KR20020088797A - Novel probiotic strain having alcohol-resistance and its use - Google Patents

Novel probiotic strain having alcohol-resistance and its use Download PDF

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KR20020088797A
KR20020088797A KR1020010027768A KR20010027768A KR20020088797A KR 20020088797 A KR20020088797 A KR 20020088797A KR 1020010027768 A KR1020010027768 A KR 1020010027768A KR 20010027768 A KR20010027768 A KR 20010027768A KR 20020088797 A KR20020088797 A KR 20020088797A
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백현동
정민용
정황영
김천제
김기태
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프로코바이오텍 주식회사
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Abstract

PURPOSE: Provided are an alcohol-resistant probiotic microorganism and the use thereof. The microorganism has high gastric juice and bile acid tolerance, produces beta-galactosidase, so that it can be useful for treatment of lactose intolerance symptom, and can inhibit the infection of Listeria. CONSTITUTION: The alcohol-resistant probiotic microorganism Lactobacillus plantarum MY4(KCCM 10266) is isolated by isolating 100 strains from the Korean feces; separating 28 strains showing similar morphological properties to Lactobacillus sp. from the 100 strains; separating anaerobic gram positive bacillus producing lactic acid from the 28 strains; and separating a strain having high activity of beta-galactosidase and no activity of beta-glucuronidase. The alcohol resistance of Lactobacillus plantarum MY4(KCCM 10266) is examined by culturing Lactobacillus plantarum MY4(KCCM 10266) in an MRS medium at optimal temperature for 12 hours; centrifuging the cultured medium at 6,000 rpm for 10 min to obtain a cell pellet; diluting the cell pellet with a 0.1M phosphate buffer solution; inoculating the diluted cell containing solution into each variable concentration of alcohol containing 0.1M phosphate buffer solution; culturing the solution at 37 deg. C for 4 hours with agitation; and determining the colony forming unit of Lactobacillus plantarum MY4(KCCM 10266) in each cultured solution.

Description

알콜에 내성있는 신규한 유용 프로바이오틱 생균 및 그의 용도{Novel probiotic strain having alcohol-resistance and its use}Novel probiotic strain having alcohol-resistance and its use

본 발명은 알콜에 내성있는 신규한 유용 프로바이오틱 생균 및 그의 용도에 관한 것으로, 위액 및 담즙산에 대한 내성이 높아 장내 생존력이 뛰어나고 알콜내성이 기존 균주에 비해 우수한 프로바이오틱 생균에 관한 것이다.The present invention relates to novel useful probiotic probiotics resistant to alcohol and uses thereof, and to probiotic probiotics having high intestinal viability and high alcohol resistance compared to existing strains due to high resistance to gastric juice and bile acids.

프로바이오틱 생균제란 살아있는 미생물 균제를 섭취함으로써 미생물이 분비하는 효소, 유기산, 비타민 및 무독성 항균물질 등에 의한 장내 균총의 정상화는 물론 장질환 치료를 통해 신체기능 개선을 목적으로 생산된 제품을 말한다(Fuller, R. 1989. Probiotics in man and animals. J. Appl. Bacteriol. 66: 365-378). 이러한 프로바이오틱 생균제는 장내균총의 안정화, 유해세균의 정착억제에 따른 부패산물 생성 감소 및 질병예방, 면역의 활성화, 간접적인 항암작용, 콜레스테롤 저하, 유당 불내증의 감소, 변비억제 및 예방과 관련되어 기능성이 인정되어 있다(Goldin, B.R. and Borbach, S.L. 1977. Alterations in fecal microflora enzymes related to diet, age, lactobacillus supplements, and dimethylhydrazine. Cancer 40: 2421-2426; Fuller, R. 1991. Probiotics in human medicine. Gut. 32: 439-442; Shah, N. 1994.Lactobacillus acidophilusand lactose intolerance, a review. ASEAN Food J. 9: 47-54; Takiguchi et al., 1997.Lactobacillus acidophilusSBT 2062 andBifidobacterium longumSBT 2928 on harmful intestinal bacteria. J. Int. Microbiol. 11: 11-17). 프로바이오틱 생균제로 많이 이용되고 있는 세균은Lactobacillus속, Streptococcus속,Bifidobacterium속 그리고 유포자 유산균인Bacillus속 등이 있다. 한편, 프로바이오틱 생균제로서 균이 가져야 할 이론적 근거는 안전성, 기능적 측면(생존성, 정착성, 서식성, 항미생물제 생성능, 면역 촉진능, antigenotoxic 활성, 병원성 세균의 억제능), 기술적 측면(관능적 특성, 안전성, bacteriophage 저항성, 제조과정중의 생존성), 그리고 GRAS(Generally Recognized As Safe)미생물로서 장내 생존력이 커야 한다는 것이다.Probiotic probiotics refers to products produced for the purpose of improving the body function through normal intestinal flora and treatment of intestinal diseases through the ingestion of live microbial fungi, enzymes, organic acids, vitamins, and non-toxic antibacterial substances secreted by microorganisms (Fuller , R. 1989. Probiotics in man and animals.J. Appl. Bacteriol. 66: 365-378). These probiotic probiotics are related to stabilizing the intestinal flora, reducing the production of decay products by inhibiting the settlement of harmful bacteria, preventing diseases, activating immunity, indirect anticancer activity, lowering cholesterol, reducing lactose intolerance, preventing constipation and preventing Functionality is recognized (Goldin, BR and Borbach, SL 1977. Alterations in fecal microflora enzymes related to diet, age, lactobacillus supplements, and dimethylhydrazine. Cancer 40: 2421-2426; Fuller, R. 1991. Probiotics in human medicine. . Gut 32: 439-442; Shah, N. 1994. Lactobacillus acidophilus and lactose intolerance, a review ASEAN Food J. 9:.. 47-54; Takiguchi et al, 1997. Lactobacillus acidophilus SBT 2062 and Bifidobacterium longum SBT 2928 on harmful intestinal bacteria.J. Int.Microbiol. 11: 11-17). Bacteria that are widely used as probiotic probiotics include Lactobacillus genus , Streptococcus genus, Bifidobacterium genus and Diffuser lactic acid bacterium Bacillus genus. On the other hand, the theoretical basis for bacteria as a probiotic probiotic should be based on safety, functional aspects (survivability, fixability, formatting, antimicrobial production ability, immune promoting ability, antigenotoxic activity, inhibitory ability of pathogenic bacteria) and technical aspects (sensory characteristics). , Safety, bacteriophage resistance, viability during manufacture), and GRAS (Generally Recognized As Safe) microorganisms require high intestinal viability.

