KR0148340B1 - Method of antibiotic bacteriocin using lactobacillus acidophilus - Google Patents
Method of antibiotic bacteriocin using lactobacillus acidophilusInfo
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- KR0148340B1 KR0148340B1 KR1019950014279A KR19950014279A KR0148340B1 KR 0148340 B1 KR0148340 B1 KR 0148340B1 KR 1019950014279 A KR1019950014279 A KR 1019950014279A KR 19950014279 A KR19950014279 A KR 19950014279A KR 0148340 B1 KR0148340 B1 KR 0148340B1
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Abstract
본 발명은 공시균주로부터 선별된, 우유배지에서의 증식이나 혐기조건에서의 성장 및 내산성, 내담즙성이 우수하며, 베타-갈락토시데이즈(β-galactosidase) 활성이 높은 락토바실루스 아시도필루스(KCTC 3145) 균주와, 전기 균주의 배양을 위한 배지 및 전기 균주를 이용하여 경제적으로 유산균 원말을 제조하는 방법을 제공한다. 본 발명의 락토바실루스 아시도필루스 균주는 프로바이오틱스에 적합하며, 본 발명의 제조방법을 이용하면 경제적으로 균체를 생산할 수 있고, 균체를 함유하는 제품중에서의 균체 생존률을 높이는 효과를 얻을 수 있다. 따라서, 본 발명은 프로바이오틱스의 제조에 이용될 수 있을 것이다.The present invention is Lactobacillus asidophilus, which is selected from the test strains, has excellent growth, acid resistance, and bile resistance in milk medium, anaerobic conditions, and high beta-galactosidase activity. (KCTC 3145) Provides a method for producing lactic acid bacteria economically using the strain, the medium for the cultivation of the electric strain and the electric strain. The Lactobacillus asidophilus strain of the present invention is suitable for probiotics, and by using the production method of the present invention, it is possible to economically produce cells and obtain an effect of increasing cell viability in products containing the cells. Thus, the present invention may be used to prepare probiotics.
Description
본 발명은 락토바실루스 아시도필루스(Lactobacillus acidophilus) 균주를 이용한 유산균 원말의 제조방법에 관한 것이다. 좀 더 구체적으로, 본 발명은 공시균주로부터 선별된, 우유배지에서의 증식이나 혐기조건에서의 성장 및 내산성, 내담즙성이 우수하며, 베타-갈락토시데이즈(β-galactosidase) 활성이 높은 락토바실루스 아시도필루스(KCTC 3145) 균주와, 전기 균주의 배양을 위한 배지 및 전기 균주를 이용하여 경제적으로 유산균 원말을 제조하는 방법에 관한 것이다.The present invention relates to a method for producing lactic acid bacteria raw material using Lactobacillus acidophilus strain. More specifically, the present invention is a lactose selected from the test strain, the growth in the milk medium or growth under anaerobic conditions, acid resistance, bile resistance, high beta-galactosidase (β-galactosidase) activity Bacillus asidophilus (KCTC 3145) strain, a medium for the cultivation of the electric strain and a method for producing a raw lactic acid bacteria economically using the electric strain.
일반적으로 미생물이 프로바이오틱스(probiotics)로 작용하기 위해서는, 다음과 같은 몇가지 성질들을 가지고 있어야 한다.In general, in order for microorganisms to act as probiotics, they must have some properties:
⑴ 원래 숙주에 존재하는 상재균(常在菌)일 것.상 Be a native flora present in the host.
⑵ 위산, 담즙산 등이 존재하는 소화관 상부의 악조건을 견딜 수 있을것.⑵ Will be able to withstand the adverse conditions of the digestive tract where stomach acid and bile acids are present.
⑶ 하부 소화관에서 증식가능할 것.증식 Proliferate in the lower digestive tract.
⑷ 정장작용 등과 같은 유용효과가 있을 것.유용 Have useful effects such as dressing.
⑸ 식품첨가제로 쓰일 경우 식품속에서 생균이 저장중 생존하고, 또한 충분한 안정성이 보장될 것.⑸ When used as a food additive, live bacteria in food survive during storage and sufficient stability is guaranteed.
⑹ 부작용이 없을 것.⑹ No side effects.
⑺ 섭취 및 음용방법이 용이할것, 및,⑺ easy to ingest and drink, and,
⑻ 경제적일 것 등이 고려되어야 한다.O Economics should be considered.
