KR20040033593A - Culture medium for lactic acid bacteria prepared from chinese cabbage wastes - Google Patents
Culture medium for lactic acid bacteria prepared from chinese cabbage wastes Download PDFInfo
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- KR20040033593A KR20040033593A KR1020020062743A KR20020062743A KR20040033593A KR 20040033593 A KR20040033593 A KR 20040033593A KR 1020020062743 A KR1020020062743 A KR 1020020062743A KR 20020062743 A KR20020062743 A KR 20020062743A KR 20040033593 A KR20040033593 A KR 20040033593A
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- lactic acid
- acid bacteria
- chinese cabbage
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- culture medium
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 68
- 239000004310 lactic acid Substances 0.000 title claims abstract description 34
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 34
- 239000002699 waste material Substances 0.000 title claims abstract description 34
- 241000894006 Bacteria Species 0.000 title claims abstract description 33
- 239000001963 growth medium Substances 0.000 title claims abstract description 15
- 235000010149 Brassica rapa subsp chinensis Nutrition 0.000 title abstract description 24
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- 241000499436 Brassica rapa subsp. pekinensis Species 0.000 title abstract description 24
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 claims abstract description 4
- 102000002322 Egg Proteins Human genes 0.000 claims abstract description 4
- 108010000912 Egg Proteins Proteins 0.000 claims abstract description 4
- 235000014103 egg white Nutrition 0.000 claims abstract description 4
- 210000000969 egg white Anatomy 0.000 claims abstract description 4
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- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 claims description 29
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Classifications
-
- 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
-
- 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
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
본 발명은 김치 제조시 대량의 폐기물로 발생되는 배추폐기물을 이용하여 제조된 유산균 배양용 배지에 관한 것이다. 보다 구체적으로, 본 발명은 김치 제조시 대량으로 발생되는 배추 폐기물을 이용하여 제조된 배지를 사용하여 유용한 유산균, 특히 루코노스탁 가시코미타툼을 다량으로 얻어내는 방법에 관한 것이다.The present invention relates to a culture medium for lactic acid bacteria prepared using cabbage waste generated as a large amount of waste during kimchi production. More specifically, the present invention relates to a method for obtaining a large amount of useful lactic acid bacteria, in particular, Lukonostock spiny comitatum, using a medium prepared using a large amount of Chinese cabbage waste generated during kimchi production.
우리의 전통식품인 김치는 한국인이 가장 즐겨먹는 음식물 중 하나일 뿐 아니라, 국제적으로 보급되어 수출이 급증하고 있으며, 대단위 유통센터의 보급 및 학교 급식의 활성화로 공장 단위의 대량 생산이 이루어지고 있다. 김치를 제조하기 위한 주원료인 배추는 전체 원료의 90 중량% 정도를 차지하며, 이 중 상당부분은 폐기물로 버려지고 있는 실정이다. 특히 대량의 배추 폐기물이 발생하는 김치 공장이나 청과물 시장 등에서는 배추 폐기물을 수집 및 처리하는데 상당한 노력, 시간, 및 비용을 소모하고 있다.Kimchi, our traditional food, is not only one of Koreans' favorite foods, but it is also widely distributed internationally, and exports are increasing rapidly. Chinese cabbage, which is the main raw material for making kimchi, accounts for about 90% by weight of the whole raw material, and a large part of it is disposed of as waste. In particular, the Kimchi plant and the fruit and vegetable market, where a large amount of cabbage waste is generated, are spending considerable effort, time, and money in collecting and processing cabbage waste.
현재 배추 폐기물을 비롯한 유기성 폐기물은 전체 폐기물의 상당량을 차지하며, 쉽게 부패하여 악취를 발생시키고, 오수를 누출시켜 처리에 어려움을 겪고 있으며, 동시에 환경문제를 유발하고 있다. 따라서, 이러한 유기성 폐기물의 처리 방법에 대한 연구가 활발하게 진행되고 있다.Organic wastes, including Chinese cabbage waste, currently make up a significant portion of the total waste, are easily decaying, causing odors, leaking sewage, causing difficulties in treatment, and causing environmental problems. Therefore, research on the treatment method of such organic waste is actively progressing.