본 발명자들은 상기와 같은 점을 착안하여 인체분변으로부터 분리된 유용 유산균 중에서 알콜내성이 시판중인 상업용 프로바이오틱 생균제에 비해 매우 우수한 균주를 선발하고, 상기 균주의 특성을 분석함으로써 본 발명을 완성하였다.The present inventors have completed the present invention by selecting strains with excellent alcohol resistance among commercially available probiotic probiotics among useful lactic acid bacteria isolated from human feces, and analyzing the characteristics of the strains.

따라서, 본 발명의 목적은 알콜에 내성있는 유용 인체용 프로바이오틱 생균을 제공함에 있다.Accordingly, it is an object of the present invention to provide useful human probiotic probiotic resistant to alcohol.

본 발명의 또 다른 목적은 상기 프로바이오틱 생균을 기능성 건강식품 및 의약품 등에 사용하는 용도를 제공함에 있다.Still another object of the present invention is to provide a use of the probiotic probiotic for functional health foods and medicines.

본 발명의 상기 목적은 우리나라 사람의 인체 분변으로부터 락토바실러스 프랜테리움(Lactobacillus plantarum) MY4 균주를 선발 분리하고, 상기 분리한 균주가 알콜에 대한 내성, 인공위액 및 인공 담즙액에 대한 내성을 확인하는 한편, 효소활성, 항생물질에 대한 내성 및 항균활성을 측정하므로써 달성하였다.The object of the present invention is to isolate and isolate the Lactobacillus plantarum MY4 strain from the human feces of the human, and to determine the resistance to alcohol, artificial gastric juice and artificial bile solution On the other hand, this was achieved by measuring enzyme activity, resistance to antibiotics and antimicrobial activity.

도 1은 본 발명 프로바이오틱 생균의 인공 위액에 대한 내성을 나타낸 그래프이다.1 is a graph showing the resistance to artificial gastric juice of the probiotic live bacteria of the present invention.

도 2는 본 발명 프로바이오틱 생균의 인공 담즙산에 대한 내성을 나타낸 그래프이다.Figure 2 is a graph showing the resistance to artificial bile acids of the probiotic live bacteria of the present invention.

도 3은 본 발명 프로바이오틱 생균의Listeria monocytogenesATCC 19111 의 생장 억제능을 조사한 결과이다.3 is a result of the growth inhibition of Listeria monocytogenes ATCC 19111 of the probiotic live bacteria of the present invention.

본 발명은 우리나라 사람의 인체 분변으로부터 락토바실러스 프랜테리움 MY4를 분리하는 단계; 여러 알콜농도에서 상기 균주와 기존 프로바이오틱 균주 PT7, PS490, PS653의 생존율을 비교하므로써 알콜내성을 조사하는 단계; Kobayashi 등의 방법에 따라 인공위액과 인공담즙산에 대한 본 발명 균주의 내성을 측정하는 단계; 그램 염색법 및 API CHL(bioMereux co., France)에 의해 49개의 탄소원에 대한 이용성을 검토함으로써 본 발명 균주의 미생물적 동정을 실시하는 단계; API ZYM 키트를 이용하여 상기 균주의 효소활성을 측정하는 단계; Paper disc방법에 따라 항생물질의 내성을 조사하는 단계 및Listeria monocytogenesATCC 19111에 대한 항균력을 측정하는 단계로 구성된다.The present invention comprises the steps of separating the Lactobacillus planterium MY4 from human feces of the human body; Investigating the alcohol resistance by comparing the survival rates of the strain with existing probiotic strains PT7, PS490, and PS653 at various alcohol concentrations; Measuring the resistance of the strain of the present invention to artificial gastric juice and artificial bile acid according to the method of Kobayashi et al .; Performing microbiological identification of the strains of the invention by examining the availability of 49 carbon sources by Gram staining and API CHL (bioMereux co., France); Measuring enzymatic activity of the strain using an API ZYM kit; Investigating antibiotic resistance according to the paper disc method and measuring the antimicrobial activity against Listeria monocytogenes ATCC 19111.

상기 알콜에 대한 내성, 인공위액에 대한 내성, 인공담즙산에 대한 내성을 측정하는 단계에 있어서, 총균수는 배양액을 0.1% 펩톤수로 10배씩 연속적인 희석을 시킨 다음, 평판배지에 0.1 ml씩 분주하여 도말한 후, 각각의 최적온도에서 배양한 후, 콜로니형성단위(colony forming unit)를 측정하여 총균수를 계산하였다. In the step of measuring the resistance to alcohol, resistance to artificial gastric juice, resistance to artificial bile acid, total bacterial count was diluted 10 times serially with 0.1% peptone water, and then 0.1 ml aliquot to the plate medium. After spreading by incubation at each optimum temperature, the colony forming unit (colony forming unit) was measured to calculate the total number of bacteria.

상기 단계에서 사용한 상업용 프로바이오틱 균주 PT77, PS490, PS653은 국내에서 시판중인 대표적인 프로바이오틱 생균을 분리하여 사용하였으며, 항균활성을 측정하는데 사용한Listeria monocytogenesATCC 19111은 ATCC사로부터 구입하여 사용하였다.Commercial probiotic strains PT77, PS490, and PS653 used in the above steps were used to isolate representative probiotic probiotics that are commercially available in Korea, and Listeria monocytogenes ATCC 19111 used to measure antimicrobial activity was purchased from ATCC.

본 발명의 프로바이토틱 생균을 락토바실러스 프랜테리움(Lactobacillus plantarum) MY4로 명명하고 이를 국제기탁기관인 한국종균협회 미생물 보존센터에 2001년 5월 10일자로 수탁하였다(수탁번호: KCCM-10266).The probiotic probiotic of the present invention was named Lactobacillus plantarum MY4 and it was deposited on May 10, 2001 to the Korea Bacterial Association Microorganism Conservation Center, an international depository institution (Accession Number: KCCM-10266).

