KR100434610B1 - Lactobacillus acidophilus 30SC - Google Patents
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Abstract
본 발명은 락토바실러스 애시도필러스에 관한 것으로서, 보다 구체적으로는 내산성, 담즙산 내성이 우수하고, 콜레스테롤 흡착 능력이 있으며, 병원성 미생물과 부패성 미생물의 생육을 억제시키는 항균 능력을 보유하여 식품, 의약품, 생활용품에 광범위하게 사용될 수 새로운 락토바실러스 애시도필러스 30SC 에 관한 것이다.The present invention relates to Lactobacillus ashdophyllus, and more specifically, has excellent anti-acid, bile acid resistance, cholesterol adsorption capacity, antimicrobial ability to inhibit the growth of pathogenic microorganisms and perishable microorganisms, A new Lactobacillus ashidophilus 30SC which can be used extensively in household goods.
인체나 동물의 장에 서식하면서 여러 가지 유익한 작용을 미생물들이 있는데 애시도필러스 유산균이 대표적인 장내 유익한 미생물 중의 하나이다. 애시도필러스는 현재, 발효유와 생균제 등에 널리 사용되고 있는 프로바이오틱 유산균이다.There are microorganisms that have various beneficial effects while living in the intestines of humans or animals. Ashdophilus lactic acid bacteria are one of the representative intestinal beneficial microorganisms. Ashdophilus is a probiotic lactic acid bacterium currently widely used in fermented milk and probiotic.
본 발명은 사람의 분변으로부터 분리되어 내산성 및 내담즙성이 우수하고, 병원성 미생물과 부패성 미생물의 생육을 억제시키는 항균능력이 있는 새로운 균주인 락토바실러스 애시도필러스(Lactobacillus acidophilus)30SC(기탁기관 : 한국종균협회, 수탁일 : 2000년 11월 22일, 수탁번호 : KFCC-11237)를 제공함에 있다.The present invention is isolated from human feces, excellent acid resistance and bile resistance, Lactobacillus acidophilus ( SC ) which is a new strain having an antimicrobial ability to inhibit the growth of pathogenic microorganisms and decaying microorganism ( SC ) Korea Seeker Association, Accession Date: November 22, 2000, Accession No .: KFCC-11237).
본 발명에 따른 락토바실러스 애시도필러스 30SC는 생리적 기능이 뛰어나 식품, 의약품, 생활용품에 광범위하게 사용될 수 있다.Lactobacillus ashidophilus 30SC according to the present invention is excellent in physiological function can be widely used in food, medicine, household goods.
Description
본 발명은 락토바실러스 애시도필러스(Lactobacillus acidophilus) 30SC(기탁기관 : 한국종균협회, 수탁일 : 2000년 11월 22일, 수탁번호 : KFCC-11237)에 관한 것으로서, 보다 구체적으로는 내산성, 담즙산 내성이 우수하고, 콜레스테롤 흡착 능력이 있으며, 병원성 미생물과 부패성 미생물의 생육을 억제시키는 항균 능력을 보유하여 식품, 의약품, 생활용품에 광범위하게 사용될 수 새로운 락토바실러스 애시도필러스에 관한 것이다.The present invention relates to Lactobacillus acidophilus 30SC (Deposit Organization: Korean spawn association, Deposit Date: November 22, 2000, Accession No .: KFCC-11237), more specifically acid resistance, bile acid It is related to a new Lactobacillus ashdophyllus which has excellent resistance, has an ability to absorb cholesterol, and has antibacterial ability to inhibit the growth of pathogenic microorganisms and perishable microorganisms, which can be widely used in food, medicine and household goods.
생균제 (프로바이오틱스:Probiotics)라는 말은 'for life'라는 뜻으로 그리스어에서 유래되었다. 생균제의 정의는 1965년 미생물이 분비하는 물질로 다른 미생물의 성장을 자극하는 물질로 정의되었고, 1874년 '숙주동물에게 유익한 효과를 주는 생물 또는 물질' 이라고 정의하고 있다.The word probiotics comes from the Greek word for for life. The definition of probiotic was defined in 1965 as a substance secreted by microorganisms and a substance that stimulates the growth of other microorganisms, and in 1874, it was defined as an organism or substance that has a beneficial effect on a host animal.
