具苯并[a]芘清除能力之乳酸菌株篩選及其特性研究

Screening and characterization of lactic acid bacterial strains possessing benzo[a]pyrene-removing ability

邱宇彤(Yu-Tung Chiu)

指導教授 ： 劉嚞睿 ； 鄭光成

國立臺灣大學/生物資源暨農學院/食品科技研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.01541

摘要

燒烤食物含苯并[a]芘（benzo[a]pyrene），經過人體代謝後容易形成致癌物。近年來的相關研究指出，可以利用乳酸菌和致癌劑之結合，進而降解或代謝成毒性較低之物質，減低對人體腸胃道的潛在危險。本研究為了篩選出具有清除苯并[a]芘之能力的乳酸菌，從嬰兒糞便中，先進行菌株之初篩選，再根據清除苯并[a]芘的能力及抗突變之分析情形，篩選出具有較高清除率之菌株，進行基本特性分析。結果顯示，初篩選出28株類乳酸菌，進一步在苯并[a]芘清除試驗中，利用添加1 ppm苯并[a]芘至不同篩選出的菌株之培養基中，進而篩選出YT 2、YT 5及YT 17乳酸菌株對苯并[a]芘具有較強之清除率，分別為97.66 %、 97.66 %及97.79 %。將活菌體經過熱處理與超音波破碎法來製備熱致死菌體、細胞壁及細胞內萃取物，結果顯示活菌體結合率大於熱致死菌體，而細胞壁結合率大於細胞內萃取物，可以推測乳酸菌株清除苯并[a]芘以吸附作用為主，細胞壁為重要的結合因子。進一步使用安氏試驗法之測試菌株Salmonella typhimurium TA100來分析乳酸菌對苯并[a]芘誘導突變之預防效果，結果顯示，三株乳酸菌對S. typhimurium TA100並不具有毒性與致突變性，並對苯并[a]芘誘突變性具有超過50%的抑制效果。連續的預反應研究顯示抗突變性的主要機制是去致突變作用。經革蘭氏染色法、API 50 CHL及16S rRNA鑑定結果顯示YT 2及YT 5屬於Enterococcus feacium，而YT 17屬於Enterococcus feaclis。以模擬宿主腸胃道環境的耐酸及膽鹽耐受性試驗顯示，此三株菌可在pH 2的環境中存活，並可於含有0.3% oxgall的MRS培養液中生長；細胞吸附試驗顯示此三株菌對人類大腸癌細胞Caco-2具有吸附能力。綜合以上結果，E. feacium YT 2、E. feacium YT 5及E. feaclis YT 17不僅具有清除苯并[a]芘之能力及抗致突變作用，且具有多項益生菌的特性，故後續將可應用於健康食品之開發。

關鍵字

益生菌 ； 乳酸菌 ； 致癌劑 ； 苯并[a]芘

並列摘要

Colorectal cancer (CRC) ranks the third most common malignant disease worldwide. The risk factors of CRC include the diet and lifestyle of people. Meat-related compounds, especially benzo(a)pyrene (BaP) is one of the best-characterised PAHs compound in the diet. Recently, studies have confirmed a correlation between probiotics and CRC that removing of carcinogens and mutagens has been considered as a possible mechanism in CRC prevention. The object of this study was to screen probiotic strains that have the BaP-removing ability. Then, the isolated strains exhibited high efficiency in removing BaP will be further evaluated for their functional properties and for its potential application as probiotic. As the results, the 28 bacterial strains were isolated from infant feces and then treated with 1 ppm BaP to analyze their BaP-removing ability. Three strains (YT 2, YT 5 and YT 17) had the best removing rate (97.66 %, 97.66 % and 97.79 %). The viability of the bacteria has significant effect on the removing of BaP. Cell walls of strains showed higher binding ability than heat-treated bacteria and intracellular extract. Therefore, we proposed that peptidoglycans played an important role in binding BaP. Antimutagenic activities of three strains were examined by Ames test. The results shows that the test strains didn’t have the cytotoxicity and mutagenicity against Salmonella typhimurium TA100 and the cultures incubated with potent mutagen BaP displayed high antimutagenic activities. After identification, YT 2 and YT 5 were classified as Enterococcus feacium, and YT 17 were Enterococcus feaclis. In the probiotic activity assay, all the test strains could survive in pH 2 solutions and grow in MRS broth containing 0.3% oxgall. In the Caco-2 cell adhesion assay, the test strains were able to adhere to Caco-2 cell. In conclusion, E. feacium YT 2, E. feacium YT 5 and E. feaclis YT 17 not only have the capable of removing BaP, but also possess antimutagenic activities, acid resistance, and bile salt tolerance ability. Therefore, E. feacium YT 2, E. feacium YT 5 and E. feaclis YT 17 have a potential to be applied in the human diet and animal feed.

並列關鍵字

probiotic ； lactic acid bacteria ； carcinogen ； benzo[a]pyrene

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