KR101744529B1 - Bacillus subtilis 1-d-5, method for growth stimulation of bifidobacteria and method for manufacturing of enzyme food using thereof - Google Patents
Bacillus subtilis 1-d-5, method for growth stimulation of bifidobacteria and method for manufacturing of enzyme food using thereof Download PDFInfo
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Abstract
본 발명은 바실러스 서브틸리스 1-D-5, 이를 이용한 비피도박테리아 증식방법 및 효소식품 제조방법을 제공한다.
신규한 균주인 바실러스 서브틸리스(Bacillus subtilis) 1-D-5를 제공하고 상기 균주와 락토바실러스 카제이(Lactobacillus casei) GW140 균주의 2단 발효를 통해 비피도박테리아를 증식시키는 방법 및 효소식품 제조방법을 제공한다.
본 발명에 따르면 죽염 된장에서 분리된, α-아밀라아제와 프로테아제 활성을 나타내는 바실러스 서브틸리스 1-D-5를 이용하여 막걸리에서 분리된, DHNA를 생산하는 락토바실러스 카제이 GW140 균주와 2단 발효를 통해 비피도박테리아를 증식시키는 효과를 제공할 수 있다.
또한 이를 이용하여 α-아밀라아제와 프로테아제 활성이 유지되면서 유산균 수가 109CFU/g 이상인 비피도박테리아 증식능이 우수한 효소식품을 제조하는 효과를 제공할 수 있다. The present invention provides Bacillus subtilis 1-D-5, a bifidobacteria propagation method using the same, and a method of producing an enzyme food.
A method for producing Bacillus subtilis 1-D-5, a novel strain, and a method for propagating Bifidobacterium through two-step fermentation of the strain and Lactobacillus casei strain GW140, ≪ / RTI >
According to the present invention, Lactobacillus casei GW140 strain producing DHNA isolated from rice wine using Bacillus subtilis 1-D-5 showing α-amylase activity and protease activity isolated from bamboo salt doenjang and 2-stage fermentation Lt; RTI ID = 0.0 > bifidobacterium. ≪ / RTI >
In addition, the present invention can provide an effect of producing an enzyme food having excellent bifidobacteria proliferative activity with a lactic acid bacterium number of 10 9 CFU / g or more while maintaining the activity of α-amylase and protease.
Description
본 발명은 바실러스 서브틸리스 1-D-5, 이를 이용한 비피도박테리아 증식방법 및 효소식품 제조방법에 관한 것으로, 더욱 상세하게는 죽염 된장에서 바실러스 서브틸리스 1-D-5를 분리하고, 분리된 바실러스 서브틸리스 1-D-5와 락토바실러스 카제이 GW140을 순차적으로 배양함으로써 비피도박테리아를 증식시키는 방법과 효소식품을 제조하는 방법에 대한 것이다. The present invention relates to a Bacillus subtilis 1-D-5, a Bacillus subtilis 1-D-5, a Bacillus subtilis 1-D-5, The present invention relates to a method for growing Bifidobacterium by sequentially culturing Bacillus subtilis 1-D-5 and Lactobacillus casei GW140, and a method for producing an enzyme food.
일반적으로 바실러스 서브틸리스 균주는 산에 대한 내성이 없기 때문에 유산균과의 동시배양을 할 경우 균이 잘 자라지 못해 효소 생성률이 낮아 효소식품을 생산하기 어려웠다. In general, since the Bacillus subtilis strain is not resistant to acid, when the co-culture with lactic acid bacteria is performed, the bacteria do not grow well and the enzyme production rate is low, making it difficult to produce the enzyme food.
또한 비피도박테리아는 인체 장 내에 서식하면서 숙주에게 이로운 역할을 하는 유익균이다. 비피도박테리아는 다양한 생리활성 예를 들면, 유해 미생물 억제, 변비 예방, 설사 예방, 면역증강 효과 등을 나타내는 것으로 알려져 있다. 그러나 사람이 나이가 들수록, 사람의 장내에서 비피도박테리아의 구성 비율이 줄어드는 것으로 나타났다. 그 결과 유해균들이 득세하여 다양한 장내 질환들을 유발한다. 이러한 문제점을 해결하기 위한 방법은 크게 두 가지인데, 하나는 외부에서 비피도박테리아를 섭취하는 것이고 다른 하나는 장내 정착되어 있는 비피도박테리아를 증식시키는 것이다. 외부에서 비피도박테리아를 섭취하는 방법은, 비피도박테리아를 포함하는 발효유 등의 식품을 섭취하는 것이다. 그러나 비피도박테리아는 편성혐기성균으로 극미량의 산소존재 하에서도 사멸하기 쉽다. 이 때문에 발효유 제품 등의 식품이 유통되는 과정에서 발효유 제품 중에 살아있는 비피도박테리아의 함량은 아주 많이 감소해 버리는 문제점이 있었다.Bifidobacteria are beneficial bacteria that live in the human intestines and play a beneficial role in the host. Bifidobacteria are known to exhibit various physiological activities such as inhibition of harmful microorganisms, prevention of constipation, prevention of diarrhea, and immunostimulating effect. However, as people age, the proportion of Bifidobacteria in the human intestinal tract decreases. As a result, harmful bacteria are infected and cause various intestinal diseases. There are two main ways to solve this problem, one is to ingest bifidobacteria from the outside, and the other is to multiply the bifidobacteria that have been colonized. A method of ingesting Bifidobacterium from the outside is to take foods such as fermented milk containing Bifidobacterium. However, Bifidobacterium is a knock-out anaerobic organism and is likely to die even in the presence of trace amounts of oxygen. Therefore, there is a problem in that the content of live bifidobacteria in fermented milk products is greatly reduced in the process of circulating foods such as fermented milk products.