이하, 본 발명의 구체적인 방법을 실시예를 들어 상세히 설명하고자 하지만,본 발명의 권리범위는 이들 실시예에만 한정되는 것은 아니다.Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

실시예 1: 본 발명 프로바이오틱 생균의 분리 및 배양Example 1 Isolation and Culture of Probiotic Probiotics of the Invention

본 발명은 프로바이오틱 생균을 분리하기 위하여 우리나라 사람의 인체분변으로부터 100여개의 균을 분리하고 이중 락토바실러스(Lactobacillussp.) 균과 유사한 형태적 특성을 갖는 균주를 28주 선발하였다. 이 균주들을 사용하여 젖산을 생산하는 혐기성 그램양성 간균을 선별하고, 이들 균주 중에서 효소활성을 측정하여 β-갈락토시다제(β-galactosidase)활성이 강하고 β-글루쿠로니다제(β-glucuronidase) 활성이 없는 균주를 선별하고 이를 MY4로 약칭하였다. 상기 분리한 젖산균은 배양배지로 lactobacilli MRS 배지(Difco Laboratories, USA)를 사용하여 배양하였다.In order to isolate probiotic live bacteria, about 100 bacteria were isolated from human feces of Koreans , and 28 strains were selected for strains having similar morphological characteristics as those of Lactobacillus sp. Using these strains, anaerobic Gram-positive bacillus producing lactic acid was selected, and the enzyme activity was measured in these strains, and thus, β-galactosidase activity was strong and β-glucuronidase. ) Inactive strains were selected and abbreviated as MY4. The isolated lactic acid bacteria were cultured using lactobacilli MRS medium (Difco Laboratories, USA) as a culture medium.

젖산균은 순수 분리되어 MRS agar plate에 보존 중인 균주를 10 ml test tube의 MRS 브로스에 한 백금이 접종하여 32℃에서 12시간 정치배양을 하였다.Lactic acid bacteria were purely isolated and preserved on a MRS agar plate, inoculated with platinum in MRS broth of a 10 ml test tube, and cultured at 32 ° C. for 12 hours.

실시예 2: 본 발명 프로바이오틱 생균의 알콜에 대한 내성 측정Example 2: Determination of Resistance to Alcohol of Probiotic Probiotics of the Invention

알콜 내성을 측정하기 위해서 각각의 균주를 최적의 온도에서 12시간 배양하고 균체를 얻기 위해서 배양액을 원심분리(6,000 rpm, 10 min)하여 상등액을 제거하였으며, 회수한 균체를 0.1 M 인산 완충용액(pH 7.0)에 희석하였다. 알콜이 각 농도별로 첨가된 0.1 M 인산 완충용액(0, 4, 8, 16, 32%) 5 ml에 상기 희석한 균체액을 접종후 37℃ 진탕 배양기에서 4시간동안 반응시킨 후 생존 활성을 조사하기 위해서 총균수를 측정하였다.In order to measure alcohol resistance, each strain was incubated at an optimal temperature for 12 hours, and the supernatant was removed by centrifugation (6,000 rpm, 10 min) to obtain the cells. The recovered cells were removed in 0.1 M phosphate buffer (pH). 7.0). After the inoculation of the diluted cell solution in 5 ml of 0.1 M phosphate buffer solution (0, 4, 8, 16, 32%) to which the alcohol was added at each concentration, the reaction was performed for 4 hours in a 37 ° C shaking incubator. In order to determine the total number of bacteria.

측정결과, 하기 표1에 나타난 바와 같이 여러 알콜농도에서 4시간 반응시킨 후 생존율을 비교·검토한 결과 MY4균주가 다른 상업용 균주에 비해 매우 높음을 알 수 있었다.As a result, as shown in Table 1, after 4 hours of reaction at various alcohol concentrations, the survival rate was compared and examined, and it was found that the MY4 strain was much higher than other commercial strains.

본 발명 균주와 상업용 균주와의 알콜내성 비교Comparison of Alcohol Tolerance between Inventive and Commercial Strains 균주명알콜농도(%)Strain Name Alcohol Concentration (%) 본 발명 균주(MY4)Inventive strain (MY4) 상업용균주1(PT77)Commercial strain 1 (PT77) 상업용균주2(PS490)Commercial strain 2 (PS490) 상업용균주3(PS653)Commercial strain 3 (PS653) 00 1.3×108 1.3 × 10 8 1.2×108 1.2 × 10 8 1.2×108 1.2 × 10 8 1.0×108 1.0 × 10 8 44 1.0×108 1.0 × 10 8 1.0×108 1.0 × 10 8 1.2×108 1.2 × 10 8 1.0×108 1.0 × 10 8 88 1.1×108 1.1 × 10 8 8.7×107 8.7 × 10 7 4.1×107 4.1 × 10 7 6.8×107 6.8 × 10 7 1616 1.0×107 1.0 × 10 7 4.0×105 4.0 × 10 5 8.5×104 8.5 × 10 4 2.0×105 2.0 × 10 5 3232 1.5×105 1.5 × 10 5 1.8×103 1.8 × 10 3 2.1×102 2.1 × 10 2 7.6×102 7.6 × 10 2 [주](단위: 콜로니형성단위/ml)[Note] (Unit: colony forming unit / ml)

실시예 3: 본 발명 프로바이오틱 생균의 인공위액에 대한 내성Example 3: Resistance to Artificial Gastric Fluid of Probiotic Probiotics of the Invention

