtggaactcc atgtgtagcg 660 gtgaaatgcg tagatatatg gaagaacacc agtggcgaag gcggctgtct ggtctgtaac 720 tgacgctgag gctcgaaagc atgggtagcg aacaggatta gataccctgg tagtccatgc 780 cgtaaacgat gaatgctagg tgttggaggg tttccgccct tcagtgccgc agctaacgca 840 ttaagcattc cgcctgggga gtacgaccgc aaggttgaaa ctcaaaggaa ttgacggggg 900 cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt 960 cttgacatct tttgatcacc tgagagatca ggtttcccct tcgggggcaa aatgacaggt 1020 ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc 1080 aacccttatg actagttgcc agcatttagt tgggcactct agtaagactg ccggtgacaa 1140 accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg ggctacacac 1200 gtgctacaat ggatggtaca acgagttgcg agaccgcgag gtcaagctaa tctcttaaag 1260 ccattctcag ttcggactgt aggctgcaac tcgcctacac gaagtcggaa tcgctagtaa 1320 tcgcggatca gcacgccgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca 1380 ccatgagagt ttgtaacacc cgaagccggt ggcgtaaccc tttagggagc gagccgtcta 1440 agtggacaaa tta 1453 <110> REPUBLIC OF KOREA <120> Probiotics for fermenting kimchi and kimchi using the same <130> FP-2107046-KR <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 1413 <212> RNA <213> Lactobacillus plantarum <400> 1 gcagtcgaac gaactctggt aatgattggt gcttgcatca tgaattacat ttgagtgagt 60 ggcgaactgg tgagtaacac gtgggaaacc tgcccagaag cgggggataa cacctggaaa 120 cagatgctaa taccgcataa caacttggac cgcatggtcc gagtttgaaa gatggcttcg 180 gctatcactt ttggatggtc ccgcggcgta ttagctagat ggtgaggtaa cggctcacca 240 tggcaatgat acgtagccga cctgagaggg taatcggcca cattgggact gagacacggc 300 ccaaactcct acgggaggca gcagtaggga atcttccaca atggacgaaa gtctgatgga 360 gcaacgccgc gtgagtgaag aagggtttcg gctcgtaaaa ctctgttgtt aaagaagaac 420 atatctgaga gtaactgttc aggtattgac ggtatttaac cagaaagcca cggctaacta 480 cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg tccggattta ttgggcgtaa 540 agcgagcgca ggcggttttt taagtctgat gtgaaagctt tcggctcaac cgaagaagtg 600 catcggaaac tgggaaactt gagtgcagaa gaggacagtg gaactccatg tgtagcggtg 660 aaatgcgtag atatatggaa gaacaccagt ggcgaaggcg gctgtctggt ctgtaactga 720 cgctgaggct cgaaagtatg ggtagcaaac aggattagat accctggtag tccataccgt 780 aaacgatgaa tgctaagtgt tggagggttt ccgcccttca gtgctgcagc taacgcatta 840 agcattccgc ctggggagta cggccgcaag gctgaaactc aaaggaattg acggggggccc 900 gcacaagcgg tggagcatgt ggtttaattc gaagctacgc gaagaacctt accaggtctt 960 gacatactat gcaaatctaa gagattagac gttcccttcg gggacatgga tacaggtggt 1020 gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac 1080 ccttattatc agttgccagc attaagttgg gcactctggt gagactgccg gtgacaaacc 1140 ggaggaaggt ggggatgacg tcaaatcatc atgcccctta tgacctgggc tacacacgtg 1200 ctacaatgga tggtacaacg agttgcgaac tcgcgagagt aagctaatct cttaaagcca 1260 ttctcagttc ggattgtagg ctgcaactcg cctacatgaa gtcggaatcg ctagtaatcg 1320 cggatcagca tgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca 1380 tgagagtttg taacacccaa agtcggtggg gta 1413 <210> 2 <211> 1466 <212> RNA <213> Lactobacillus fermentum <400> 2 gtgctataca tgcagtcgaa cgcgttggcc caattgattg atggtgcttg cacctgattg 60 attttggtcg ccaacgagtg gcggacgggt gagtaacacg taggtaacct gcccagaagc 120 gggggacaac atttggaaac agatgctaat accgcataac aacgttgttc gcatgaacaa 180 cgcttaaaag atggcttctc gctatcactt ctggatggac ctgcggtgca ttagcttgtt 240 ggtggggtaa tggcctacca aggcgatgat gcatagccga gttgagagac tgatcggcca 300 caatgggact gagacacggc ccatactcct acgggaggca gcagtaggga atcttccaca 360 atgggcgcaa gcctgatgga gcaacaccgc gtgagtgaag aagggtttcg gctcgtaaag 420 ctctgttgtt aaagaagaac acgtatgaga gtaactgttc atacgttgac ggtatttaac 480 cagaaagtca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgtta 540 tccggattta ttgggcgtaa agagagtgca ggcggttttc taagtctgat gtgaaagcct 600 tcggcttaac cggagaagtg