한편, 유산균중 위를 통과하여 장내에 도달할 수 있고, 또한 장내에서 정착할 수 있는 미생물로는 락토바실루스 아시도필루스(Lactobacillus acidophilus), 락토바실루스 카자이(L. casei) 및 비피도박테리움 종(Bifidobacterium spp.)등이 알려져 있다. 그러나, 현재 시판되고 있는 균주들은 전술한 프로바이오틱스로 사용되기 위해 요구되는 조건 중 내산성 및 내담즙성이 약하여 소화관 상부의 악조건을 견지디 못하는 등의 조건을 충분히 만족시키지 못하는 문제점이 있었다.Among the lactic acid bacteria, microorganisms that can reach the intestine through the stomach and can also settle in the intestine are Lactobacillus acidophilus, Lactobacillus kazai (L. casei) and Bifidobacterium spp. (Bifidobacterium spp.) Are known. However, currently commercially available strains have a problem in that they do not sufficiently satisfy conditions such as not being able to withstand the adverse conditions of the upper digestive tract due to weak acid and bile resistance among the conditions required for use as the aforementioned probiotics.
이에, 본 발명자들은 공시균주로부터 우유배지에서의 증식, 혐기적 조건에서의 성장, 내산성, 내담즙성 면에서 우수할 뿐 아니라, 병원성 균주인 클로스트리디움 퍼프링겐스(Clostridium perfringens)의 생육을 억제하고, 시판되고 있는 유산균 정장제와 비교하여 베타-갈락토시데이즈(β-galactosidase) 활성도가 높은 성질을 가지고 있는 균주를 선별하고자 예의 연구 노력한 결과, 한국과학기술원 생명공학 연구소 유전자은행(KCTC)에 기탁되어 있는 공식균주인 락토바실루스 아시도필루스(KCTC 3145)가 이러한 조건에 부합되는 것을 발견하고, 본 발명을 완성하게 되었다.Therefore, the inventors of the present invention not only have excellent growth in milk medium, growth in anaerobic conditions, acid resistance, and bile resistance from test strains, but also inhibit the growth of Clostridium perfringens, a pathogenic strain. As a result of careful research to select strains having higher beta-galactosidase activity compared to commercially available lactic acid bacterium formants, they were deposited in the KCTC. The formula strain Lactobacillus asidophilus (KCTC 3145) was found to meet these conditions, the present invention was completed.
본 발명자들은 프로바이오틱스의 제조에 있어 가장 관건이 되는 프로바이오틱 효과를 보유하는 균체를 선별하고, 전기 균체의 경제적 배양을 위한 값싼 배지 및 생산된 제품 중의 균체의 생존률을 높이기 위한 수확방법을 개발하고자 하였다.The inventors of the present invention aimed to select a cell having the probiotic effect which is the most important factor in the production of probiotics, and to develop a cheap medium for economic cultivation of electric cells and a harvesting method for increasing the survival rate of the cells in the produced product. .
결국 본 발명의 목적은 프로바이오틱스에 적합한 락토바실루스 아시도필루스 균주를 경제적으로 배양하기 위한 배지조성물을 제공하는 것이다. 본 발명의 다른 목적은 전기균주를 이용하여 유산균 원말을 제조하는 방법을 제공하는 것이다.After all, an object of the present invention is to provide a medium composition for economically culturing Lactobacillus asidophilus strains suitable for probiotics. It is another object of the present invention to provide a method for preparing lactic acid bacteria raw material using an electric strain.
이하, 본 발명을 보다 상세히 설명하고자 한다.Hereinafter, the present invention will be described in more detail.
우선, 본 발명의 락토바실루스 아시도필루스 균주가 소화관 상부의 악조건을 견딜 수 있는 프로바이오틱스로서의 조건에 적합한지를 알아보았다. 즉, 본 발명의 균주가 소화관 상부의 강산성 조건을 견디어 낼 수 있는지의 여부를 확인하기 위해, 전기의 균주를 pH 1,2, 및 3에서 각각 3시간동안 배양한 결과, pH 2에서 3시간까지 높은 생존률을 보여, 본 발명의 균주가 내산성이 매우 높은 균주임을 확인하였다. 또한, 병원성 균주인 클로스트리디움 퍼프링겐스에 대한 항균성을 알아본 결과, 본 발명의 균주가 전기 균주에 대하여 길항작용을 나타내는 것으로 밝혀져, 유효한 항균력을 보유함을 알 수 있었다.First, it was examined whether the Lactobacillus asidophilus strain of the present invention was suitable for conditions as probiotics capable of withstanding the adverse conditions of the upper digestive tract. That is, in order to confirm whether the strain of the present invention can withstand the strong acid conditions of the upper digestive tract, the previous strain was incubated for 3 hours at pH 1,2, and 3, respectively, until pH 2 to 3 hours By showing a high survival rate, it was confirmed that the strain of the present invention is a very high acid resistance strain. In addition, as a result of examining the antimicrobial activity against the pathogenic strain Clostridium perpringens, it was found that the strain of the present invention exhibits an antagonistic action against the electric strain, and has an effective antimicrobial activity.