식품용 프로바이오틱 생균으로서 가져야 할 특성이 여러 가지가 있는데, 이 중에서 가장 중요한 특성 중 하나가 생존력이 높아야 한다는 점이다(Fuller, R. 1989. Probiotics in amn and animals. J. Appl. Bacteriol. 66: 365-378). 구강을 통하여 섭취되는 균은 위와 십이지장을 통해 생존하여 최종 목적 부위인 장에 도달하기 때문에 프로바이오틱 생균제로서의 기능을 발휘하기 위해서는 강산성의 위액을 통과하여야 한다. 순수한 위액의 pH는 1.4∼2.0 정도로 거의 대부분 미생물은 여기에서 사멸하게 된다. 하지만 섭취한 음식물의 완충작용으로 인해 다소 pH가 높아져 미생물의 사멸율이 어느 정도 감소하게 된다(Conway et al., 1987. Survival of lactic acid bacteria in the human stomach and adhesion to intestinal cells. J. Dairy Sci. 70: 1-12). 내산성 실험으로는 in vivo에서 직접 생존율을 확인하는 방법과 인공위액을 이용한 간접적으로 측정하는 방법이 알려져 있다. 프로바이오틱 생균의 사멸은 주로 낮은 pH에 의한 것이며 in vitro와 in vivo에서의실험결과가 거의 일치한다는 사실이 보고되어 있다(Conway et al., 1987. Survival of lactic acid bacteria in the human stomach and adhesion to intestinal cells. J. Dairy Sci. 70: 1-12; Giannella et al., 1972. Gastric acid barrier to ingested microorganisms in man: Studiesin vivoandin vitro. Gut 13: 251-256; Berrada et al., 1991.Bifidobacteriumfrom fermented milks: Survival during gastric transit. J. Dairy Sci. 74: 409-413).There are a number of characteristics that must be taken as probiotic probiotics for food, and one of the most important characteristics is the high viability (Fuller, R. 1989. Probiotics in amn and animals. J. Appl. Bacteriol. 66 : 365-378). Bacteria ingested through the oral cavity survive through the stomach and duodenum and reach the final target site of the intestine, so they must pass strong acidic gastric juice to function as a probiotic probiotic. Pure gastric juice has a pH of 1.4-2.0, and most of the microbes die from it. However, due to the buffering effect of the ingested food, the pH is increased to some extent, and the killing rate of microorganisms is reduced to some extent (Conway et al., 1987. Survival of lactic acid bacteria in the human stomach and adhesion to intestinal cells. 70: 1-12). Acid resistance experiments are known to determine the survival rate in vivo directly and indirectly using artificial gastric juice. It has been reported that the killing of probiotic probiotics is mainly due to low pH and that the experimental results in vitro and in vivo are almost identical (Conway et al., 1987. Survival of lactic acid bacteria in the human stomach and adhesion to intestinal cells.J. Dairy Sci. 70: 1-12; Giannella et al., 1972.Gastric acid barrier to ingested microorganisms in man: Studies in vivo and in vitro.Gut 13: 251-256; Berrada et al., 1991 Bifidobacterium from fermented milks: Survival during gastric transit.J. Dairy Sci. 74: 409-413).

본 실시예에서는 상기와 같은 점을 감안하여 pH 2.5로 조정된 인공위액에서의 본 발명 균주의 내성을 측정하였다.In this example, the resistance of the strain of the present invention in artificial gastric juice was adjusted to pH 2.5 in view of the above.

인공위액은 Kobayashi 등의 방법에 따라 1N HCl을 사용하여 pH 2.5로 조정한 각각의 브로스에 펩신 1%를 첨가하여 사용하였다(Kobayashi et al., 1974. Tolerance of the multiple antibiotic resistant strain,L. caseiPSR 3002, to artificial digestive fluids. Jpn. J. Microbiol. 29: 691-197). 인공위액에 대한 내성을 실험하기 위해 각 균주의 배양액을 에펜돌프 튜브에 1 ml 넣은 후 6000 rpm에서 10분간 원심분리한 후 배양 상등액을 버리고 균체만을 회수하였다. 회수된 균체는 다시 인공위액을 상등액과 동량으로 첨가하여 9 ml 테스트 튜브 인공위액에 넣어 2시간 동안 37℃에서 배양하여 총균수를 측정하였다. 대조구는 pH를 조절하지 않고 또한 펩신을 첨가하지 않은 각각의 브로스를 사용하여 위와 같은 동일한 방법을 거쳤다.Artificial gastric fluid was used by adding 1% pepsin to each broth adjusted to pH 2.5 using 1N HCl according to the method of Kobayashi et al. (Kobayashi et al., 1974. Tolerance of the multiple antibiotic resistant strain, L. casei PSR 3002, to artificial digestive fluids.Jpn. J. Microbiol. 29: 691-197). In order to test the resistance to artificial gastric juice, the culture solution of each strain was placed in 1 ml of an eppendorf tube, centrifuged at 6000 rpm for 10 minutes, and the culture supernatant was discarded and only the cells were recovered. The recovered cells were added in the same amount as the supernatant and added to the 9 ml test tube artificial gastric juice and incubated at 37 ° C. for 2 hours to measure the total bacterial count. The control was subjected to the same method as above using each broth without adjusting pH and adding pepsin.

실험결과, 도 1에 나타난 바와 같이 MY4균은 2시간 경과후 73.3%의 균이 생존하는 매우 높은 생존율을 보여 인공위액에 대한 내성을 지니고 있음을 알 수 있었다.As a result, as shown in Figure 1 MY4 bacteria showed a very high survival rate of survival of 73.3% of the bacteria after 2 hours, it can be seen that it is resistant to artificial gastric juice.

실시예 4: 본 발명 프로바이오틱 생균의 인공담즙산에 대한 내성Example 4 Resistance to Artificial Bile Acids of Probiotic Probiotics of the Invention

섭취된 프로바이오틱 생균이 장에 도달하기 위해서 위를 거쳐 췌장과 십이지장을 통과하게 된다. 이 부위에서 분비되는 담즙액에 대한 내성 또한 프로바이오틱 생균이 가져야 할 중요한 특성이다. Gilliland 등은 프로바이오틱 생균이 가져야 할 담즙액에 대한 내성은 oxgall이 0.3% 함유된 배지에서 성장할 수 있어야 한다고 보고했다(Gilliland et al. 1984. Importance of bile tolerance ofLactobacillus acidophilusused as a dietary adjunct. J. Dairy Sci. 67: 3045-3051).Ingested probiotic bacteria pass through the stomach and pancreas and duodenum to reach the intestines. Resistance to the bile secreted from this site is also an important property of probiotic probiotics. Gilliland et al. Reported that the resistance to bile for probiotic probiotics should be able to grow in medium containing 0.3% oxgall (Gilliland et al. 1984. Importance of bile tolerance of Lactobacillus acidophilus used as a dietary adjunct. J. Dairy Sci. 67: 3045-3051).