그 후에 풀러 (Fuller)는 1989년에 생균제를 정의하기를 '장내균총을 개선시킴으로서 숙주에게 유익한 영향을 주는 생균제제'라고 하여 살아있는 미생물에 대한 개념이 도입되었다.Fuller then introduced the concept of living microorganisms in 1989, defining probiotics as 'probiotics that have beneficial effects on the host by improving the intestinal flora.'
최근에 와서 장내세균에 대한 연구가 계속되면서 장내균총에 대한 중요성이 새롭게 인식되기 시작하였는데, 무균동물이 정상적인 균총을 형성하고 있는 동물에 비해 살로넬라와 클로스트리디움속의 미생물 감염에 의해 쉽게 사망하였거나 발병률이 높았다고 하였다.With the recent research on enterobacterial bacteria, the importance of enterobacterial flora has been newly recognized. Aseptic animals are more likely to be killed or affected by microbial infections in Salonella and Clostridium than those that form normal flora. Was high.
사람의 장내 구성 균총에 대한 연구 결과 비피더스균과 락토바실러스속의 유산균 구성비율이 높게 나타났으며 그중 건강한 사람의 장내에서 분리되는 주요 유산간균들은 락토바실러스 애시도필러스, 락토바실러스 카제이, 락토바실러스 훠멘텀, 락토바실러스 플랜타럼 등인 것으로 보고되고 있고, 이러한 유산균들은 현재 전 세계적으로 발효유제품의 종균, 생균제제 등으로 널리 이용되고 있다.As a result of research on the human intestinal constituent flora, the ratio of bifidus and lactobacillus was high in Lactobacillus, among which the major lactic acid bacterium isolated from the intestine of healthy people was Menthol, Lactobacillus plantarum, etc. are reported, these lactic acid bacteria are now widely used as spawn, probiotic agent, etc. of fermented milk products worldwide.
이중에서도 락토바실러스 애시도필러스 균은 콜레스테롤 억제기능을 비롯해서 사람이나 동물에게 유익한 효과를 준다는 여러 연구 보고가 있다.Among them, Lactobacillus ashdophilus is reported to have a beneficial effect on humans and animals, including cholesterol suppression function.
이러한 유산균이 생균제로서의 역할을 수행하기 위해서는,In order for these lactic acid bacteria to act as probiotics,
첫째, 장내에 정상적으로 존재하는 균주 이어야 하고,First, the strain must be normally present in the intestines,
둘째, 소화기관을 통과하면서 생존해야 하며,Second, they must survive through the digestive system,
셋째, 숙주에게 유익한 효과를 주어야 하고,Third, it should have a beneficial effect on the host,
넷째, 장내에서 증식 또는 정착할 수 있어야 하며,Fourth, it must be able to proliferate or settle in the intestines,
다섯째, 식품 제조 시에 식품의 품질에 손상을 주어서는 안 된다.Fifth, food quality should not be impaired in food manufacturing.
이와 같은 조건 이외에도 기본적으로 병원성 미생물이 아니어야하며 독성이 없어야 한다.In addition to these conditions, they should be basically non-pathogenic and non-toxic.
그러나, 지금까지 개발된 유산균들은 대부분 사람이 아닌 자돈 등의 가축이나 동물에서 분리되었으나, 사람에게 사용할 경우 안전성 문제가 있었고,However, most of the lactic acid bacteria developed so far have been isolated from livestock or animals such as piglets, but not humans, but there are safety problems when used in humans.
특히, 내산성과 내즙산성이 미약하여 생균제로서의 역할은 기대하기 어려운 실정이었다.In particular, the role as a probiotic was difficult to expect due to the weak acid resistance and acid resistance.
본 발명자들은 종래의 유산균의 기능성 문제에 관하여 오랜 시간 연구해 오던 중 사람의 분변으로부터 살아있는 균주를 분리함으로서 종래의 락토바실러스 애시도필러스균에 비하여 위산 및 담즙에서 생존할 수 있으며 사람에게 안전하여 생리적 효능이 우수하고 항균물질도 생산하는 균주를 얻는데 성공하였다.The present inventors have been living for a long time on the functional problems of conventional lactic acid bacteria by separating the living strain from human feces can survive in gastric acid and bile compared to the conventional Lactobacillus ashdophilus bacteria and is safe for human physiological efficacy It was successful to obtain a strain that produces excellent and antimicrobial substances.