현재까지, 배추 폐기물은 대부분 매립되어 토양 미생물에 의한 분해에 의존하고 있으나 매립지에 한계가 있으며, 배추는 수분을 약 80%이상 함유하고 있어 부패가 빠르게 진행되기 때문에 악취가 발생하고, 폐기물의 분해산물이 수계로 유입될 경우 부영양화의 원인이 되기도 한다. 따라서, 매립 방법을 대체할 만한 다른처리 방법이 모색되어 오고 있다.To date, most of Chinese cabbage waste is reclaimed and rely on soil microorganisms, but there is a limit to landfill. Chinese cabbage contains more than 80% of moisture, so decay develops rapidly, and odor is generated. Inflows to this water system may cause eutrophication. Accordingly, other treatment methods have been sought to replace the landfill method.
본 발명자는 배추 폐기물을 처리하는 동시에 김치 유산균 생체량을 대량으로 생산할 수 있는 유산균 배양용 배지를 개발하게 되었다.The present inventors have developed a culture medium for lactic acid bacteria that can produce a large amount of kimchi lactic acid bacteria biomass while simultaneously processing the Chinese cabbage waste.
도 1은 배추 폐기물을 15℃에서 21일 동안 발효시키는 과정에서 24시간 간격으로 측정한 김치액의 pH 변화를 나타낸 그래프이다.Figure 1 is a graph showing the pH change of kimchi solution measured at 24 hours interval in the process of fermenting cabbage waste for 21 days at 15 ℃.
도 2는 배추 폐기물을 15℃에서 21일 동안 발효시키는 과정에서 24시간 간격으로 측정한 김치액의 산도 변화를 나타낸 그래프이다.Figure 2 is a graph showing the change in the acidity of kimchi solution measured at intervals of 24 hours in the process of fermenting cabbage waste at 15 ℃ for 21 days.
도 3은 배추 폐기물을 15℃에서 21일 동안 발효시키는 과정에서 24시간 간격으로 측정한 김치액 내 유산균 세포수의 변화를 나타낸 그래프이다.Figure 3 is a graph showing the change in the number of lactic acid bacteria cells in kimchi solution measured at 24 hours in the process of fermenting cabbage waste for 21 days at 15 ℃.
도 4는 본 발명에 따라 김치액에서 분리된 JN3 균주의 생장 곡선을 나타낸 그래프로서, 배추 배지를 사용하여 25℃에서 배양하여 시간별로 흡광도를 측정하여 세포수로 환산하였다.4 is a graph showing the growth curve of the JN3 strain isolated from kimchi solution according to the present invention, incubated at 25 ℃ using a Chinese cabbage medium was measured by the absorbance for each time was converted into cell number.
도 5는 도 4의 생장곡선으로부터 계산된 JN3의 세포수 변화를 나타낸 생장곡선이다.5 is a growth curve showing the change in cell number of JN3 calculated from the growth curve of FIG.
도 6은 발효조, 혼합기, 배지와 배양액 저장고, pH 조절장치로 이루어지는 연속배양 발효조의 구성도이다.6 is a block diagram of a continuous culture fermenter consisting of a fermenter, a mixer, a medium and a culture medium reservoir, and a pH adjusting device.
본 발명에서는 배추 폐기물을 이용한 배양용 배지를 제조하기에 앞서 우선 배추 폐기물을 이용한 김치를 발효시켜 유산균을 생산한다.In the present invention, prior to producing a culture medium using the Chinese cabbage waste, firstly fermented kimchi using the Chinese cabbage waste to produce lactic acid bacteria.
A. 김치유산균의 분리를 위한 김치발효A. Kimchi Fermentation for Isolation of Kimchi Lactic Acid Bacteria
배추 폐기물의 발효 공정은 일반적인 김치 담금 공정을 이용한다. 사용되는 재료는 배추 폐기물 100 중량부, 고춧가루, 마늘, 및 파 각각 1 내지 3 중량부, 양파 2 내지 5 중량부, 타이로신 0.01 내지 0.1 중량부이다. 배추는 3 내지 5㎝ 길이로 절단하여 사용하며, 나머지 재료는 적당한 크기로 분쇄한 다음 모두 혼합하고 2 내지 5% 농도의 소금물 30 내지 70 중량부와 혼합한다. 상기 혼합물을 폴리에틸렌 또는 폴리프로필렌 재질 반응기에서 13 내지 17℃ 온도를 유지하면서 15일 내지 25일 동안 발효시킨다. 상기 재료 중 타이로신은 발효과정 중 성장하는 유산균 중 특정 유산균만이 선택적으로 성장되도록 하는 역할을 한다.The fermentation process of Chinese cabbage waste uses a conventional kimchi soaking process. Materials used are 100 parts by weight of Chinese cabbage waste, 1 to 3 parts by weight of red pepper powder, garlic, and leeks, 2 to 5 parts by weight of onion, and 0.01 to 0.1 parts by weight of tyrosine. Chinese cabbage is cut into 3 to 5 cm in length, and the remaining ingredients are ground to a suitable size, mixed together, and mixed with 30 to 70 parts by weight of brine at a concentration of 2 to 5%. The mixture is fermented in a polyethylene or polypropylene material reactor for 15 to 25 days while maintaining a temperature of 13 to 17 ° C. Tyrosine in the material serves to selectively grow only the specific lactic acid bacteria among the lactic acid bacteria growing during the fermentation process.