상기와 같은 점을 감안하여, 인공담즙액은 멸균된 각각의 브로스에 멸균한 10% oxgall (Difco) 용액을 1% 첨가하여 사용하였다. 인공담즙산 내성을 실험하기 위해서 인공위액을 거친 배양액을 인공담즙액이 있는 test tube에 첨가하여 37℃에서 24시간 동안 배양한 후 생존력을 조사하기 위해서 인공위액과 마찬가지로 총균수를 측정하였다. 대조구는 oxgall이 첨가되지 않은 각각의 브로스에 위와 같은 동일한 방법을 사용하여 실험하였다.In view of the above, the artificial bile solution was used by adding 1% sterilized 10% oxgall (Difco) solution to each sterilized broth. In order to test the resistance of artificial bile acid, the culture medium subjected to artificial gastric juice was added to a test tube containing artificial bile and incubated for 24 hours at 37 ° C. Controls were tested using the same method as above for each broth without oxgall added.

인공담즙산에 대한 내성을 실험한 결과, 젖산균의 인공위액을 거친 인공담즙산의 내성은 도 2에서 보는 바와 같이 인공위액을 통과한 인공담즙산에 대한 내성은 control에 비해 다소 낮은 생존율을 보였지만 거의 대부분 108CFU/ml 수준을 상회하고 있고 초기 접종 균수에 비해 높은 수치이다.After a test for resistance to artificial bile acid, resistance to artificial bile acid that has passed through the artificial gastric juice as shown in the resistance of a rough artificial bile acid the lactic acid bacteria of the artificial gastric juice Figure 2 showed a somewhat lower survival rate compared to control most 10 8 It is above the CFU / ml level and is higher than the initial cell count.

따라서 본 발명 프로바이오틱 생균은 인공위액 뿐 아니라 인공 담즙산에 대하여도 내성을 지니고 있어 목적부위인 장에 도달하여 프로바이오틱 생균으로서의 역할을 수행할 수 있을 것이다.Therefore, the probiotic probiotic of the present invention is resistant not only to artificial gastric juice but also to artificial bile acids, thus reaching the target site of the intestinal tract and may serve as a probiotic probiotic.

실시예 5 : 본 발명 프로바이오틱 생균의 미생물학적 동정Example 5 Microbiological Identification of Probiotic Probiotics of the Invention

현재 널리 이용되는 세균의 분류 방식은 미국의 Bergey식의 분류법(Bergey's Manual of Determinative Bacteriology)과 소련 및 동구지역에서 이용되는 Krassilinikov (1949)의 분류법 그리고 프랑스를 비롯한 유럽 및 아프리카의 일부 등지에서 쓰이는 Prevot (1961)의 분류법 등이 있다.Currently widely used bacterial classification methods include Bergy's Manual of Determinative Bacteriology in the United States, Krassilinikov's (1949) classification used in the Soviet Union and Eastern Europe, as well as Prevot (used in France, parts of Europe and Africa). 1961).

우리 나라에서는 Begey식의 분류법을 따르고 있는데 Bergey 편람은 여러 학자들이 분담 저술한 것을 미국 미생물학회의 편집위원회에서 편집한 것으로 장점도 있는 반면에 내용에 통일성이 없다는 단점도 있다.In our country, Begey's taxonomy is used. Bergey's manual is edited by the American Microbiological Editor's Committee, which has been shared by many scholars.

본 실시예에서는 그람 염색법 및 API CHL(bioMereux Co., France)에 의해 49개의 탄소원에 대한 이용성을 검토함으로써 젖산균 MY4 균주를 동정하였다(Kim, W.J. 1996. Screening of bacteriocinogenic lactic acid bacteria and their antagonistic effects in sausage fermentation. J. Microbiol. Biotechnol. 6: 461-467).In this example, Lactobacillus MY4 strains were identified by examining the availability of 49 carbon sources by Gram staining and API CHL (bioMereux Co., France) (Kim, WJ 1996. Screening of bacteriocinogenic lactic acid bacteria and their antagonistic effects in sausage fermentation.J. Microbiol.Biotechnol. 6: 461-467).

API CHL 키트에 의해 탄소원 이용성을 살펴본 결과 표 2처럼 MY4 균주는 76.2%의 유사성으로Lactobacillus plantarum으로 동정되었다.As a result of examining carbon availability by API CHL kit, MY4 strain was identified as Lactobacillus plantarum with a similarity of 76.2% as shown in Table 2.

API 50 CHL kit을 이용한 프로바이오틱 균주 MY4의 미생물학적 동정Microbiological Identification of Probiotic Strain MY4 Using API 50 CHL kit Test itemsTest items MY4MY4 Test itemsTest items MY4MY4 대조구Control -- 에스큘린(Esculin)Esculin ++ 글리세롤(Glycerol)Glycerol ++ 살리신(Salicin)Salincin ++ 에리트리톨(Erythritol)Erythritol -- 셀로비오스(Cellobiose)Cellobiose ++ D-아라비노스(D-Arabinose)D-Arabinose -- 말토오스(Maltose)Maltose ++ L-아라비노스(L-Arabinose)L-Arabinose ++ 락토오스(Lactose)Lactose ++ 리보오스(Ribose)Ribose ++ 멜리비오스(Melibiose)Melibiose ++ D-크실로스(D-Xylose)D-Xylose -- 사카로즈(Saccharose)Saccharose ++ L-크실로스(L-Xylose)L-Xylose -- 트레할로스(Trehalose)Trehalose ++ 아도니톨(Adonitol)Adonitol -- 이눌린(Inulin)Inulin -- β-메틸크실로시드(β-Methylxyloside)β-methylxyloxide (β-Methylxyloside) ++ 멜레지토스(Melezitose)Melezitose -- 갈락토스(Galactose)Galactose ++ 라피노스(Raffinose)Raffinose -- 글루코스(Glucose)Glucose ++ 녹말(Starch)Starch ++ 플라토스(Fructose)Fructose ++ 글리코겐(Glycogen)Glycogen -- 만노스(Mannose)Mannose ++ 자일리톨(Xylitol)Xylitol -- 소르보스(Sorbose)Sorbose -- 겐티오비아제(Gentiobiose)Gentiobiose ++ 람노스(Rhamnose)Rhamnose ++ D-투라노스(D-Turanose)D-Turanose -- 둘시톨(Dulcitol)Dulcitol -- D-릭소스(D-Lyxose)D-Lyxose -- 이노시톨(Inositol)Inositol -- D-타카토스(D-Tagatose)D-Tagatose ++ 만니톨(Mannitol)Mannitol ++ D-푸코스(D-Fucose)D-Fucose -- 솔비톨(Sorbitol)Sorbitol -- L-푸코스(L-Fucose)L-Fucose -- α-메틸-D-만노시드(α-Methyl-D-mannoside)α-Methyl-D-mannoside -- D-아라비톨(D-Arabitol)D-Arabitol -- α-메틸-d-글루코시드(α-Methyl-D-glucoside)α-Methyl-d-glucoside -- L-아라비톨(L-Arabitol)L-Arabitol -- N-아세틸글루코사민(N-Acetylglucosamine)N-acetylglucosamine ++ 글루코네이트(Gluconate)Gluconate -- 아미그달린(Amygdaline)Amygdaline ++ 2-케토글루코네이트(2-Ketogluconate)2-ketogluconate -- 아르부틴(Arbutin)Arbutin ++ 5-케토글루코네이트(5-Ketogluconate)5-ketogluconate -- [주] +: utilized, -: not utilized[Note] +: utilized,-: not utilized