즉, 본 발명은 사람의 분변으로부터 분리되어 내산성 및 내담즙성이 우수하고, 병원성 미생물과 부패성 미생물의 생육을 억제시키는 항균능력이 있는 새로운 균주인 락토바실러스 애시도필러스 30SC(기탁기관 : 한국종균협회, 수탁일 : 2000년 11월 22일, 수탁번호 : KFCC-11237호)를 제공함에 있다.That is, the present invention is isolated from human feces and excellent acid resistance and bile resistance, Lactobacillus ashdophyllus 30SC is a new strain having an antimicrobial ability to inhibit the growth of pathogenic microorganisms and perishable microorganisms (deposit institution: Korean spawn) Association, Date of Trust: November 22, 2000, Accession No .: KFCC-11237).
이하, 본 발명에 따른 락토바실러스 애시도필러스 30SC의 분리과정과 그 특성에 대하여 상세히 살펴보기로 한다.Hereinafter, the separation process of Lactobacillus ashidophilus 30SC and its characteristics according to the present invention will be described in detail.
〈 락토바실러스 애시도필러스 30SC의 분리 〉〈Isolation of Lactobacillus ashidophilus 30SC〉
본 발명에 따른 유산균은 인체의 분변을 채취하여 생리 식염수로 연속적으로 희석한 후 브로모크레졸 퍼플이 함유된 앰알에스 고체배지에 도말하여 37℃ 배양기에서 24시간 배양한 후 노란색을 나타내는 집락을 얻었다.Lactobacillus according to the present invention was collected by diluting the feces of the human body continuously with physiological saline and then plated in a solid medium containing bromocresol purple and cultured in a 37 ℃ incubator for 24 hours to obtain a yellow colony.
그 결과, 본 발명의 애시도필러스 30SC균주는 내산성이 우수하였으며, 0.35. 0.5%의 담즙산염이 함유된 배지에서도 성장이 가능하였다.As a result, the Ashdophilus 30SC strain of the present invention was excellent in acid resistance, 0.35. Growth was also possible in medium containing 0.5% bile salts.
또한, 높은 콜레스테롤 흡착성질을 가지고 있을 뿐 아니라 유해 미생물을 사멸시킬 수 있는 항균성 물질을 분비하는 것으로 밝혀졌다.In addition, it has been found to have high cholesterol adsorbents and secrete antimicrobial substances that can kill harmful microorganisms.
본 발명에 따른 락토바실러스 애시도필러스 30SC 균주의 특성은 다음과 같으며 당 발효 특성은 표 1에 나타난 바와 같다.The characteristics of the Lactobacillus ashidophilus 30SC strain according to the present invention are as follows and the sugar fermentation characteristics are as shown in Table 1.
〈균주의 특성〉<Characteristic of strain>
1. 균의 형태 : 막대형 2. 집락 색깔 : 유백색1. Fungal form: rod 2. Colony color: milky white
3. 생육 최적 온도 : 37℃ 4. 운동성 : 없음3. Optimum temperature for growth: 37 ℃ 4. Mobility: None
5. 그람염색 : 양성 6. 카탈라제 : 음성5. Gram staining: positive 6. Catalase: negative
7. 산소 영향 : 편성 혐기성7. Oxygen Effects: Organized Anaerobic
이하, 본 발명에 대하여 실시 예에 의하여 더욱 상세히 설명하고자 한다.Hereinafter, the present invention will be described in more detail with reference to Examples.
[실시예 1]Example 1
내산성 평가Acid resistance evaluation
페하를 2.5로 조정하여 멸균된 앰알에스 액체배지에 0.22 마이크론 필터로 제균시킨 펩신을 배지 밀리리터당 1000 유닛이 되도록 첨가하여 내산성 측정용 인공위액으로 사용하였다.The pH was adjusted to 2.5, and pepsin, which was sterilized with a 0.22 micron filter, was added to the sterilized AMAL liquid medium to be 1000 units per milliliter of the medium, and used as an artificial gastric juice solution for measuring acid resistance.