이에 본 발명자는 바실러스 서브틸리스 균주의 배양을 통한 효소식품을 개발하던 중 2단 발효를 통해 바실러스 서브틸리스 균주와 유산균의 동시 배양을 해결하고, 이때 DHNA를 생산하는 유산균을 도입하여 비피도박테리아 증식능이 우수한 효소식품을 생산하는 방법을 개발하기에 이르렀다. Therefore, the present inventor has developed enzyme food through the cultivation of Bacillus subtilis strain, and simultaneously solved the simultaneous cultivation of Bacillus subtilis strain and lactic acid bacterium through two-stage fermentation. At this time, lactic acid bacteria producing DHNA were introduced, And to develop a method for producing an enzyme food having excellent ability to proliferate.
본 발명은 신규한 균주인 바실러스 서브틸리스(Bacillus subtilis) 1-D-5를 제공한다.The present invention provides a novel strain, Bacillus subtilis 1-D-5.
또한 상기 균주와 락토바실러스 카제이(Lactobacillus casei) GW140 균주의 2단 발효를 통해 비피도박테리아를 증식시키는 방법 및 효소식품 제조방법을 제공한다. Also provided is a method for growing Bifidobacterium and a method for producing an enzyme food through two-step fermentation of the strain and Lactobacillus casei strain GW140.
본 발명은 α-아밀라아제와 프로테아제 활성을 갖는 바실러스 서브틸리스 1-D-5(기탁번호 KCTC 12813BP)를 제공한다. 상기 바실러스 서브틸리스 1-D-5는 죽염 된장에서 분리하는 것이 바람직하다. The present invention provides? -Amylase and Bacillus subtilis 1-D-5 (accession number KCTC 12813BP) having protease activity. The Bacillus subtilis 1-D-5 is preferably isolated from bamboo salt doenjang.
또한 본 발명은 바실러스 서브틸리스 1-D-5(기탁번호 KCTC 12813BP) 및 락토바실러스 카제이 GW140을 순차적으로 접종하여 배양하는 단계를 포함하는 비피도박테리아 증식방법을 제공한다. 상기 바실러스 서브틸리스 1-D-5는 죽염 된장에서 분리하는 것이 바람직하고, α-아밀라아제와 프로테아제 활성을 가질 수 있다. 상기 락토바실러스 카제이 GW140는 막걸리에서 분리하는 것이 바람직하고, 1,4-디하이드록시-2-나프토익 애시드(1,4-Dihydroxy-2-Naphthoic acid; DHNA)를 생산할 수 있다. The present invention also provides a method for propagating Bifidobacterium bacteria comprising the steps of sequentially inoculating Bacillus subtilis 1-D-5 (Accession No. KCTC 12813BP) and Lactobacillus casei GW140 in succession. The Bacillus subtilis 1-D-5 is preferably isolated from bamboo salt doenjang, and may have an α-amylase activity and a protease activity. The Lactobacillus casei GW140 is preferably isolated from rice wine and can produce 1,4-dihydroxy-2-naphthoic acid (DHNA).
상기 바실러스 서브틸리스 1-D-5의 배양은 20 내지 28시간 이루어지고 상기 락토바실러스 카제이 GW140의 배양은 10 내지 14 시간 이루어질 수 있다. 바람직하게는 24시간 동안 바실러스 서브틸리스 1-D-5를 배양한 후 락토바실러스 카제이 GW140를 추가적으로 12시간 동안 배양한다. 이때 유산균의 배양시간이 더 길어질 경우 효소의 활성이 줄어들 수 있다. The Bacillus subtilis 1-D-5 may be cultured for 20 to 28 hours, and the Lactobacillus casei GW140 may be cultured for 10 to 14 hours. Bacillus subtilis 1-D-5 is preferably cultured for 24 hours, and then Lactobacillus casei GW140 is cultured for an additional 12 hours. At this time, the longer the culture time of the lactic acid bacteria, the less the activity of the enzyme can be.