본 발명에 따르면, 자원 재활용 및 환경 친화적 관점에서 배추 폐기물을 이용하여 김치를 발효시키는 것이 바람직하다. 그러나, 본 발명에서 얻고자 하는 루코노스탁 가시코미타툼균은 김치 발효 과정에서 얻어지는 유산균이므로, 배추 폐기물 대신 일반 배추를 이용하여 상기와 같은 방법으로 김치를 발효시켜 동일한 결과를 얻을 수 있다.According to the present invention, it is preferable to ferment kimchi using cabbage waste from the viewpoint of resource recycling and environmental friendliness. However, since the Lukonosstock spiny comitatum bacteria to be obtained in the present invention are lactic acid bacteria obtained during the kimchi fermentation process, the same results can be obtained by fermenting kimchi in the same manner as above using general cabbage instead of cabbage waste.
B. 유산균의 분리 및 동정B. Isolation and Identification of Lactic Acid Bacteria
상기 과정에서 발효된 김치로부터 24시간 단위로 김치액을 채취하여 멸균증류수로 10배 희석하여 희석액을 MRS-BPB(de Man, Rgosa and Sharpe-bromophenol blue)배지에 접종하고 도말한다. 15℃에서 약 48시간 배양하여 생성된 집락의 형태와 색상이 다른 집락을 접종침을 이용하여 순수분리하여 MRS(de Man, Rgosa and Sharpe) 사면 배지에서 순수배양한다. 분리균주는 15일마다 MRS 사면 배지에 계대배양하여 보존하며 균주를 동정한다.Kimchi solution is collected from fermented kimchi 24 hours in the above process and diluted 10 times with sterile distilled water to inoculate the diluted solution in MRS-BPB (de Man, Rgosa and Sharpe-bromophenol blue) medium and smear. Colonies produced by incubating at 15 ° C. for about 48 hours are separated from the colony by pure inoculation using pure inoculation needles and purely cultured in MRS (de Man, Rgosa and Sharpe) slope medium. The isolates are preserved by passage in MRS slope medium every 15 days and the strains are identified.
C. 배추 배지의 제조C. Preparation of Chinese Cabbage Medium
배추 폐기물을 이용하여 배추배지를 제조하기 위하여, 배추 폐기물을 분쇄기로 분쇄하여 고액분리를 한다. 배추즙 내의 고형물을 제거하기 위하여 80℃까지 가열한 후 배추즙 100 중량부에 응집제로 난백 0.5 내지 3 중량부를 혼합한 다음 상온으로 냉각시킨다. 냉각된 배추즙은 거즈를 이용하여 고형물을 여과시킨 후 여액을 여과지로 여과한다. 여액을 막여과(Φ, 0.45㎛)로 멸균하여 배지로 사용한다.In order to produce the cabbage medium using the cabbage waste, the cabbage waste is pulverized with a grinder to separate the liquid. After heating to 80 ℃ to remove the solids in the cabbage juice mixed 0.5 to 3 parts by weight of egg white with a flocculant to 100 parts by weight cabbage juice and then cooled to room temperature. The cooled cabbage juice is filtered through a gauze and the filtrate is filtered through a filter paper. The filtrate is sterilized by membrane filtration (Φ, 0.45㎛) and used as a medium.