catcggaaac tggataactt gagtgcagaa gagggtagtg 660 gaactccatg tgtagcggtg gaatgcgtag atatatggaa gaacaccagt ggcgaaggcg 720 gctacctggt ctgcaactga cgctgagact cgaaagcatg ggtagcgaac aggattagat 780 accctggtag tccatgccgt aaacgatgag tgctaggtgt tggagggttt ccgcccttca 840 gtgccggagc taacgcatta agcactccgc ctggggagta cgaccgcaag gttgaaactc 900 aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc gaagctacgc 960 gaagaacctt accaggtctt gacatcttgc gccaacccta gagatagggc gtttccttcg 1020 ggaacgcaat gacaggtggt gcatggtcgt cgtcagctcg tgtcgtgaga tgttgggtta 1080 agtcccgcaa cgagcgcaac ccttgttact agttgccagc attaagttgg gcactctagt 1140 gagactgccg gtgacaaacc ggaggaaggt ggggacgacg tcagatcatc atgcccctta 1200 tgacctgggc tacacacgtg ctacaatgga cggtacaacg agtcgcgaac tcgcgagggc 1260 aagcaaatct cttaaaaccg ttctcagttc ggactgcagg ctgcaactcg cctgcacgaa 1320 gtcggaatcg ctagtaatcg cggatcagca tgccgcggtg aatacgttcc cgggccttgt 1380 acacaccgcc cgtcacacca tgagagtttg taacacccaa agtcggtggg gtaacctttt 1440 aggagccagc cgcctaaggt gacaga 1466 <210> 3 <211> 1453 <212> RNA <213> Lactobacillus paracasei <400> 3 atgcaagtcg acgagttctc gttgatgatc ggtgcttgca ccgagattca acatggaacg 60 agtggcggac gggtgagtaa cacgtgggta acctgccctt aagtggggga taacatttgg 120 aaacagatgc taataccgca tagatccaag aaccgcatgg ttcttggctg aaagatggcg 180 taagctatcg cttttggatg gacccgcggc gtattagcta gttggtgagg taatggctca 240 ccaaggcgat gatacgtagc cgaactgaga ggttgatcgg ccacattggg actgagacac 300 ggcccaaact cctacgggag gcagcagtag ggaatcttcc acaatggacg caagtctgat 360 ggagcaacgc cgcgtgagtg aagaaggctt tcgggtcgta aaactctgtt gttggagaag 420 aatggtcggc agagtaactg ttgtcggcgt gacggtatcc aaccagaaag ccacggctaa 480 ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttatccggat ttattgggcg 540 taaagcgagc gcaggcggtt ttttaagtct gatgtgaaag ccctcggctt aaccgaggaa 600 gcgcatcgga aactgggaaa cttgagtgca gaagaggaca gtggaactcc atgtgtagcg 660 gtgaaatgcg tagatatatg gaagaacacc agtggcgaag gcggctgtct ggtctgtaac 720 tgacgctgag gctcgaaagc atgggtagcg aacaggatta gataccctgg tagtccatgc 780 cgtaaacgat gaatgctagg tgttggaggg tttccgccct tcagtgccgc agctaacgca 840 ttaagcattc cgcctgggga gtacgaccgc aaggttgaaa ctcaaaggaa ttgacggggg 900 cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt 960 cttgacatct tttgatcacc tgagagatca ggtttcccct tcggggggcaa aatgacaggt 1020 ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc 1080 aacccttatg actagttgcc agcatttagt tgggcactct agtaagactg ccggtgacaa 1140 accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg ggctacacac 1200 gtgctacaat ggatggtaca acgagttgcg agaccgcgag gtcaagctaa tctcttaaag 1260 ccattctcag ttcggactgt aggctgcaac tcgcctacac gaagtcggaa tcgctagtaa 1320 tcgcggatca gcacgccgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca 1380 ccatgagagt ttgtaacacc cgaagccggt ggcgtaaccc tttagggagc gagccgtcta 1440 agtggacaaaa tta 1453
Claims (10)
상기 락토바실러스 플란타럼 (L. plantarum) HJ-Ki(기탁번호 : KACC 92324P) 균주는 서열번호 1로 표시되는 16s rRNA 염기서열을 포함하고, 상기 락토바실러스 퍼멘텀(L. fermentum) HJ-C1(기탁번호 : KACC 92322P) 균주는 서열번호 2로 표시되는 16s rRNA 염기서열을 포함하고, 상기 락토바실러스 파라카세이(L. paracasei) MO-C1(기탁번호 : KACC 92325P) 균주는 서열번호 3으로 표시되는 16s rRNA 염기서열을 포함하는 김치 발효용 프로바이오틱스 제제.According to claim 1,
The Lactobacillus plantarum ( L. plantarum ) HJ-Ki (accession number: KACC 92324P) strain contains a 16s rRNA base sequence represented by SEQ ID NO: 1, and the Lactobacillus fermentum ( L. fermentum ) HJ-C1 (Accession number: KACC 92322P) The strain contains a 16s rRNA base sequence represented by SEQ ID NO: 2, and the Lactobacillus paracasei ( L. paracasei ) MO-C1 (Accession number: KACC 92325P) strain is represented by SEQ ID NO: 3. A probiotic preparation for kimchi fermentation containing the 16s rRNA base sequence.