본 발명은, 기질에 바실러스 서브틸리스 1-D-5(기탁번호 KCTC 12813BP)를 접종하고 배양하는 단계(a 단계); 및 상기 a 단계 이후 락토바실러스 카제이 GW140를 접종하고 배양하는 단계(b 단계)를 포함하는 효소식품 제조방법을 제공한다. The present invention relates to a method for producing a recombinant bacillus subtilis 1-D-5 (step a) of inoculating and culturing a substrate with Bacillus subtilis 1-D-5 (accession number KCTC 12813BP); And a step (b) of inoculating and culturing Lactobacillus casei GW140 after step (a).
상기 바실러스 서브틸리스 1-D-5는 죽염 된장에서 분리될 수 있고, α-아밀라아제와 프로테아제 활성을 가질 수 있다. 상기 락토바실러스 카제이 GW140는 막걸리에서 분리될 수 있고, 1,4-디하이드록시-2-나프토익 애시드(1,4-Dihydroxy-2-Naphthoic acid; DHNA)를 생산할 수 있다. The Bacillus subtilis 1-D-5 may be isolated from bamboo salt doenjang, and may have an a-amylase activity and a protease activity. The Lactobacillus casei GW140 can be isolated from rice wine and can produce 1,4-Dihydroxy-2-naphthoic acid (DHNA).
상기 a 단계의 배양은 20 내지 28시간 이루어지고 상기 b 단계의 배양은 10 내지 14 시간 이루어질 수 있다. 바람직하게는 24시간 동안 바실러스 서브틸리스 1-D-5를 배양한 후 락토바실러스 카제이 GW140를 추가적으로 12시간 동안 배양한다. 이때 유산균의 배양시간이 더 길어질 경우 효소의 활성이 줄어들 수 있다. The cultivation in step a may be performed for 20 to 28 hours, and the cultivation in step b may be performed for 10 to 14 hours. Bacillus subtilis 1-D-5 is preferably cultured for 24 hours, and then Lactobacillus casei GW140 is cultured for an additional 12 hours. At this time, the longer the culture time of the lactic acid bacteria, the less the activity of the enzyme can be.
상기 기질은 현미분말, 쌀눈, 미강분말 및 대두분말로 이루어진 군에서 선택되는 하나 또는 둘 이상의 혼합일 수 있고, 바람직하게는 현미분말 50wt%, 쌀눈 30wt% 및 대두분말 20wt%의 혼합이다. The substrate may be a mixture of one or more selected from the group consisting of brown rice powder, rice gruel, rice gruel powder and soybean powder, preferably a mixture of brown rice powder 50wt%, rice gruel 30wt% and soybean powder 20wt%.
상기 바실러스 서브틸리스 균주와 락토바실러스 카제이 GW140 균주의 2단 발효를 통해 제조된 효소 식품은, 효소의 활성을 유지하면서 109CFU/g 이상의 유산균 수를 보유할 수 있고, 비피도박테리아 증식능을 가질 수 있다.The enzyme food prepared through the two-stage fermentation of the Bacillus subtilis strain and Lactobacillus casei GW140 strain can retain the lactic acid bacteria count of 10 9 CFU / g or more while maintaining the activity of the enzyme, and the bifidobacterial growth ability Lt; / RTI >
본 발명에 따르면 α-아밀라아제와 프로테아제 활성을 나타내는, 신규한 균주 바실러스 서브틸리스(Bacillus subtilis) 1-D-5와, 락토바실러스 카제이(Lactobacillus casei) GW140 균주의 2단 발효를 통해 비피도박테리아를 증식시킬 수 있다.According to the present invention, a novel strain Bacillus subtilis 1-D-5 and a strain Lactobacillus casei GW140, which exhibit? -Amylase and protease activity, Lt; / RTI >
또한 이를 이용하여 α-아밀라아제와 프로테아제 활성이 유지되면서 유산균 수가 109CFU/g 이상이고, 비피도박테리아 증식능이 우수한 효소식품을 제조할 수 있다.Also, it is possible to produce an enzyme food having a lactic acid bacterium number of 10 9 CFU / g or more and an excellent bifidobacteria proliferative activity while keeping the activity of α-amylase and protease.
도 1은 1-D-5 균주의 α-아밀라아제와 프로테아제 활성을 나타낸 이미지이다.
도 2는 B. subtilis 1-D-5 균주와 L. casei GW140균주의 혼합배양 조건을 다르게 하여 시간에 따른 pH를 측정한 그래프이다.
도 3은 B. subtilis 1-D-5 균주와 L. casei GW140균주의 혼합배양 조건을 다르게 하여 시간에 따른 바실러스균 수를 측정한 그래프이다.
도 4는 B. subtilis 1-D-5 균주와 L. casei GW140균주의 혼합배양 조건을 다르게 하여 시간에 따른 유산균 수를 측정한 그래프이다.