실시예 6: 본 발명 프로바이오틱 생균의 효소활성 측정Example 6 Determination of Enzymatic Activity of Probiotic Probiotics of the Invention

프로바이오틱 생균이 가져야 할 특성 중에 이들 균이 생산하는 효소 또한 중요한 부분을 차지하고 있다. 예를 들어 발암효소로 알려진 β-글루쿠로니다제(β-glucuronidase)이다. 벤존피렌(Benzo(a)pyrene) 등의 발암물질이 체내에 들어오면 간에서 glucuronic acid와 포합되어 해독작용이 이루어지지만, 이것이 담즙과 함께장내에 배설되어 장내 세균의 β-글루쿠로니다제에 의해 탈포합되면 다시 독성을 가지게 된다(Cole et al. 1989. Effect of probiotic supplements of Lactobacillus acidophilus and Bifidobacterium adolescentis 2204 on β-glucosidase and β-glucuronidase activity in the lower gut of rats associated with a human fecal flora. Microb. Ecol. Health Dis. 2: 223-225). 따라서 프로바이오틱 생균으로서 사용되는 균들이 발암효소인 β-글루쿠로니다제의 활성을 갖고 있다는 것은 바람직하지 않다. 그리고 프로바이오틱 생균들의 유익한 효소는 사람이나 동물이 영양분을 섭취하였을 때 위와 장을 통해 흡수되지 않거나 분해되지 않아서 장내에서 이상발효를 일으킬 수 있는 방지해 준다.Among the properties that probiotic probiotics have, the enzymes they produce are also an important part. For example, β-glucuronidase, known as a carcinase. When carcinogens such as Benzo (a) pyrene enter the body, they are combined with glucuronic acid in the liver to detoxify them, but they are excreted in the intestines together with bile to induce β-glucuronidase of intestinal bacteria. Decontamination by toxic acid is re-toxic (Cole et al. 1989. Effect of probiotic supplements of Lactobacillus acidophilus and Bifidobacterium adolescentis 2204 on β-glucosidase and β-glucuronidase activity in the lower gut of rats associated with a human fecal flora.Microb Ecol Health Dis. 2: 223-225). Therefore, it is not preferable that the bacteria used as probiotic live bacteria have the activity of β-glucuronidase, a carcinogenic enzyme. In addition, the beneficial enzymes of probiotic probiotics prevent humans and animals from ingesting or disintegrating the stomach and intestines, causing abnormal fermentation in the intestines.

효소활성을 측정하기 위해서 선별된 균주를 각각의 브로스에 3회 계대배양하여 배양액을 원심분리하여 균체만을 회수하여 멸균수로서 2회 반복하여 세척하였다. 세척한 후 멸균수에 현탁하여 106CFU/ml 정도의 농도가 되도록 하였다. 이 현탁액을 API ZYM(bioMerieux Co.)에 접종하여 어두운 곳에서 37℃, 4시간 동안 배양하였다. 표현활성 증가와 용해를 돕기 위해 ZYM A, B 시약을 각각의 큐플에 한 방울씩 떨어뜨린 후 밝은 곳에서 약 5분간 반응시킨 후 색깔의 변화를 관찰하여 효소활성을 측정하였다.In order to measure the enzyme activity, the selected strains were passaged three times in each broth, and the culture medium was centrifuged to recover only the cells and washed twice with sterile water. After washing, it was suspended in sterile water to a concentration of about 10 6 CFU / ml. The suspension was inoculated with API ZYM (bioMerieux Co.) and incubated in the dark at 37 ° C. for 4 hours. ZYM A and B reagents were added dropwise to each chapel to help increase expression activity and to dissolve, and then reacted in a bright place for about 5 minutes.

본 연구에서 선택되어진 젖산균 MY4균을 API ZYM kit에 의해 확인한 결과 하기 표 3에서와 같이 발암 효소인 β-글루쿠로니다제에 대한 활성이 없는 것으로 나타났다. 이러한 결과는 프로바이오틱 생균으로 인해 발생할 수 있는 발암 유발의위험성이 없다는 것이다. 한편 유당불내증을 완화시킬 수 있는 β-갈락토시데이즈(β-galactosidase)를 생산하므로 프로바이오틱 생균으로서의 좋은 특성을 가지고 있다.As a result of confirming the Lactobacillus MY4 bacteria selected in this study by the API ZYM kit, it was found that there was no activity against the oncogenic enzyme β-glucuronidase as shown in Table 3 below. These results indicate that there is no risk of carcinogenesis caused by probiotic live bacteria. Meanwhile, β-galactosidase, which can alleviate lactose intolerance, produces good properties as a probiotic probiotic.