D. 배추 배지에서의 유산균의 생장D. Growth of Lactic Acid Bacteria in Chinese Cabbage Medium
A 단계에서 분리된 균주를 C 단계에서 제조된 배추 배지에서 배양시켜 생장 여부를 확인하고 생장율이 가장 높은 유산균을 선별한 다음, 배양시켜 대량의 유산균 생체량을 얻는다.The strain isolated in step A is cultured in the cabbage medium prepared in step C to check the growth, select the highest growth rate lactic acid bacteria, and then culture to obtain a large amount of lactic acid bacteria biomass.
본 발명은 하기의 실시예에 의하여 보다 더 잘 이해될 수 있으며, 하기의 실시예는 본 발명을 예시하기 위한 것이며 첨부된 특허청구범위에 의하여 한정되는보호범위를 제한하고자 하는 것은 아니다.The invention can be better understood by the following examples, which are intended to illustrate the invention and are not intended to limit the scope of protection defined by the appended claims.
실시예Example
유산균의 동정Identification of Lactobacillus
배추 폐기물 100g, 고춧가루, 마늘, 및 파 각각 1.5g, 양파 3g, 및 타이로신 0.05g을 준비하여, 2.5% 소금물 50g과 혼합하였다. 배추 폐기물은 3㎝ 길이로 절단하여 사용하였고, 고춧가루, 마늘, 파, 양파는 분쇄기로 적당한 크기로 분쇄하여 사용하였다. 상기 혼합물을 폴리프로필렌 재질 반응기에서 15℃ 온도를 유지하면서 21일 동안 발효시켰다.100 g of Chinese cabbage waste, 1.5 g of red pepper powder, garlic, and green onion, 1.5 g of onion, 3 g of onion, and 0.05 g of tyrosine were prepared and mixed with 50 g of 2.5% brine. Chinese cabbage waste was cut to 3cm in length, and red pepper powder, garlic, green onion, and onion were ground to a suitable size using a grinder. The mixture was fermented for 21 days at 15 ° C. temperature in a polypropylene reactor.
발효 과정 동안 24시간 간격으로 김치액의 pH, 산도, 및 유산균 세포수를 측정하였다.During the fermentation process, the pH, acidity, and lactic acid cell number of kimchi were measured at 24 hour intervals.
pH는 pH 미터(ORION 920A)를 사용하여 측정하였으며, 그 결과를 도 1에 나타내었다. pH는 김치 담금 후 24시간까지는 일시적으로 증가하다가 이후 5일 동안 급격히 감소하여 이후부터는 pH 3.6 내지 4.2를 유지하였다.pH was measured using a pH meter (ORION 920A), the results are shown in FIG. The pH was increased temporarily up to 24 hours after kimchi immersion, and then drastically decreased for 5 days to maintain pH 3.6 to 4.2 thereafter.
산도는 0.1N-NaOH로 적정하여 유산으로 환산시켜 측정하였으며, 그 결과를 도 2에 나타내었다. 산도는 pH와는 달리 7일째까지 증가하여 0.6-0.7%를 유지하였다.Acidity was measured in terms of lactic acid by titration with 0.1 N-NaOH, and the results are shown in FIG. 2. Unlike pH, acidity increased up to 7 days and maintained 0.6-0.7%.
유산균 세포수 측정은 생균수 측정법을 이용하였다. 시료(김치액)를 멸균 증류수로 10배 희석법으로 희석한 다음 0.1㎖의 희석액을 유산균 배양용 배지인 MRS-BPB 배지에 접종 도말한 후 15℃에서 48시간 동안 배양하였다. 배지에 형성된 집락을 계수하여 세포수로 환산하고, 그 결과를 도 3에 나타내었다. 세포수의 변화는 4일째까지 급격히 증가하여 1012세포/㎖를 나타내었으며, 그 이후는 파동 현상을 보였다.Lactobacillus cell count was measured by viable cell count. After diluting the sample (kimchi solution) by 10-fold dilution with sterile distilled water, 0.1 ml dilution was inoculated into MRS-BPB medium, which is a culture medium for lactic acid bacteria, and then cultured at 15 ° C. for 48 hours. The colonies formed in the medium were counted and converted into cell numbers, and the results are shown in FIG. 3. The number of cells increased rapidly until day 4, indicating 10 12 cells / ml, and thereafter, a wave phenomenon.