도 5는 B. subtilis 1-D-5 균주와 L. casei GW140균주의 혼합배양 조건을 다르게 하여 시간에 따른 α-아밀라아제 활성을 측정한 이미지이다.
도 6은 B. subtilis 1-D-5 균주와 L. casei GW140균주의 혼합배양 조건을 다르게 하여 시간에 따른 프로테아제 활성을 측정한 이미지이다.
도 7은 대조군, B. subtilis 1-D-5 균주 단독 발효 및 B. subtilis 1-D-5 균주와 L. casei GW140 균주의 2단 발효에 의해 생산된 효소식품의 비피도박테리아 증식능을 나타낸 그래프이다. Fig. 1 is an image showing the activity of? -Amylase and protease of 1-D-5 strain.
FIG. 2 is a graph showing pH values measured over time by varying the mixed culture conditions of B. subtilis 1-D-5 and L. casei GW140.
FIG. 3 is a graph showing the number of Bacillus bacteria counted over time under different mixed culture conditions of B. subtilis 1-D-5 and L. casei GW140.
FIG. 4 is a graph showing the number of lactic acid bacteria counted over time with different culturing conditions of B. subtilis 1-D-5 and L. casei GW140.
FIG. 5 is an image showing the activity of α-amylase activity over time with different culture conditions of B. subtilis 1-D-5 and L. casei GW140.
FIG. 6 is an image obtained by measuring the protease activity with time in the mixed culture conditions of B. subtilis 1-D-5 and L. casei GW140.
7 is a control group, B. subtilis 1-D-5 strain alone and B. subtilis fermentation bifidobacteria graph showing the proliferation of bacteria in the 1-D-5 strain and the enzyme products produced by the two-stage fermentation of the strains L. casei GW140 to be.
이하, 실시예를 통하여 본 발명을 상세하게 설명한다. 본 발명의 목적, 특징, 장점은 이하의 도면 및 실시예를 통하여 쉽게 이해될 것이다. 본 발명은 여기서 설명되는 도면 및 실시예에 한정되지 않고, 다른 형태로 구체화될 수 있다. 여기서 소개되는 도면 및 실시예는 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에게 본 발명의 사상이 충분히 전달될 수 있도록 하기 위하여 제공되는 것이다. 따라서. 이하의 도면 및 실시예에 의하여 본 발명의 권리범위가 제한되어서는 안 된다.Hereinafter, the present invention will be described in detail by way of examples. The objects, features and advantages of the present invention will be readily understood through the following drawings and examples. The present invention is not limited to the drawings and embodiments described herein, but may be embodied in other forms. The drawings and embodiments are provided so that those skilled in the art can fully understand the spirit of the present invention. therefore. The scope of the present invention should not be limited by the following drawings and examples.
<< 실시예Example 1> α-아밀라아제와 프로테아제 활성을 가진 1 > α-amylase and protease activity Bacillus Bacillus subtilissubtilis 1-D-5 균주의 분리 및 동정 Isolation and Identification of 1-D-5 Strain
1) 균주 분리1) Strain isolation
지역의 한 장류 업체로부터 숙성된 죽염 된장을 얻어 α-아밀라아제 및 프로테아제 활성이 우수한 바실러스 균주들을 선발하였다. 먼저 된장 시료 3.5g을 펩톤수(0.1%, w/v) 31.5㎖에 넣고 현탁액을 제조하여 십진희석법(ten-fold dilution method)으로 영양 한천(nutrient agar; Difco, Sparks, MD, USA) 평판배지에 도말하였다. 그 다음 형성된 콜로니 430개를 확보하여 3분획법으로 순수분리 후 5㎖의 영양배지(nutrient broth; Difco)에 배양한 후 글리세롤(최종농도 40%)을 첨가하여 -78℃에 보관하였다. 보관된 균주들은 영양배지에서 계대배양 한 후 0.5%(w/v) 녹말과 1%(w/v) 탈지 우유(skim milk)가 각각 포함된 영양 한천 평판배지에 접종하여 하룻밤 배양 후 기질 분해 환(substrate degradation zone)의 크기로 효소활성을 측정하여 도 1에 나타내었다. 측정 결과, 1-D-5 균주에서 두 효소 모두 높은 활성을 보여 이를 효소식품 제조를 위한 스타터 균주로 선발하였다.Bacillus strains with excellent α - amylase activity and protease activity were selected from mature bamboo salt soybean paste obtained from a local soybean maker. First, 3.5 g of the doenjang sample was added to 31.5 ml of the peptone water (0.1%, w / v) to prepare a suspension. The suspension was prepared by a ten-fold dilution method using a nutrient agar (Difco, Sparks, . Next, 430 colonies were obtained and purified by 3 fractionation method. Then, the cells were cultured in 5 ml nutrient broth (Difco), and glycerol (final concentration 40%) was added and stored at -78 ° C. The stored strains were subcultured in nutrient broth and inoculated on nutrient agar plate medium containing 0.5% (w / v) starch and 1% (w / v) skim milk, The enzyme activity was measured by the size of the substrate degradation zone and is shown in Fig. As a result, both enzymes in the 1-D-5 strain showed high activity and were selected as a starter strain for the production of enzyme foods.