양념을 준비하는 단계;
상기 양념에 락토바실러스 플란타럼 (L. plantarum) HJ-Ki(기탁번호 : KACC 92324P) 균주, 락토바실러스 퍼멘텀(L. fermentum) HJ-C1(기탁번호 : KACC 92322P) 균주, 및 락토바실러스 파라카세이(L. paracasei) MO-C1(기탁번호 : KACC 92325P) 균주 중 적어도 하나를 포함하는 프로바이오틱스 제제를 혼합하는 단계;
상기 소금물에 절여둔 상기 배추를 세척하고 물기를 제거하는 단계; 및
혼합된 상기 양념을 배추에 버무리는 단계;를 포함하는 김치 제조방법.Pickling cabbage in salt water;
Preparing seasoning;
In the above seasoning, Lactobacillus plantarum ( L. plantarum ) HJ-Ki (accession number: KACC 92324P) strain, Lactobacillus fermentum ( L. fermentum ) HJ-C1 (accession number: KACC 92322P) strain, and Lactobacillus para. Mixing a probiotic preparation containing at least one of the L. paracasei MO-C1 (accession number: KACC 92325P) strains;
Washing the cabbage pickled in salt water and removing moisture; and
A kimchi manufacturing method comprising mixing the mixed seasoning with cabbage.
상기 양념에 버무린 상기 배추를 발효시키는 단계를 더 포함하며, 상기 발효는 김치 내 유산균을 제외한 일반 세균 대비 유산균 함량이 100배 이상이 되도록 6주 이내로 수행되는 김치 제조 방법.According to clause 3,
A kimchi manufacturing method further comprising fermenting the cabbage mixed with the seasoning, wherein the fermentation is performed within 6 weeks so that the content of lactic acid bacteria in kimchi is 100 times or more compared to general bacteria excluding lactic acid bacteria.
상기 발효하는 단계는 1℃ 내지 10℃에서 수행되는 김치 제조방법.According to clause 4,
A kimchi manufacturing method wherein the fermentation step is performed at 1°C to 10°C.
양념을 준비하는 단계;
상기 양념에 락토바실러스 플란타럼 (L. plantarum) HJ-Ki(기탁번호 : KACC 92324P) 균주 또는 락토바실러스 파라카세이(L. paracasei) MO-C1(기탁번호 : KACC 92325P) 균주를 포함하는 프로바이오틱스 제제를 혼합하는 단계;
상기 소금물에 절여둔 상기 배추를 세척하고 물기를 제거하는 단계;
혼합된 상기 양념을 배추에 버무리는 단계; 및
상기 양념에 버무린 배추를 2주 내지 8주 동안 발효시키는 단계를 포함하는 신김치 제조방법.Pickling cabbage in salt water;
Preparing seasoning;
A probiotic preparation containing Lactobacillus plantarum ( L. plantarum ) HJ-Ki (accession number: KACC 92324P) strain or Lactobacillus paracasei ( L. paracasei ) MO-C1 (accession number: KACC 92325P) strain in the above seasoning. mixing;
Washing the cabbage pickled in salt water and removing moisture;
Mixing the mixed seasoning with cabbage; and
A method of producing sour kimchi comprising fermenting cabbage mixed with the seasoning for 2 to 8 weeks.
상기 발효시키는 단계는 4주 내지 8주 동안 수행되며, 상기 신김치의 총산도(%)는 0.7% 이상인 신김치 제조방법.According to clause 7,
The fermentation step is performed for 4 to 8 weeks, and the total acidity (%) of the sour kimchi is 0.7% or more.
상기 발효하는 단계는 1℃ 내지 10℃에서 수행되는 신김치 제조방법.According to clause 8,
The fermentation step is a method of producing sour kimchi wherein the fermentation step is performed at 1°C to 10°C.
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