김치유산균을 분리하기 위하여, MRS-BPB 배지에 생성된 집락의 형태와 색상이 다른 집락을 접종침을 이용하여 순수 분리하여 MRS 사면 배지에 접종하였다. 그 다음 15일마다 MRS 사면 배지에 계대배양하여 사용하였다. 분리된 균주는 총 17개였다.In order to separate the kimchi lactic acid bacteria, colonies having different forms and colors of colonies generated in MRS-BPB medium were inoculated into MRS slope medium by pure separation using an inoculation needle. Every 15 days thereafter, subcultured to MRS slope medium was used. A total of 17 strains were isolated.
배추 배지의 제조Preparation of Chinese Cabbage Medium
상기 과정과는 별도로, 배추 배지를 제조하기 위하여, 배추를 분쇄기로 분쇄하여 배추즙을 제조하고, 즙내의 고형물을 제거하기 위하여 상기 배추즙을 80℃까지 가열한 후 배추즙 100g에 응집제인 난백 1g을 혼합하고 상온으로 냉각시켰다. 냉각된 배추즙은 거즈를 이용하여 고형물을 여과시킨 후 여액을 여과지(Whatman NO. 1 USA)로 재차 여과하였다. 여액은 막여과법(Φ, 0.45㎛)으로 멸균하여 배지로 사용하였다.Apart from the above process, in order to prepare the cabbage medium, to make a cabbage juice by grinding the cabbage with a grinder, in order to remove the solids in the juice, the cabbage juice is heated to 80 ℃ and 100g cabbage juice egg white 1g as a flocculant Were mixed and cooled to room temperature. The cooled cabbage juice was filtered through gauze and the filtrate was again filtered through filter paper (Whatman NO. 1 USA). The filtrate was sterilized by membrane filtration (Φ, 0.45㎛) was used as a medium.
유산균의 배양Culture of Lactic Acid Bacteria
상기에서 분리된 17종의 유산균을 제조된 배추 배지에서 배양시켰다. 이 중 생장률이 높은 유산균 4종을 분리하였으며, 이를 JN1 내지 JN4로 지명하였다. 상기 4종의 균주는 모두 구균이었으며, 그램염색, 수크로오즈 배지에서 덱스트란의 형성, 및 MRS 배지에서 가스 생성시험이 모두 양성 반응이었다. 온도별 생장실험에서는 5-37℃에서 모두 생장하였으나, NaCl 농도별 생장 시험에서는 1% NaCl 첨가 배지에서는 생장이 일부 억제되었으며, 2.5% NaCl 첨가 배지에서는 생장이 억제되었다. 이러한 결과를 종합해보면, 분리균주는 생장 온도 면에서는 중온성 세균에 해당되며, 저온(5℃)에서도 생장하였고, NaCl 농도가 높아짐에 따라 생장이 억제됨을 알 수 있었다. 각 균주의 형태 및 생태학적 특징을 표 1에 나타내었다.17 kinds of lactic acid bacteria isolated above were cultured in the prepared cabbage medium. Among them, 4 kinds of lactic acid bacteria with high growth rate were isolated, which were designated as JN1 to JN4. All four strains were cocci, and gram staining, dextran formation in sucrose medium, and gas production test in MRS medium were all positive. In the growth test by temperature, all growth was performed at 5-37 ° C., but growth was inhibited in 1% NaCl medium and 2.5% NaCl medium, respectively. Taken together, these isolates corresponded to mesophilic bacteria in terms of growth temperature, were grown at low temperature (5 ℃), and growth was inhibited as NaCl concentration increased. The morphology and ecological characteristics of each strain are shown in Table 1.
+: 양성, -: 음성, (+): 약한 양성+: Positive,-: negative, (+): weak positive
균주의 생화학적 특성 측정Determination of Biochemical Properties of Strains
균주의 생화학적 특성을 측정하기 위하여 다중시험체계를 이용하여 당 발효시험을 하였다. 배지는 MRS 배지를 사용하였으며, 글루코오즈 대신 시험하고자 하는 19개의 당을 막 여과법을 이용하여 멸균하여 최종 농도가 1% 되게 첨가하여 시험배지로 사용하였다. 균주를 배지에 접종하여 25℃에서 2일간 배양 후 복합 pH 지시약을 사용하여 발효 유무를 판단하였다. 그 결과는 표 2에 나타내었다.In order to measure the biochemical properties of the strain, the sugar fermentation test was carried out using a multi-test system. MRS medium was used as a medium, and instead of glucose, 19 sugars to be tested were sterilized using membrane filtration to add a final concentration of 1% and used as a test medium. After inoculating the strain into the medium and incubated at 25 ° C. for 2 days, the presence or absence of fermentation was determined using a complex pH indicator. The results are shown in Table 2.