2) 균주 동정2) Identification of strain
1-D-5 균주의 동정을 위해 16S rRNA 유전자 염기서열을 이용한 분석법을 사용하였다. 16S rRNA 유전자 염기서열의 분석은 국내 생명공학회사(마크로젠)에 의뢰하여 수행하였다. 1-D-5 균주의 16S rRNA 유전자 염기서열은 서열번호 1에 나타내었다. GenBank 염기서열 데이터베이스를 이용하여 1-D-5 균주의 16S rRNA 염기서열 상동성(homology)을 분석한 결과 Bacillus subtilis 균주들(GenBank accession no. CP007173.1, CP011115.1 등)과 가장 높은 일치율(상동성 99%)을 보여 1-D-5 균주를 B. subtilis 1-D-5로 명명하였다. For the identification of 1-D-5 strains, 16S rRNA gene sequences were used. Analysis of the 16S rRNA gene sequence was performed by a domestic biotechnology company (Macrogen). The 16S rRNA gene sequence of the strain 1-D-5 is shown in SEQ ID NO: 1. The 16S rRNA nucleotide sequence homology of 1-D-5 strains was analyzed using the GenBank sequence database and the highest concordance rate with Bacillus subtilis strains (GenBank accession no. CP007173.1, CP011115.1, etc.) 99%). The strain 1-D-5 was named B. subtilis 1-D-5.
<< 실시예Example 2> 2> B. B. subtilissubtilis 1-D-5 균주를 이용한 효소식품 기질 선발 Selection of enzyme food substrate using 1-D-5 strain
단독 혹은 혼합기질에 B. subtilis 1-D-5 균주를 접종하여 최고의 효소활성도를 보이는 기질 조합을 선발하였다. 즉, 현미분말(100wt%), 미강분말(100wt%), 대두분말(100wt%), 현미분말(60wt%) + 미강분말(40wt%), 현미분말(50wt%) + 미강분말(30wt%) + 대두분말(20wt%), 현미분말(50wt%) + 대두분말(50wt%), 현미분말(50wt%) + 쌀눈(30wt%) + 대두분말(20wt%) 등의 조합을 만든 후 100℃에서 1시간 증숙하고 여기에 B. subtilis 1-D-5 균주 0.5wt%를 접종한 후 수분함량을 최종농도 60%로 맞추고 30℃에서 72시간 배양하였다. 배양을 하면서 경시적으로(0, 12, 24, 36, 48, 60, 72시간) α-아밀라아제와 프로테아제의 활성을 측정하여 최고의 활성을 나타내는 기질 조합을 선발하였다. 단독 혹은 혼합기질의 종류에 상관없이 전반적으로 비슷한 효소의 활성을 보였으며 특히, 현미분말(50wt%) + 쌀눈(30wt%) + 대두분말(20wt%) 조합에서 다소 높은 효소활성이 나타났다. 따라서 효소의 활성 및 영양학적인 가치를 고려하여 B. subtilis 1-D-5 균주를 위한 기질로써 현미분말(50wt%) + 쌀눈(30wt%) + 대두분말(20wt%) 조합을 최종적으로 선택하였다.Substrate combinations showing the highest enzymatic activity were selected by inoculating B. subtilis 1-D-5 to single or mixed substrates. (100wt%), brown rice powder (100wt%), brown rice powder (100wt%), brown rice powder (60wt%), rice bran powder (40wt%), brown rice powder (50wt%) and rice bran powder , A mixture of soybean powder (20wt%), brown rice powder (50wt%) + soybean powder (50wt%), brown rice powder (50wt%) + rice grain (30wt%) + soybean powder (20wt% After 1 hour of incubation, 0.5wt% of B. subtilis 1-D-5 strain was inoculated and the moisture content was adjusted to 60% of final concentration and cultured at 30 ° C for 72 hours. The activity of α-amylase and protease was measured over time (0, 12, 24, 36, 48, 60, 72 hours) while culturing to select substrate combinations showing the highest activity. (50wt%) + rice husk (30wt%) + soybean powder (20wt%) showed higher enzymatic activity regardless of the kind of the mixture. Therefore, the combination of brown rice powder (50wt%) + rice husk (30wt%) + soybean powder (20wt%) was finally selected as a substrate for the B. subtilis 1-D-5 strain considering the activity and nutritional value of the enzyme.
