식품 및 천연물을 섭취한 후 소화관내의 장내미생물총 대사에 의해 독성 및 효능이 나타날 수 있으므로 in vitro에서 식품의 독성 및 효능 평가가 이루어져야 한다. 본 연구의 목적은 첫째, 당분해효소활성이 우수한 장내미생물을 선별하여 fecal microbial enzyme mix(FM)를 제조하고 이를 fecalase대신 Ames test에 사용하여 대두의 항돌연변이원성 측정에 이용할 수 있는지를 알아보고자 한다. 둘째, FM을 이용한 Ames test에 서 안전성이 확인된 대두의 첨가량을 달리한 대두 막걸리의 발효특성을 파악하고 발효하는 과정에 isoflavone의 비배당체(aglycone)로의 전환을 확인하여 생리활성이 우수한 대두 막걸리를 개발하고자 한다.
본 연구에서는 건강한 성인의 분변을 수집하여 장내 미생물을 분리하였고 14개의 당분해 효소활성을 측정하여 효소활성이 강한 4개의 균주 (Megasphaera elsdenii, Klebsiella pneumonia, Eubacterium rectale, Parabacteroides distasonis)를 선택하여 fecalase의 단점을 보완한 FM을 제조하였다. FM은 동결건조하여 4℃에 저장한 경우가 효소활성이 가장 안정하였으므로 동결건조한 FM을 4℃에 저장하면서 Ames test에 사용하였다. FM을 사용한 Ames test에서 천연 양성대조물질인 rutin과 화학 양성대조물질인 2-AA은 fecalase와 동일한 복귀돌연변이 집락수의 증가가 나타나 FM을 소화관 대사계로 사용할 수 있을 것으로 사료되었다. 그러므로 FM을 사용하여 대두 추출물과 genistein의 소화관 대사에 의한 돌연변이성을 측정한 결과 S. typhimurium TA98, TA100, TA102, TA1535, TA1537 균주에서 복귀돌연변이 집락수가 증가하지 않아 대두는 소화관 대사에 의한 항돌연변이원성을 보였다.
소화관 대사에 의해 안전성이 확보된 대두의 첨가량을 달리하여 대두 막걸리를 제조하였다. 생균수는 밑술은 발효 2일에 총균수, 유산균수가 최대로 나타났고 발효 4일에 효모수가 최대로 나타났다. 효모수가 최대인 발효 4일째의 밑술을 이용하여 덧술을 제조하여 15일간 발효하는 동안 품질특성을 측정하였다. 덧술의 amylase 활성은 발효 3일까지 증가하다가 감소하였고 환원당의 함량은 발효기간이 지날수록 유의적으로 감소하였다. 덧술은 15일 발효시킨 후 체에 걸러서 65℃에서 30분간 저온살균하여 4℃에서 12주 동안 저장하면서 품질 평가를 하였다. 살균한 대두막걸리의 총균수, 유산균수, 효모수는 저장 초기에 거의 증가하지 않았으나 그 이후 서서히 증가하여 저장 8주차에 가장 크게 증가하는 것으로 나타났다. 살균한 대두 막걸리를 4℃에서 2일동안 저장한 후 20세 이상 성인 남녀 42명을 대상으로 색, 맛, 풍미(냄새), 전반적인 선호도에 대해 7점 척도법으로 관능검사를 실시한 결과 맛과 전반적인 선호도에서 3% 첨가군이 우수하였다. 대두 첨가량을 달리하여 제조한 대두 막걸리를 덧술 발효 0, 6, 9, 15일에 시료를 취하여 isoflavone 함량 변화를 측정한 결과 발효가 진행되면서 누룩에 포함되어 있는 미생물들에 의해 대두의 배당체(glycosides)가 비배당체(aglycone)로 전환되어 발효 15일에는 생리활성이 우수한 daidzein과 genistein의 함량이 높게 나타났다. 그러나 시판중인 A주조의 검은콩 막걸리는 막걸리의 발효가 끝난 후 대두 추출물을 첨가하여 제조하므로 daidzein과 genistein이 검출되지 않았다.
본 연구에서 개발된 대두 막걸리는 3% 첨가군이 관능평가 결과 맛과 전반적인 선호도가 높았고 시판 검은콩 막걸리보다 생리활성이 우수한 daidzein과 genistein을 포함하고 있었다. 저장 8주째에 대두 막걸리의 생균수가 크게 증가하였으므로 6주까지 냉장 보관하여 유통할 수 있을 것으로 판단되었다.