분리균주는 L-아라비노스, 멜리비오스, 라피노스, 리보스, D-크실로스, 말토스, 수크로스, 프락토스, 멜레지토스를 발효시켰으나, 갈락토스, 락토스, 만니톨, 살리신, 람노스, 솔비톨은 발효시키지 못하였다. 이상의 결과는 루코노스탁 가시코미타툼(Leuconostoc gasicomitatum)과 유연관계가 높은 것으로 나타났다.The isolates fermented L-arabinose, melibiose, raffinose, ribose, D-xylose, maltose, sucrose, fructose and melezitose, while galactose, lactose, mannitol, salicycin, rhamnose and sorbitol fermented. I could not let you. The above results were found to be highly related to Leuconostoc gasicomitatum.
보다 정확한 동정을 위하여, 루코노스탁 종 특이성 프라이머를 이용하여 PCR(polymerase chain reaction)법으로 동정하였으며, 그 결과로 루코노스탁 가시코미타툼임을 확인하였다. 루코노스탁 가시코미타툼 균은 2000년 최초로 발견되어 아직은 그 구체적 용도에 대하여 보고된 바 없으나, 유용한 유산균으로서 생균제(동물사료 첨가제), 정장제(인체 의약품용), 건강보조식품(건강식품용), 및 화장품 첨가제 등의 용도로 사용될 것이 기대된다.For more accurate identification, it was identified by PCR (polymerase chain reaction) method using the Lukonostach species specific primers, and as a result, it was confirmed that it is Lukonostek chycomattum. Luconotstock Kashikomitum was first discovered in 2000 and has not yet been reported for its specific use. However, it is a useful lactic acid bacterium that is useful for probiotics (animal feed additives), dressing agents (for human medicines), health supplements (for health foods), And cosmetic additives.
JN1-4 : 분리 균주JN1-4: Isolated Strains
L.m :루코노스탁 메센테로이드스sp.메센테로이드스,Lm: Lukonotstock mesenteroroids sp. Mesenteroids ,
L.g :루코노스탁 겔라디움 Lg: Lukonot Stock Geladium
L.c :루코노스탁 시트륨 Lc: Lukonotstock Citrium
L.l :루코노스탁 락티스 Ll: Lukonostact lactis
L.ga :루코노스탁 가시코미타툼 L.ga: Lukonok Stark Komitatum
+: 90% 또는 그 이상의 균주가 양성임;+: 90% or more strains are positive;
D: 89-11%의 균주가 양성임;D: 89-11% of the strains are positive;
-: 90% 또는 그 이상의 균주가 음성임;-: 90% or more strains are negative;
ND: 감지되지 않음.ND: not detected.
배추 배지에서의 생체량 측정Biomass Measurement in Chinese Cabbage Medium
배추 배지에서 분리한 균주를 25℃에서 3일간 배양한 후 분광 광도계를 이용하여 550nm에서 흡광도를 측정하고, 생체량은 배양액 20㎖를 막여과하여 105℃에서 4 시간 동안 건조하여 건조중량으로 측정하였다. 그 결과를 표 3에 나타내었다. 결과적으로 흡광도와 건조 중량은 무관하게 나타났다. 이는 세포의 형태와 크기의 차이에 따른 것으로 판단되며, 건조 중량은 JN3가 가장 높게 나타났다.After culturing the strain isolated from the cabbage medium for 3 days at 25 ℃ was measured for absorbance at 550nm using a spectrophotometer, the biomass was measured by dry weight after filtration of 20ml culture medium and dried at 105 ℃ for 4 hours. The results are shown in Table 3. As a result, absorbance and dry weight were irrelevant. This is judged to be due to the difference in the shape and size of the cells, the dry weight was the highest in JN3.
건조 중량이 가장 높은 JN3의 생장 곡선을 측정하였다. 생장 곡선 측정은 배추 배지를 사용하여 25℃에서 배양하여 시간별로 흡광도를 측정하여 세포수로 환산하였다. 흡광도와 세포수의 상관관계는 도 5에 나타내었으며, 루코노스탁 가시코미타툼(JN3)의 생장 곡선은 도 6에 나타내었다.The growth curve of JN3 with the highest dry weight was measured. Growth curve measurement was incubated at 25 ℃ using cabbage medium to measure the absorbance by time and converted into cell number. Correlation between absorbance and cell number is shown in FIG. 5, and the growth curve of Lukonstock Takkomitatum (JN3) is shown in FIG. 6.