한편, 본 발명의 균주는 아시아인 중 성인의 약 90% 이상이 겪고 있는 유당불내증(lactose intolerance)을 완화시킬 수 있는 베타-갈락토시데이즈 효소를 생성할 수 있는 것으로 알려지고 있다. 이와 관련하여, 본 발명에서는 시판되고 있는 생균제로부터 분리, 동정된 락토바실루스 아시도필루스 균주와 본 발명의 균주에 대하여 베타-갈락토시데이즈 효소의 활성을 비교하였는데, 본 발명의 균주가 매우 높은 베타-갈락토시데이즈 활성을 지니는 것을 확인할 수 있었다.On the other hand, the strain of the present invention is known to be able to produce beta-galactosidase enzyme that can alleviate lactose intolerance suffered by more than about 90% of adults among Asians. In this regard, the present invention compared the activity of beta-galactosidase enzyme against the Lactobacillus asidophilus strain isolated from the commercial probiotic and the strain of the present invention, the strain of the present invention is very high It was confirmed that it has beta-galactosidase activity.
본 발명의 균주는 바람직하게는 증류수 1000ml에 유청분말(whey powder) 1 내지 10%(w/v), 싸이오톤(Thiotone) 0.1 내지 1.0%(w/v), 트윈(Tween) 80 0.01 내지 0.1%(w/v), 및 탄산칼슘 0.1 내지 0.5%(w/v)을 함유하는 배지에서 배양된다. 이때, 탄산칼슘의 첨가는 락토바실러스 아시도필러스균이 산에 의하여 생육저해를 받는 것으로 알려져 있으므로 균주의 배양 중 일어나는 pH의 저하를 막는 역할을 한다. 본 발명자들은 배지에 탄산칼슘을 첨가하여 균주의 배양 중 pH의 저하를 억제하는 실험을 수행한 결과, pH 값이 4.2에서 4.9까지 상승하고, 세포농도가 증가함을 확인하였다.The strain of the present invention is preferably 1000 ml of distilled water, 1 to 10% (w / v) whey powder, 0.1 to 1.0% (w / v) thiotone, Tween 80 0.01 to 0.1 culture in a medium containing% (w / v) and 0.1 to 0.5% (w / v) calcium carbonate. At this time, the addition of calcium carbonate is known to be inhibited by Lactobacillus asidophilus growth by acid serves to prevent the decrease in pH during the culture of the strain. The present inventors conducted experiments to suppress the lowering of the pH during the culture of the strain by adding calcium carbonate to the medium, it was confirmed that the pH value is increased from 4.2 to 4.9, the cell concentration is increased.
또한, 본 발명의 균주를 이용하여 유산균 원말을 제조하는 과정에서 동결건조시에 탄산나트륨을 0.01 내지 0.1(w/v) 첨가한 결과, 배양액 1000ml당 얻어진 동결건조된 균체의 건조 중량 및 동결된 균체의 건조중량당 생균수에는 영향을 미치지 않으나, 동결건조된 균체의 g당 생균수를 현저히 증가시켜 건조과정에서의 세포 생존률을 높이는 효과를 나타내는 것을 확인하였다.In addition, as a result of the addition of 0.01 to 0.1 (w / v) of sodium carbonate during lyophilization during the preparation of the lactic acid bacteria powder using the strain of the present invention, the dry weight of the lyophilized cells obtained per 1000 ml of the culture medium and the frozen cells It did not affect the number of viable cells per dry weight, but it was confirmed that the increase in viable cells per gram of lyophilized cells to increase the cell survival rate during the drying process.
따라서, 본 발명의 락토바실루스 아시도필루스(KCTC 3145)는 우유배지에서의 증식, 혐기적 조건에서의 성장, 내산성, 내담즙성 면에서 우수할 뿐 아니라, 병원성 균주인 클로스트리디움 퍼프링겐스(Clostridium perfringens)의 생육을 억제하고, 유당불내증을 완화시킬 수 있는 매우 높은 베타-갈락토시데이즈 활성을 보유하고 있어, 프로바이오틱스로서의 조건에 부합하므로 유산균 제제로 널리 이용될 수 있을 것이다.Therefore, Lactobacillus asidophilus (KCTC 3145) of the present invention is excellent in terms of growth in milk medium, growth in anaerobic conditions, acid resistance, and bile resistance, as well as the pathogenic strain Clostridium ppringen It has a very high beta-galactosidase activity that can inhibit the growth of (Clostridium perfringens) and alleviate lactose intolerance, and can be widely used as a lactic acid bacterium preparation because it meets the conditions as a probiotic.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 국한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.