<< 실시예Example 3> 3> B. B. subtilissubtilis 1-D-5와 1-D-5 and L. L. caseicasei GW140 균주의 혼합배양 조건 탐색 실험 Experiments on the culture conditions of GW140 strain
상기 실시예 2에서 선발된 혼합기질(100g; 현미분말 50g + 쌀눈 30g + 대두분말 20g)을 제조하여 B. subtilis 1-D-5 균주와 L. casei GW140 균주의 혼합배양이 가능한 조건을 탐색하였다(표 1). The mixed substrate (100 g; brown rice powder 50 g + rice gruel 30 g + soybean powder 20 g) selected in Example 2 was prepared to investigate conditions capable of culturing the mixture of B. subtilis 1-D-5 and L. casei GW140 (Table 1).
이때 L. casei GW140 균주는 -76℃ 극저온 냉동고에 보관중인 글리세롤 스톡을 MRS broth(Difco)에서 계대배양하여 사용하였다.At this time, L. casei GW140 strain was used by subculturing glycerol stock stored in a -76 ° C cryogenic freezer with MRS broth (Difco).
두 균주를 동시에 접종해서 배양하는 방법과 순차적으로(B. subtilis 1-D-5 균주 접종 후 L. casei GW140 균주 접종) 접종해서 배양하는 방법을 비교 분석(효소활성 및 유산균 수 측정)하여 도 2 내지 도 6에 나타내었다. 2), and the method of culturing inoculated with the two strains simultaneously and the method of successively culturing inoculation (inoculation with L. casei GW140 strain after inoculation with B. subtilis 1-D-5 strain) 6 and Fig.
[표 1][Table 1]
그 결과 동시에 접종한 경우, 유산균 증식에 의한 pH 저하로 바실러스의 생육이 억제되어 효소의 활성이 나타나지 않았으며 순차적으로 접종한 경우, 바실러스 증식에 의해 효소생산이 이루어지고 난 후 유산균이 접종되어 효소의 활성이 유지되면서 유산균 수도 확보되는 조건을 찾았다. 즉, 바실러스 접종 후 24시간 배양 후 유산균을 접종하여 추가적으로 12시간 배양하는 것이 최적의 조건으로 확인되었다(도 2 내지 도 6). 유산균의 배양시간이 더 길어질 경우 효소의 활성이 줄어드는 결과를 초래한다. As a result, in the case of simultaneous inoculation, the growth of bacillus was inhibited by the pH drop due to the growth of the lactic acid bacteria, and the enzyme activity was not exhibited. In case of successive inoculation, the enzyme production was carried out by bacillus growth and the lactic acid bacteria were inoculated, The activity was retained and the conditions for securing lactic acid bacteria were found. In other words, culturing for 24 hours after inoculation with Bacillus, followed by inoculation with lactic acid bacteria, and culturing for an additional 12 hours were confirmed as optimal conditions (FIGS. 2 to 6). A longer incubation time of the lactic acid bacteria results in a decrease in the activity of the enzyme.
<< 실시예Example 4> 4> B. B. subtilissubtilis 1-D-5와 1-D-5 and L. L. caseicasei GW140 균주의 2단 발효를 통해 생산된 효소식품의 Of enzyme food produced through the two-stage fermentation of strain GW140 비피도박테리아Bifidobacterium 증식능Proliferative ability 확인 Confirm
DHNA 생산 유산균의 접목으로 기대될 수 있는 비피도박테리아 증식능을 검증하기 위해 비피도박테리움 락티스(Bifidobacterium lactis) BL750 균주(Culture systems Inc., USA)를 RCM (Reinforced Clostridial Medium) 배지에서 활성화시킨 다음 배지 성분이 1/10로 줄어든 RCM 배지에 1% 접종 한 후 시료 100㎕를 첨가하여 경시적으로 비피도박테리아 균수를 측정하였다. 균수 측정은 배양액을 10진 희석법으로 희석한 후 RCM 고체 배지에 도말하여 혐기 용기(jar)에서 배양하면서 자란 콜로니를 개수하여 나타내었다. 사용한 시료는 대조구(균주 무 접종 시료), 1-D-5(B. subtilis 1-D-5 균주만 접종한 효소식품), 1-D-5 + GW140(B. subtilis 1-D-5 균주와 L. casei GW140 균주의 2단 발효를 통해 생산한 효소식품) 3가지였으며 대조구 및 발효물 10g에 90㎖의 펩톤수를 첨가하여 현탁한 후 6,000g에서 10분간 원심분리하여 상등액을 취해 주사필터(0.45um, Sartorius Co.)로 여과하여 비피도박테리아 증식능 검증을 위한 시료로 사용하였다. 시간대별 균수는 3반복 실험을 통하여 평균 ± 표준편차로 나타내어 도 7에 나타내었다. 값들 위에 표시한 서로 다른 알파벳은 Duncan's multiple range test에 의한 유의적 차이를 나타낸다(p<0.05).DHNA BP can be expected to incorporate the production of lactic acid bacteria also Bifidobacterium lactis in order to verify the proliferation of bacteria (Bifidobacterium lactis ) BL750 (Culture systems Inc., USA) was activated in RCM (Reinforced Clostridial Medium) medium. Then, 1% of the medium was inoculated into RCM medium containing 1/10 of the culture medium, 100 μl of the sample was added, The germ cell count was measured. The cultures were diluted with decanter, diluted with RCM, and cultured in an anaerobic jar to recover colonies. The samples used were 1-D-5 (an enzyme food only inoculated with B. subtilis 1-D-5 strain), 1-D-5 + GW140 ( B. subtilis 1-D-5 strain, And L. casei GW140), and 90 g of peptone was added to 10 g of the control and fermented product, and the suspension was centrifuged at 6,000 g for 10 minutes to obtain a supernatant. (0.45um, Sartorius Co.) and used as a sample for the verification of bifidobacterial proliferative capacity. The number of bacteria by time period is shown by the mean ± standard deviation through three repeated experiments and is shown in FIG. The different alphabets displayed above the values indicate a significant difference by Duncan's multiple range test (p <0.05).