API ZYM 키트에 의한 프로바이오틱 MY4 균주의 효소활성 측정Determination of Enzyme Activity of Probiotic MY4 Strains by API ZYM Kit 효소enzyme 균주Strain MY4MY4 대조구Control 00 알카라인 포스파타제(Alkaline phosphatase)Alkaline phosphatase 22 에스테라제 (Esterase; C4)Esterase (C 4 ) 44 에스테라제 리파제 (Esterase Lipase; C8)Esterase Lipase (C 8 ) 44 리파제(Lipase; C14)Lipase (C 14 ) 22 로이신 아릴아미다제(Leucine arylamidase)Leucine arylamidase 55 발린 아릴아미다제(Valine arylamidase)Valine arylamidase 33 크리스틴 아릴아미다제(Crystine arylamidase)Christine arylamidase 22 트립신(Trypsin)Trypsin 00 α-키모트립신(α-Chymotrypsin)α-chymotrypsin 1One 에시드 포스파타아제(Acid phosphatase)Acid phosphatase 55 나프톨-AS-BI-포스포하이드로아제(Naphthol-AS-BI-phosphohydrolase)Naphthol-AS-BI-phosphohydrolase 1One α-갈락토시다제(α-Galactosidase)α-galactosidase 00 β-갈락토시다제(β-Galactosidase)β-galactosidase 1One β-글루쿠로니다제(β-Glucuronidase)β-glucuronidase 00 α-글루쿠로니다제 (α-Glucosidase)α-glucuronidase (α-Glucosidase) 00 β-글루쿠로니다제(β-Glucosidase)β-glucuronidase (β-Glucosidase) 00 N-아세틸-β-글루코사민다제( N-Acetyl-β-glucosaminidase)N-Acetyl-β-glucosaminidase 00 α-만노시다제(α-Mannosidase)α-Mannosidase 00 α-푸코시다제(α-Fucosidase)α-fucosidase 00 [주]0: 0 nmole, 1: 5 nmole, 2: 10 nmole, 3: 20 nmole,4: 30 nmole, 5: ≥40 nmole0: 0 nmole, 1: 5 nmole, 2: 10 nmole, 3: 20 nmole, 4: 30 nmole, 5: ≥ 40 nmole

실시예 7: 본 발명 프로바이오틱 생균의 항생물질에 대한 내성조사Example 7 Investigation of Resistance of Antibiotic to Probiotic Probiotics of the Invention

항생물질 내성을 측정하기 위해서 paper disc 방법을 사용하였다. 즉, 각각의 균주를 최적의 온도에서 12시간 배양하고, 그리고 각각의 소프트 아가(0.7%)에 배양액을 100 ㎕로 접종하여 아가플레이트에 overlay한다. Overlay된 플레이트에 각 농도별의 항생물질(nisin, kanamycin, streptomycin, rifamycin)을 충분히 적신paper disc를 올려서 24시간 배양하여 항생물질 내성을 측정하였다.The paper disc method was used to measure antibiotic resistance. That is, each strain is incubated at the optimal temperature for 12 hours, and 100 μl of the culture solution is inoculated on each soft agar (0.7%) and overlayed on the agar plate. Antimicrobial resistance was measured by incubating a paper disc sufficiently wetted with antibiotics of each concentration (nisin, kanamycin, streptomycin, and rifamycin) on the overlayed plate for 24 hours.

하기 표 4에서 보는 바와 같이 MY4 균주가 테트라사이클린 20 ㎍/ml의 농도에서 저해를 받았고 나머지 3종류의 항생물질에 대해서는 내성을 가지고 있다. 이러한 결과들은 이 등이 보고한 항생물질에 대한 내성보다도 높은 내성을 가지고 있었다(Lee, S.H. and No, M.J. 1997. Viability in artificial gastric and bile juice and antimicrobial activity of some lactic acid bacteria isolated from Kimchi. J. Appl. Microbiol. Biotechnol. 6: 617-622).As shown in Table 4, MY4 strain was inhibited at a concentration of 20 ㎍ / ml of tetracycline and has resistance to the other three antibiotics. These results were higher than those reported by Lee et al. (Lee, SH and No, MJ 1997. Viability in artificial gastric and bile juice and antimicrobial activity of some lactic acid bacteria isolated from Kimchi. Appl. Microbiol.Biotechnol. 6: 617-622).

프로바이오틱 MY4 균주의 항생물질에 대한 내성조사Antibiotic Resistance of Probiotic MY4 Strains 항생물질(㎍/ml)Antibiotic (µg / ml) 균 주Strain MY4MY4 니신( Nisin)Nisin 00 ++ 2525 ++ 5050 ++ 100100 ++ 스트렙토바이신(Streptomycin)Streptomycin 00 ++ 55 ++ 1010 ++ 2020 ++ 테트라사이클린(Tetracycline)Tetracycline 00 ++ 55 ++ 1010 ++ 2020 -- 리팝피신(Rifamycin)Ripopmycin 00 ++ 55 ++ 1010 ++ 2020 ++ [주]+: growth, -: no growthㅡ[Note] +: growth,-: no growth

실시예 8: 본 발명 프로바이오틱 생균의 항균활성 측정Example 8 Determination of Antimicrobial Activity of Probiotic Probiotics of the Invention

본 발명 균주의 병원성 세균에 대한 항균력을 측정하기 위해ListeriamonocytogenesATCC 19111를 사용하였다. Listeriamonocytogenes ATCC 19111 was used to measure the antimicrobial activity of pathogenic bacteria of the strain of the present invention.

병원성 세균인 리스테리아(listeria)는 그램 음성 무포자 간균으로서 가축(소, 말 양, 돼지, 산양 등) 및 가금(닭, 오리 등)에 감염되는 질환을 일으키고 때로는 사람에게 감염된다. 사람은 감염동물과 직접 접촉 및 오염된 식육이나 유제품 등에 의한 경구감염이 되는 경우가 주이며, 때로는 오염된 먼지에 의한 호흡을 통하여 감염되기도 한다. 잠복기는 3일에서 수주일이며 증상은 수막염, 패혈증 및 자궁내막염(임산부)등을 일으킨다(오승희, 김동원. 1997. 최신식품위생학. pp. 241).Listeria, a pathogenic bacterium, is a Gram-negative despoiler bacillus that causes livestock (cows, horses, pigs, goats, etc.) and poultry (chicken, ducks, etc.) and sometimes humans. Humans are often in direct contact with infected animals and orally infected with contaminated meat or dairy products, sometimes through contaminated dust. The incubation period is three to several weeks, with symptoms causing meningitis, sepsis and endometritis (pregnant women) (Oh Seung-hee, Kim Dong-won, 1997. The latest food hygiene, pp. 241).

항균활성 측정에 사용된 리스테리아는 TSB 배지에서 37℃, 24시간 동안 3회 계대배양하여 사용하였으며, 선택배지로서는 영국 Unipath사 제품의 Listeria Selective Agar base를 사용하였다.Listeria used to measure antimicrobial activity was used in three passages for 24 hours at 37 ℃ in TSB medium, Listeria Selective Agar base manufactured by Unipath UK was used as the selection medium.