[실시예 1]Example 1
내산성의 확인Confirmation of acid resistance
본 발명의 락토바실루스 아시도필루스(KCTC 3145)균주가 강산성 조건인 pH 1,2, 및 3에서 3시간동안 얼마나 생존하는지를 조사하였다. 그 결과 pH 2에서 3시간까지 82%의 생존률을 가지고 있어, 내산성이 매우 높음을 알 수 있었다(참조; 표 1).Lactobacillus asidophilus (KCTC 3145) strains of the present invention were examined for survival for 3 hours at pH 1, 2, and 3, which are strongly acidic conditions. As a result, it has a survival rate of 82% from pH 2 to 3 hours, it was found that the acid resistance is very high (see Table 1).
[실시예 2]Example 2
클로스트리디움 퍼프링겐스 균주에 대한 길항작용의 확인Identification of antagonism against Clostridium ppringen strains
본 발명의 락토바실루스 아시도필루스(KCTC 3145)균주가 병원성 균주인 클로스트리디움 퍼프링겐스(ATCC 13124) 균주에 대해서 길항작용을 나타내는지를 조사하였다(참조; 표 2). 하기 표 2에서 보듯이, 본 발명의 균주가 한천배지 위에서 전기 클로스트리디움 퍼프링겐스 균주에 대해 길항작용을 나타내는 것을 확인하였으며, 더우기 0.15%의 옥스갈(oxgall)이 포함된 인체의 장과 유사한 배양조건에서도 항균력이 있음을 확인하였다.It was examined whether the Lactobacillus asidophilus (KCTC 3145) strain of the present invention showed antagonistic activity against the Clostridium puffingens (ATCC 13124) strain, which is a pathogenic strain (see Table 2). As shown in Table 2, it was confirmed that the strain of the present invention exhibits an antagonism against the Clostridium puffingen strain strain on agar medium, moreover, similar to the human intestine containing 0.15% of oxgall (oxgall) It was confirmed that there is an antimicrobial activity in culture conditions.
[실시예 3]Example 3
베타-갈락토시데이즈 효소활성에 대한 확인Confirmation of Beta-galactosidase Enzyme Activity
락토바실루스 아시도필루스가 생성하는 베타-갈락토시데이즈 효소의 효과를 본 발명의 균주와 시판되고 있는 생균제로부터 분리, 동정된 락토바실루스 아시도필루스 균주를 이용하여 비교하였다. 그 결과 본 발명의 균주는 매우 높은 베타-갈락토시데이즈 활성을 나타내었다(참조; 표 3).The effects of beta-galactosidase enzyme produced by Lactobacillus asidophilus were compared using the strain of the present invention and the identified Lactobacillus asidophilus strains from commercially available probiotics. As a result, the strain of the present invention showed very high beta-galactosidase activity (see Table 3).
[실시예 4]Example 4
균주의 배양Cultivation of Strains
증류수 1000ml속에 유청분말(whey powder) 50g, 트윈(Tween) 801g, 싸이오톤(Thiotone) 5g을 함유하는 배지에서 락토바실루스 아시도필루스(KCTC 3145) 균주를 37。C에서 배양하였을때, 생장곡선은 15내지 20시간 사이에 대수생장기에서 정지기로 바뀌었다. 대수생장기의 후반부 또는 정지기 초기의 세포가 동결건조시 생존률이 높다고 알려져 있기 때문에, 본 발명의 균주의 배양시간을 15내지 20시간으로 제한하였다.Growth curves of Lactobacillus asidophilus (KCTC 3145) at 37 ° C Changed from algebraic to stationary in 15 to 20 hours. Since the cells in the late or early stages of algebraic growth are known to have a high survival rate during lyophilization, the culture time of the strain of the present invention was limited to 15 to 20 hours.