도 7을 살펴보면 1-D-5와 1-D-5 + GW140 모두 대조구에 비해 비피도박테리아 증식능이 우수하였으며 특히 유산균이 첨가된 경우 최대 0.81Log 차이를 보였다(도 7). 이는 L. casei GW140 균주가 생산하는 DHNA를 포함하는 대사산물들이 비피도박테리아 증식에 기여했음을 의미한다. FIG. 7 shows that 1-D-5 and 1-D-5 + GW140 showed superior Bifidobacterium proliferative potency compared to the control. Especially, when lactic acid bacteria were added, the maximum difference was 0.81 log (FIG. This means that metabolites containing DHNA produced by the L. casei GW140 strain contributed to Bifidobacteria proliferation.
<110> Korea National University of Transportation <120> BACILLUS SUBTILIS 1-D-5, METHOD FOR GROWTH STIMULATION OF BIFIDOBACTERIA AND METHOD FOR MANUFACTURING OF ENZYME FOOD USING THEREOF <130> P15-0216 <160> 1 <170> KoPatentIn <210> 1 <211> 1486 <212> DNA <213> Bacillus subtilis <400> 1 gaacaggctc aggacgaacg ctggcggcgt gcctaataca tgcaagtcga gcggacagat 60 gggagcttgc tccctgatgt tagcggcgga cgggtgagta acacgtgggt aacctgcctg 120 taagactggg ataactccgg gaaaccgggg ctaataccgg atggttgttt gaaccgcatg 180 gttcaaacat aaaaggtggc ttcggctacc acttacagat ggacccgcgg cgcattagct 240 agttggtgag gtaacggctc accaaggcga cgatgcgtag ccgacctgag agggtgatcg 300 gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagta gggaatcttc 360 cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt gatgaaggtt ttcggatcgt 420 aaagctctgt tgttagggaa gaacaagtac cgttcgaata gggcggtacc ttgacggtac 480 ctaaccagaa agccacggct aactacgtgc cagcagccgc ggtaatacgt aggtggcaag 540 cgttgtccgg aattattggg cgtaaagggc tcgcaggcgg tttcttaagt ctgatgtgaa 600 agcccccggc tcaaccgggg agggtcattg gaaactgggg aacttgagtg cagaagagga 660 gagtggaatt ccacgtgtag cggtgaaatg cgtagagatg tggaggaaca ccagtggcga 720 aggcgactct ctggtctgta actgacgctg aggagcgaaa gcgtggggag cgaacaggat 780 tagataccct ggtagtccac gccgtaaacg atgagtgcta agtgttaggg ggtttccgcc 840 ccttagtgct gcagctaacg cattaagcac tccgcctggg gagtacggtc gcaagactga 900 aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc 960 aacgcgaaga accttaccag gtcttgacat cctctgacaa tcctagagat aggacgtccc 1020 cttcgggggc agagtgacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg 1080 ggttaagtcc cgcaacgagc gcaacccttg atcttagttg ccagcattca gttgggcact 1140 ctaaggtgac tgccggtgac aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc 1200 ccttatgacc tgggctacac acgtgctaca atggacagaa caaagggcag cgaaaccgcg 1260 aggttaagcc aatcccacaa atctgttctc agttcggatc gcagtctgca actcgactgc 1320 gtgaagctgg aatcgctagt aatcgcggat cagcatgccg cgggtgaata cgttcccggg 1380 ccttgtacac accgcccgtc acaccacgaa gagtttgtaa cacccgaagt cggtgaggta 1440 accttttagg agccagccgc cgaaggtggg acagatgatt ggggtg 1486 <110> Korea National University of Transportation <120> BACILLUS SUBTILIS 1-D-5, METHOD FOR GROWTH STIMULATION OF BIFIDOBACTERIA AND METHOD FOR MANUFACTURING OF ENZYME FOOD USING THEREOF <130> P15-0216 <160> 1 <170> KoPatentin <210> 1 <211> 1486 <212> DNA <213> Bacillus subtilis <400> 1 gaacaggctc aggacgaacg ctggcggcgt gcctaataca tgcaagtcga gcggacagat 60 gggagcttgc tccctgatgt tagcggcgga cgggtgagta acacgtgggt aacctgcctg 120 taagactggg ataactccgg gaaaccgggg ctaataccgg atggttgttt gaaccgcatg 180 gttcaaacat aaaaggtggc ttcggctacc acttacagat ggacccgcgg cgcattagct 240 agttggtgag gtaacggctc accaaggcga