유산균을 18℃에서 18시간동안 항온수조에서 배양한 다음 원심분리기를 이용하여 셀을 침전시킨 후 생리적 식염수로 침전된 셀을 2회 세척하였다.The lactic acid bacteria were cultured in a constant temperature water bath at 18 ° C. for 18 hours, and then the cells were precipitated using a centrifuge, followed by washing the cells precipitated with physiological saline twice.
이렇게 얻어진 셀을 미리 제조된 인공위액에서 1, 2, 및 3시간 배양시킨 후 생균수를 측정하였다. 생균수 측정 시에는 2중량부 베타-글리세로포스페이트가 함유된 엠알에스 배지를 이용하였다.The cells thus obtained were incubated for 1, 2, and 3 hours in a prepared gastric juice solution and the viable cell count was measured. When measuring the number of viable cells was used MS medium containing 2 parts by weight of beta-glycerophosphate.
그래프 1은 애시도필러스 30SC 와 현재 발효유 제조에 사용되는 애시도필러스 NCFM, 그리고 프로바이오틱 유산균으로 사용되는 ATCC 43121, 애시도필러스 GP1B, 애시도필러스 ATCC 4356 균주들의 내산성을 비교한 결과를 나타낸 것이다.Graph 1 compares the acid resistance of Ashdophilus 30SC, Ashdophilus NCFM currently used in the manufacture of fermented milk, and ATCC 43121, Ashdophilus GP1B, and Ashdophilus ATCC 4356 strains used as probiotic lactic acid bacteria. The results are shown.
그 결과, 본 발명의 애시도필러스 30SC 균주는 페하 2.5에서 3시간 처리했을 때에도 사멸하지 않는 것으로 확인되었다.As a result, it was confirmed that the Ashdophilus 30SC strain of the present invention did not die even when treated for 3 hours at 2.5.
또한, 본 발명자들이 선발한 애시도필러스 30SC균주의 내산성은 기존의 프로바이오틱 유산균으로 사용되는 애시도필러스 NCFM이나 애시도필러스 ATCC 43121 등과 비교했을 때 커다란 차이가 없는 것으로 나타났다.In addition, the acid resistance of the Ashdophilus 30SC strain selected by the present inventors was found to be no significant difference compared to the Ashdophilus NCFM and the Ashdophilus ATCC 43121 and the like used as a conventional probiotic lactic acid bacteria.
[실시예 2]Example 2
내담즙산성 평가Bile Acid Resistance Evaluation
담즙산에 대한 내성은 0.3%의 담즙산 (옥스갈, oxgall)이 함유된 앰알에스 배지에서의 생육 상태를 조사하였다.Tolerance to bile acids was examined for growth in AM medium containing 0.3% bile acid (oxgall).
즉, 유산균을 18℃에서 18시간동안 항온수조에서 배양한 다음 원심분리기를 이용하여 셀을 침전시킨 후 생리적 식염수로 침전된 셀을 2회 세척한 후 담즙산이 함유된 배지에서 생육을 관찰하였다.That is, lactic acid bacteria were incubated in a constant temperature water bath at 18 ° C. for 18 hours, and then the cells were precipitated using a centrifuge. After washing the cells precipitated with physiological saline twice, growth was observed in a medium containing bile acid.
그래프 2는 담즙산에 대한 생육 결과를 나타낸 것으로 애시도필러스 30SC는0.3% 담즙산을 함유한 배지에서도 사멸하지 않아 우수한 담즙산 내성을 보였고, 기존의 프로바이오틱 유산균으로 사용되는 유산균들과 비교했을 때에도 유사한 생육을 보였다.Graph 2 shows the growth results for bile acids. Ashdophilus 30SC did not die in the medium containing 0.3% bile acid, which showed excellent bile acid resistance, similar to that of lactic acid bacteria used as a conventional probiotic lactic acid bacterium. It showed growth.