[실시예 5]Example 5
균체의 성장에 대한 탄산칼슘의 영향Effect of Calcium Carbonate on Growth of Cells
락토바실루스 아시도필루스균은 산에 의하여 생육저해를 받는 것으로 알려져 있다. 따라서, 균주의 배양 중 일어나는 pH의 저하를 막는다면 세포의 농도를 더욱 높일 수 있을 것이므로, 배지에 탄산칼슘을 첨가하여 균주의 배양 중 pH의 저하를 억제하도록 하였다(참조; 표 4). 표 4에서 보듯이, 배지 1000ml 당 2.5g의 탄산칼슘을 첨가함으로써, 20시간의 배양중 pH가 4.2에서 4.9까지 올라갔으며, 세포 농도 또한 18%가 증가하였다.Lactobacillus asidophilus is known to be inhibited by acid growth. Therefore, if the pH of the strain is prevented from lowering, the concentration of the cells may be further increased. Therefore, calcium carbonate was added to the medium to suppress the lowering of pH during the culture of the strain (see Table 4). As shown in Table 4, by adding 2.5 g of calcium carbonate per 1000 ml of medium, the pH rose from 4.2 to 4.9 during 20 hours of culture, and the cell concentration also increased by 18%.
[실시예 6]Example 6
균주의 동결 및 동결건조시 첨가되는 탄산나트륨의 균체수와 중량에 대한 효과Effect of Sodium Carbonate Added on Freeze and Lyophilization on Cell Count and Weight
탄산나트륨이 -70。C에서 10시간 보관되는 균체의 생균수와 동결건조되는 균체의 생균수 및 중량에 미치는 영향을 조사하였다(참조; 표 5). 그 결과, 배지에 0.01 내지 0.1%(w/v) 범위의 탄산나트륨의 첨가는 배양액 1000ml당 얻어진 동결건조된 균체의 건조 중량에 영향을 주지 않는 것으로 확인 되었으며, 동결된 균체의 건조중량당 생균수에는 영향을 미치지 못했으나, 동결건조된 균체의 g당 생균수를 현저히 증가시켜 건조과정에서의 세포 생존률을 8%까지 높이는 효과를 나타내는 것을 알 수 있었다.The effect of sodium carbonate on the viable cell number and weight of the cells stored at -70 ° C for 10 hours and the lyophilized cells (see Table 5). As a result, it was confirmed that the addition of sodium carbonate in the range of 0.01 to 0.1% (w / v) did not affect the dry weight of the lyophilized cells obtained per 1000 ml of culture medium. Although it did not affect, it was found that the viable cells per gram of the lyophilized cells increased significantly to increase the cell survival rate to 8% during the drying process.
이상에서 상세히 설명하고 입증하였듯이, 본 발명은 프로바이오틱스에 적합한 락토바실루스 아시도필루스 균주 및 이를 이용하여 유산균 원말을 제조하는 방법을 제공한다. 본 발명의 락토바실루스 아시도필루스 균주는 우유배지에서의 증식, 혐기적 조건에서의 성장, 내산성, 내담즙성 면에서 우수할 뿐 아니라, 병원성 균주인 클로스트리디움 퍼프링겐스(Clostridium perfringens)의 생육을 억제하고, 유당불내증을 완화시킬 수 있는 매우 높은 베타-갈락토시데이즈 활성을 보유하고 있으므로, 프로바이오틱스로서의 제조에 적합하다. 아울러, 본 발명의 제조방법을 이용하면 경제적으로 프로바이오틱스 균체를 생산할 수 있고, 전기 균체를 함유하는 제품 중에서의 균체의 생존률을 높이는 효과를 얻을 수 있다. 따라서, 본 발명은 프로바이오틱스의 제조에 이용될 수 있을 것이다.As described and demonstrated in detail above, the present invention provides a Lactobacillus asidophilus strain suitable for probiotics and a method for producing lactic acid bacteria raw material using the same. Lactobacillus asidophilus strains of the present invention are excellent in terms of growth in milk medium, growth in anaerobic conditions, acid resistance and bile resistance, as well as the pathogenic strain Clostridium perfringens (Clostridium perfringens) It possesses very high beta-galactosidase activity that can inhibit growth and alleviate lactose intolerance, making it suitable for production as probiotics. In addition, by using the production method of the present invention it is possible to economically produce probiotics cells, it is possible to obtain the effect of increasing the survival rate of the cells in the product containing the cells. Thus, the present invention may be used to prepare probiotics.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20030097235A (en) * | 2002-06-20 | 2003-12-31 | (주) 피엘바이오 | Medium for lactic acid producing bacteria not affecting taste, color and flavour of product |
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| KR20030097235A (en) * | 2002-06-20 | 2003-12-31 | (주) 피엘바이오 | Medium for lactic acid producing bacteria not affecting taste, color and flavour of product |
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