생장곡선으로부터 생장률 상수(K)를 구하면 다음과 같다.Obtaining the growth rate constant (K) from the growth curve is as follows.
(상기의 X t 는 세포수/㎖, t 는 시간임)( X t is the number of cells / ml, t is the time)
K = 1/ t gen 이므로, t gen = 1/0.77 = 1.3(시간)이다. Since K = 1 / t gen , t gen = 1 / 0.77 = 1.3 (hours).
(상기의 t gen 는 배가시간(doubling time)임.)( T gen is the doubling time.)
따라서, 비생장률(μ)는 0.693× K 이므로 μ는 0.53임.Therefore, the specific growth rate (μ) is 0.693 × K, so μ is 0.53.
연속배양 발효조Continuous culture fermenter
본 발명에 따라 김치를 배양하기 위한 연속배양 발효조는 발효조와 혼합기, 배지와 배양액 저장고, pH 조절장치로 나뉘어지며, 발효조의 모식도는 도 6에 나타내었다.Continuous culture fermenter for cultivating kimchi according to the present invention is divided into a fermenter and a mixer, a medium and a culture medium reservoir, a pH adjusting device, the schematic diagram of the fermenter is shown in FIG.
1) 발효조와 혼합기1) Fermenter and Mixer
발효조는 멸균 가능한 유리 재질의 1ℓ 용기를 사용한다. 배지와 배양액, pH 조절액의 혼합을 위하여 마그네틱 스터러를 설치하여 사용하며, pH 측정을 위한 pH전극을 설치하였다.The fermenter uses a 1 L container of sterile glass. A magnetic stirrer was used to mix the medium, the culture medium, and the pH control solution, and a pH electrode for pH measurement was installed.
2) 배지와 배양액 저장고2) Medium and culture reservoir
배지와 배양액 저장고도 멸균 가능한 용기를 사용하였으며, 배지와 배양액이 정량적으로 유출입시키기 위하여 연동 펌프를 설치하였다.A sterilizable container was also used for the medium and the culture reservoir, and a peristaltic pump was installed to quantitatively flow the medium and the culture medium.
3) pH 조절장치3) pH controller
유산균이 생장하면서 생성되는 유산에 의하여 pH가 낮아져 생장이 저해되는 것을 방지하기 위하여 pH 조절장치를 설치하였으며, 조절장치로 pH가 6.5 이하이면 작동하는 연동 펌프(2)를 설치하여 0.1N-NaOH를 주입케 하였다.In order to prevent the pH from being lowered by the lactic acid produced by lactic acid bacteria growth and inhibiting the growth, a pH control device was installed. When the pH is less than 6.5, a peristaltic pump (2) is installed to operate 0.1N-NaOH. Injection.
4) 배지의 유량4) flow rate of the medium
배지의 저장고로부터 발효조로 유입되는 배지의 유량은 루코노스탁 가시코미타툼의 배가시간이 1.3시간이며, 발효조의 용량이 1ℓ이므로 환산하면 유량이 13㎖/min였다.The flow rate of the medium flowing into the fermenter from the reservoir of the medium was 1.3 hours for the doubling time of the Lukonstock Kashikomitatum, and the capacity of the fermenter was 1 L, so that the flow rate was 13 ml / min.
본 발명은 배추 폐기물을 이용하여 고부가가치의 유산균 생체량을 대량생산할 수 있는 배양용 배지를 제공하는 효과를 가진다. 상기 배지에서 가장 대량으로 배양될 수 있는 균주는 루코노스탁 가시코미타툼이다. 본 발명에 따르면, 폐기물 처리가 문제되던 대량의 배추 폐기물을 처리함과 동시에 유산균 생체량을 대량으로 얻을 수 있게 된다.The present invention has the effect of providing a culture medium capable of mass production of high value-added lactic acid bacteria biomass using cabbage waste. The strain that can be cultured in the bulk in the medium is Lukonostach spycomitatum. According to the present invention, a large amount of lactic acid bacteria biomass can be obtained while treating a large amount of cabbage waste, which has been a problem of waste treatment.
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