이상의 연구결과를 토대로 FM을 이용한 Ames test에서 안정성이 확보된 대두를 사용하여 개발된 대두 막걸리가 대량 생산과정에서도 품질을 유지하여 대두 추출물을 첨가한 기존의 시판 대두 막걸리보다 생리활성이 우수한 daidzein과 genistein이 포함된 우수한 품질의 기능성 막걸리가 시판되기를 기대한다.

After intaking of food and natural products, toxicity or efficacy may occur by metabolization of the intestinal microflora in the digestive tract. Therefore, toxicity and efficacy of food should be evaluated in vitro. There are two purposes of this study. The first purpose of this study is to examine if it is possible to use fecal microbial enzyme mix (FM) on Ames test instead of fecalase in order to measure antimutagenic activities of the soybeans. By selecting intestinal flora with superior glycolytic enzyme activity, fecal microbial enzyme mix (FM) is produced. The second purpose is that we want to develop soybean makgeolli that has high biological activity by observing conversion from isoflavone to aglycone during the fermentation process. We find out fermentation characteristics of soybean makgeolli groups which have different amounts of the soybeans. And the safety of the soybeans is confirmed by Ames test using FM.
By collecting the feces of healthy adults, the intestinal floras were isolated. The activities of the 14 glycolytic enzymes were measured, and FM covering weak points of fecalase was made by choosing four strains(Megasphaera elsdenii, Klebsiella pneumonia, Eubacterium rectale, Parabacteroides distasonis) with strong enzyme activities. FM was used for Ames test within its most stable condition which was being freeze-dried and stored at 4℃. In Ames test using FM, rutin that is a positive control natural substance and 2-AA that is a positive control chemical substance showed the same amounts of increased reverse mutation colonies as fecalase. Therefore, it seems possible that the FM will be able to be used as digestive tract metabolites. Therefore, mutagenic activities by intestinal metabolism of soy extracts and genistein were measured with FM, and it did not show increased numbers of reverse mutation colonies in S. typhimurium TA98, TA100, TA102, TA1535, TA1537 strains. Soybeans showed anti-mutagenic characteristics by the digestive tract metabolism.
We made the soybean makgeolli by putting different amounts of the soybeans of which safety of intestinal metabolism had been confirmed. Considering the viable cell counts of the first mashes, total cell and lactic acid bacteria were maximal on the second day of fermentation, and the yeast counts were maximal on the fourth day of fermentation. The quality of makgeolli was measured as the second mash was produced by using the first mash from the fourth day of fermentation that had the highest number of the yeasts. The amylase activity of the second mash was increased until the third day of fermentation and then decreased. The amount of reducing sugars was significantly reduced while the fermentation duration becomes longer. After fermenting the second mash for 15 days, the second mash was filtered through a sieve. The filtrate was pasteurized at 65℃ for 30 minutes and stored at 4℃ for 12 weeks, and its quality was assessed during this period. The viable cell counts(total cell, lactic acid bacteria, and yeast counts) in sterilized makgeolli were hardly increased during the early storage period. However, they were increased gradually, and the maximal increase was shown on the eighth week of the storage. Sterilized makgeolli was stored at 4℃ for 2 days and then it was evaluated by 42-adults. Seven-point hedonic scales(from 1=dislike extremely to 7=like extremely) for color, taste, flavor, and overall preference were used to evaluate the makgeolli. The results of the sensory evaluation showed that makgeolli that had 3% proportion of the soy beans had high scores at taste and overall preference. At 0, 6, 9, 15 days of fermentation of the second mash, isoflavone content was measured in makgeolli with different amounts of soybeans. As a result, during fermentation, glycosides of the soy beans were converted to aglycone by microorganisms in the yeast, and at 15days, there were high proportions of daidzein and genistein that had excellent biological activities. However, dadizein and genistein were not detected in the commercial makgeolli, produced by “A” manufacturer because it was produced by putting soybean extracts after the end of makgeolli fermentation.
In this study, makgeolli that has 3% of soy beans had highest scores at taste and overall preference from the sensory evaluation of the soybean makgeolli and had greater proportions of highly bioactive daidzein and genistein than the commercial makgeolli. It seems that makgeolli may be refrigerated and distributed for sale till the sixth week because of a significant increase in the number of viable microorganism counts on the eighth week of the storage.
Based on the above findings, the soybean makgeolli was made from the soybeans of which safety had been confirmed by Ames test using FM. And the soybean makgeolli had greater proportions of highly bioactive daidzein and genistein than the conventional commercial soybean makgeolli made from the soybean extracts. We hope to see the soybean makgeolli that has higher quality than the conventional commercial soybean makgeolli keep its quality even through mass production and finally come to the market.