항균활성을 측정하기 위해서 MY4균주와L. monocytogenesATCC 19111을 각각 12시간 동안 배양한 다음 균수를 105∼106CFU/mL 수준으로 희석하고 1:1 부피비율로 각각 테스트 튜브에 5 mL를 혼합하여 37℃ shaking water bath에서 배양하였다. 배양을 하면서 1, 2, 4, 8, 24 시간 간격으로 sampling 하여 리스테리아 선택배지를 사용하여 균수를 측정하였다.In order to measure the antimicrobial activity, MY4 and L. monocytogenes ATCC 19111 were incubated for 12 hours, respectively, and the bacteria were diluted to 10 5-10 6 CFU / mL and mixed in 5 mL of test tubes in a 1: 1 volume ratio. And incubated in 37 ℃ shaking water bath. During incubation, the cells were sampled at 1, 2, 4, 8, and 24 hour intervals, and the number of bacteria was measured using Listeria selective medium.

젖산균인 MY4균주는 도 3에서 보는 바와 같이 4시간까지는 리스테리아의 균수가 증가하다가 8시간 이후부터는 감소하기 시작하였으며 24시간 후에는 대조구에 비해 상당히 많은 감소를 보였다(log 2 이하까지 감소). 이러한 저해의 주된 원인은 젖산균에 의해서 생성된 유기산과 젖산에 의한 pH 저하와 박테리오신과 같은 물질에 기인한 것으로 보인다(Hong et al. 1996. Growth ofLactobacillus acidophilusin whey-based medium and preparation of cell concentrate for production of probiotics. J. Appl. Microbiol. Biotechnol. 2: 128-131).As shown in FIG. 3, the MY4 strain, lactic acid bacteria, increased the number of bacteria of Listeria until 4 hours and began to decrease after 8 hours. After 24 hours, Lactobacillus MY4 showed a significant decrease compared to the control (reduced to less than log 2). The main cause of this inhibition appears to be due to organic acids produced by lactic acid bacteria and lowering of pH by lactic acid and substances such as bacteriocin (Hong et al. 1996. Growth of Lactobacillus acidophilus in whey-based medium and preparation of cell concentrate for production of probiotics.J. Appl.Microbiol.Biotechnol. 2: 128-131).

위와 같은 결과에 의해 만약 리스테리아에 감염이 되었을 경우 장내에 상주하는 프로바이오틱 균주들에 의해 어느 정도 억제할 수 있을 것이라 판단된다.According to the above results, if the infection of Listeria, it is judged that it can be suppressed to some extent by the probiotic strains resident in the intestine.

이상, 상기 실시예를 통하여 설명한 바와 같이 본 발명 프로바이오틱 생균인Lactobacillus plantarumMY4는 위액 및 담즙산에 대한 내성이 높아 장내 생존력이 뛰어나고, β-갈락토시데이즈를 생산하므로 유당불내증을 감소시킬 수 있는 효과가 있으며 리스테리아 감염을 억제할 수 있는 뛰어난 효과가 있고, 알콜내성이 기존 균주에 비해 우수하여 현대인이 음주시 섭취하는 기능성 식품으로의 이용가능성이 있으므로 식품산업상 및 의학 산업상 매우 유용한 발명인 것이다.As described above, the present invention probiotic probiotic Lactobacillus plantarum MY4 has a high resistance to gastric juice and bile acids, has excellent intestinal viability, and produces β-galactosidase, thereby reducing lactose intolerance. It is effective and has an excellent effect of suppressing Listeria infection, and has excellent alcohol resistance compared to existing strains, so that it can be used as a functional food consumed by modern people when drinking, which is a very useful invention in the food industry and the medical industry.

Claims (3)

알콜에 내성있는 프로바이오틱 생균인 락토바실러스 프랜테리움(Lactobacillus plantarum) MY4 균주(기탁번호 KCCM 10266). Lactobacillus plantarum MY4 strain (accession number KCCM 10266), a probiotic probiotic resistant to alcohol. 제 1항의 프로바이오틱 생균을 유효성분으로 함유함을 특징으로 하는 유당불내증 치료용 조성물.The composition for treating lactose intolerance, comprising the probiotic probiotic of claim 1 as an active ingredient. 제 1항의 프로바이오틱 생균을 유효성분으로 함유함을 특징으로 하는 리스테리아 감염 억제용 조성물.A composition for inhibiting Listeria infection, comprising the probiotic probiotic of claim 1 as an active ingredient.
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KR100585391B1 (en) * 2002-01-24 2006-06-01 주식회사 프로바이오닉 Acid tolerant probiotic Lactobacillus plantarum Probio-38 that can suppresses the growth of pathogenic microorganisms and TGE coronavirus
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KR100911115B1 (en) * 2007-06-15 2009-08-11 주식회사 씨티씨바이오 Novel lactic acid bacteria having acid resistance, bile-salt resistance and anti-bacterial effect and composition containing the same
KR101960189B1 (en) * 2017-10-25 2019-03-19 건국대학교 산학협력단 NOVEL STRAIN OF Lactobacillus plantarum AND USE THEREOF
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KR100585391B1 (en) * 2002-01-24 2006-06-01 주식회사 프로바이오닉 Acid tolerant probiotic Lactobacillus plantarum Probio-38 that can suppresses the growth of pathogenic microorganisms and TGE coronavirus
KR100723843B1 (en) * 2005-04-29 2007-05-31 (주)엠지인투바이오 Novel strains, Lactobacillus plantarum IB 213KCTC 18106P and its application as probiotics
KR100715730B1 (en) * 2005-11-15 2007-05-08 연세대학교 산학협력단 24A novel Bifidobacterium longum A24 with anti-bacterial activity against toxigenic pathogens
KR100911115B1 (en) * 2007-06-15 2009-08-11 주식회사 씨티씨바이오 Novel lactic acid bacteria having acid resistance, bile-salt resistance and anti-bacterial effect and composition containing the same
KR101960189B1 (en) * 2017-10-25 2019-03-19 건국대학교 산학협력단 NOVEL STRAIN OF Lactobacillus plantarum AND USE THEREOF
CN111944712A (en) * 2020-07-13 2020-11-17 天津科技大学 Lactobacillus plantarum with excellent alcohol tolerance and application thereof

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