cgatgcgtag ccgacctgag agggtgatcg 300 gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagta gggaatcttc 360 cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt gatgaaggtt ttcggatcgt 420 aaagctctgt tgttagggaa gaacaagtac cgttcgaata gggcggtacc ttgacggtac 480 ctaaccagaa agccacggct aactacgtgc cagcagccgc ggtaatacgt aggtggcaag 540 cgttgtccgg aattattggg cgtaaagggc tcgcaggcgg tttcttaagt ctgatgtgaa 600 agcccccggc tcaaccgggg agggtcattg gaaactgggg aacttgagtg cagaagagga 660 gagtggaatt ccacgtgtag cggtgaaatg cgtagagatg tggaggaaca ccagtggcga 720 aggcgactct ctggtctgta actgacgctg aggagcgaaa gcgtggggag cgaacaggat 780 tagataccct ggtagtccac gccgtaaacg atgagtgcta agtgttaggg ggtttccgcc 840 ccttagtgct gcagctaacg cattaagcac tccgcctggg gagtacggtc gcaagactga 900 aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc 960 aacgcgaaga accttaccag gtcttgacat cctctgacaa tcctagagat aggacgtccc 1020 cttcgggggc agagtgacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg 1080 ggttaagtcc cgcaacgagc gcaacccttg atcttagttg ccagcattca gttgggcact 1140 ctaaggtgac tgccggtgac aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc 1200 ccttatgacc tgggctacac acgtgctaca atggacagaa caaagggcag cgaaaccgcg 1260 aggttaagcc aatcccacaa atctgttctc agttcggatc gcagtctgca actcgactgc 1320 gtgaagctgg aatcgctagt aatcgcggat cagcatgccg cgggtgaata cgttcccggg 1380 ccttgtacac accgcccgtc acaccacgaa gagtttgtaa cacccgaagt cggtgaggta 1440 accttttagg agccagccgc cgaaggtggg acagatgatt ggggtg 1486
Claims (12)
상기 1 단계 이후, 락토바실러스 카제이 GW140를 접종하고 배양하는 단계(2 단계)를 포함하는 효소식품 제조방법으로서,
상기 기질은 현미분말, 쌀눈, 미강분말 및 대두분말로 이루어진 군에서 선택되는 하나 또는 둘 이상을 혼합한 것이고,
상기 효소식품은 효소의 활성이 유지되면서 109CFU/g 이상의 유산균 수를 보유하는 것을 특징으로 하고,
상기 효소식품은 2단 발효에 의해 생산된 것으로, α-아밀라아제와 프로테아제 활성을 갖는 것을 특징으로 하는 효소식품 제조방법. Inoculating and culturing the Bacillus Subtilis 1-D-5 (Accession No. KCTC 12813BP) having α-amylase activity and protease activity (step 1) to the substrate; And
A method for producing an enzyme food comprising the step of inoculating and culturing Lactobacillus casei GW140 (step 2) after the above step 1,
The substrate is a mixture of one or more selected from the group consisting of brown rice powder, rice husk, rice bran powder and soybean powder,
The enzyme food is characterized by having the number of lactic acid bacteria of 10 9 CFU / g or more while maintaining the activity of the enzyme,
The enzyme food is produced by two-stage fermentation, and is characterized by having? -Amylase and protease activity Enzyme food manufacturing method.
상기 락토바실러스 카제이 GW140는
1,4-디하이드록시-2-나프토익 애시드를 생산하는 것을 특징으로 하는 효소식품 제조방법.The method of claim 5,
The Lactobacillus casei GW140
1,4-dihydroxy-2-naphthoic acid.
상기 1 단계의 배양은 20 내지 28 시간 배양하는 것을 특징으로 하는 효소식품 제조방법.The method of claim 5,
Wherein the culture of step 1 is cultured for 20 to 28 hours.
상기 2 단계의 배양은 10 내지 14 시간 배양하는 것을 특징으로 하는 효소식품 제조방법.The method of claim 5,
Wherein the culture in the second step is cultured for 10 to 14 hours.
상기 효소식품은 비피도박테리움 락티스(Bifidobacterium lactis) BL750 증식능이 있는 것을 특징으로 하는 효소식품 제조방법.
The method of claim 5,
Wherein said enzyme food is capable of replicating Bifidobacterium lactis BL750.
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