[실시예 3]Example 3
콜레스테롤 분해능력 평가Cholesterol Degradation Evaluation
엠알에스 액체배지에 thioglycolic acid(티오글리콜릭 엑시드)와 옥스갈 또는 taurocholic acid(타우로콜릭 엑시드)을 각각 0.2 - 0.3 중량% 수준으로 첨가하여 121℃에서 15분간 멸균한 다음 1㎖ 콜레스테롤 미셀을 9㎖ 제조된 배지에 첨가하여 최종 배지에 콜레스테롤 수준이 100㎍/㎖이 되도록 하였다.Thioglycolic acid (thioglycolic acid) and oxgal or taurocholic acid (taurocholic acid) were added to the liquid liquid medium at 0.2-0.3 wt%, respectively, and sterilized for 15 minutes at 121 ° C. It was added to the prepared medium so that the cholesterol level in the final medium was 100 μg / ml.
이 배지에 락토바실러스 애시도필러스 30SC를 배양시켰으며 배양 후 상징액 내에 잔존하는 콜레스테롤 미셀 함량을 다음과 같이 측정하였다.Lactobacillus ashidophilus 30SC was cultured in this medium, and the cholesterol micelle content remaining in the supernatant after culture was measured as follows.
즉, 9.1㎎ 콜레스테롤과 0.2 ㎖ L-α-lecithin(레시틴)를 1㎖의 클로로포름에 용해하여, 질소가스로 건조 후 0.4 M, 10㎖ 슈크로스를 첨가하였고 15분씩 초음파 파쇄를 실시하였다.That is, 9.1 mg cholesterol and 0.2 ml L-α-lecithin (lecithin) were dissolved in 1 ml of chloroform, dried with nitrogen gas, 0.4 M and 10 ml sucrose were added, and ultrasonic fracturing was performed for 15 minutes.
잔류 콜레스테롤의 분석은 0.5㎖ 상징액에 3㎖ 97% 에탄올과 2㎖ 50중량% 수산화칼륨을 첨가한 후 60℃에서 10분간 가열하였으며 상온에서 냉각된 용액에 5㎖ 헥산과 함께 30초간 잘 섞어 콜레스테롤을 추출하였다.Residual cholesterol was analyzed by adding 3ml 97% ethanol and 2ml 50wt% potassium hydroxide to 0.5ml supernatant, and heating at 60 ℃ for 10 minutes, and mixing well with 5ml hexane in a solution cooled at room temperature for 30 seconds. Extracted.
추출된 헥산 2.5㎖을 시험관에 분주하여 질소가스로 건조하였으며, 60℃ 가열을 통해 건조를 촉진하였다.2.5 ml of extracted hexane was dispensed into a test tube and dried with nitrogen gas, and drying was promoted by heating at 60 ° C.
건조된 시험관에 4㎖ o-phtalaldehyde(프탈알데히드)(50㎎/100㎖ 초산) 용액을 첨가한 후 원활한 발색을 위해 10분간 정치하였으며, 정치 후 2㎖ 농황산을 첨가하여 발색을 유도하였다. 농황산 첨가 10분 후 550㎚에서 흡광도를 측정하여 콜레스테롤을 정량 하였다.4 ml o-phtalaldehyde (phthalaldehyde) (50 mg / 100 ml acetic acid) solution was added to the dried test tube and allowed to stand for 10 minutes for smooth color development. After standing, 2 ml concentrated sulfuric acid was added to induce color development. Cholesterol was quantified by measuring the absorbance at 550 nm after 10 minutes of concentrated sulfuric acid.
표 2에서 나타난 바와 같이, 애시도필러스 30SC균주는 29.05%의 콜레스테롤을 흡착하는 것을 보여 중도의 흡착성을 가지고 있었다.As shown in Table 2, the Ashdophilus 30SC strain showed moderate adsorption, showing 29.05% of the adsorption of cholesterol.
이와 반면에 애시도필러스 에이시티시 43121균주는 57%로 가장 높은 흡착성을 보였고 락티스 ATCC 4797균주는 16.26%로 가장 낮게 나타났다.On the other hand, the Ashdophilus acysi 43121 strain showed the highest adsorption with 57% and the Lactis ATCC 4797 strain showed the lowest with 16.26%.
[실시예 4]Example 4
항균활성 측정Antimicrobial Activity
항균활성 평가는 다음과 같이 수행하였다. 애시도필러스 30SC를 앰알에스 액체배지에서 37℃ 항온수조에서 18시간 배양한 다음, 원심분리(6000 g, 30분, 4℃)를 하여 셀을 제거한 다음 페하를 7.0으로 조정하였다.Antimicrobial activity was evaluated as follows. Ashdophilus 30SC was incubated in an AM liquid medium for 18 hours in a 37 ° C. constant temperature water bath, centrifuged (6000 g, 30 minutes, 4 ° C.) to remove the cells, and the pH was adjusted to 7.0.
이를 0.45 마이크론 필터를 이용하여 여과시켜 얻어진 상징액을 앰알에스 고체배지와 티에스 고체배지에 각각 20 마이크로리터식 분주하여 지시균을 중층하였다.The supernatant obtained by filtration with a 0.45 micron filter was 20 microliters each of an AM solid medium and a TS solid medium to superimpose the indicator bacteria.
각 지시균들의 최적 성장 온도에서 18 내지 24시간 배양한 후, 억제환 생성 여부로 항균활성을 측정하였다.After culturing for 18 to 24 hours at the optimum growth temperature of each indicator, the antimicrobial activity was measured by the inhibition of the production of ring.
이때, 유산균은 앰알에스 배지를 기타 미생물은 티에스 배지를 각각 사용하였으며 그 배지 조성물은 다음과 같다.At this time, the lactic acid bacteria were used as the AM medium, and other microorganisms as the TS medium, the medium composition is as follows.
표 3은 항균 스펙트럼을 나타낸 것으로 기존의 유산균이 생산하는 박테리오신과 다른 스펙트럼을 보였으며, 바실러스와 같은 포자형성균과 화농을 일으키는 스타필로코거스 오레우스 그리고 리스테리아 이바노비 등 병원성 미생물의 생육을 억제시키는 것으로 나타났다.Table 3 shows the antibacterial spectrum, which is different from the bacteriocin produced by conventional lactic acid bacteria, and inhibits the growth of pathogenic microorganisms such as spore-forming bacteria such as Bacillus, staphylococcus oreus, and Listeria ivanobi. Appeared.
또한, 락토바실러스 카제이, 락토바실러스 랙티스 ATCC 4797, 락토바실러스훠멘텀 ATCC 11931, 락토바실러스 플랜타럼과 같은 유산간균의 생육을 억제시켰으며, 김치에서 분리한 류코노스톡 스피시스 K2도 억제시키는 것으로 나타났다.In addition, it inhibited the growth of lactobacillus such as Lactobacillus casei, Lactobacillus lactis ATCC 4797, Lactobacillus spmentum ATCC 11931, and Lactobacillus plantarum, and also inhibited leuconosstock spissis K2 isolated from kimchi. appear.
(+) 저해됨; (-) 저해되지 않음(+) Inhibited; (-) Not inhibited
이상에서 상세히 살펴본 바와 같이, 본 발명의 락토바실러스 애시도필러스 30SC는 내산성이 우수하고, 내담즙산성이 뛰어남은 물론, 병원성 미생물과 부패성 미생물의 생육을 억제시키는 항균능력이 기존의 프로바이오틱 유산균 보다 뛰어나고, 또한 콜레스테롤을 저하시키는 능력을 가능하여 인체의 생균제로서의 활용이 가능하고, 발효제품, 유산균 식품에 유용한 균주로서 활용이 제공되며, 동물사료제로서의 첨가제 등으로 활용이 가능하여 산업 상에 매우 폭넓게 활용될 수 있는 효과가 있다.As described in detail above, the Lactobacillus ashidophilus 30SC of the present invention is excellent in acid resistance, excellent in bile acid resistance, as well as the existing probiotic lactic acid bacteria have an antimicrobial ability to inhibit the growth of pathogenic microorganisms and spoilage microorganisms. It can be used as a probiotic of the human body because it is superior to the ability to lower cholesterol, and can be used as a strain useful for fermented products and lactic acid bacteria food, and as an additive as an animal feed agent. There is an effect that can be widely used.
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