地衣芽孢桿菌CK1清除黴菌毒素zearalenone之機制及其益生功能的研究

Study of the zearalenone removal mechanism and evaluation of the probiotic functions of Bacillus licheniformis CK1

徐翠君(Tsui-Chun Hsu)

指導教授 ： 劉?睿

國立臺灣大學/生物資源暨農學院/動物科學技術學研究所/博士(2018年)

https://doi.org/10.6342/NTU201802279

摘要

根據聯合國糧食及農業組織的調查，全球約50％的穀物被黴菌毒素污染。黴菌毒素造成人類、動物和作物產生疾病以及經濟損失。在飼料當中添加黴菌毒素去除劑是被經常使用的解決方案。先前的研究顯示，本實驗室篩選到的地衣芽孢桿菌CK1（Bacillus licheniformis CK1，CK1），在與含有黴菌毒素zearalenone（ZEN）的玉米粉培養基中共培養36小時後，降低了98％以上的zearalenone，顯示CK1具有成為黴菌毒素去除劑的潛力。另外，在評估CK1作為益生菌的研究中也證明，CK1對胃酸和膽鹽具有抗性、可以粘附於人類腸道上皮細胞（Caco-2）、並降低病原菌如enterohemorrhagic Escherichia coli 與 Salmonella typhimurium對Caco-2細胞表面的粘附，這些試驗的結果均顯示CK1均有做為食品益生菌的潛力。
由於生物性的黴菌毒素去除劑可能具有吸附毒素與分解毒素的兩種作用機制，各有優缺點，因此，有必要深入了解CK1對zearalenone毒素清除的作用機制。利用熱處理或酸處理去除酵素活性的CK1死菌及其活菌進行毒素清除試驗，分析0小時及24小時後的zearalenone含量比例，結果顯示，僅僅在10分鐘內，活菌和死菌狀態的CK皆可以去除液體中約75%的zearalenone；培養24小時後，僅僅再微幅增加0.5-2.2%的去除率。由於生物性分解反應需要較長的作用時間才能完成，因此推測此清除機制可能主要來自於吸附作用。同時，將CK1與兩款市售黴菌毒素去除劑進行比較，濃度1010 CFU/ ml的CK1與建議添加量1%礦物性來源的吸附劑Omniguard®相比，提高了25%的清除效率。CK1與相同濃度生物性來源的去毒劑Lactobacillus rhamnosus GG相比，提高了約30％的清除效率，證實CK1在zearalenone的去除上，優於此兩項市售商品。進一步模擬在腸道中影響CK1吸附的各項因子發現：在0.5-200 ppm的毒素濃度下，CK1均能夠去除約70%的zearalenone；在模擬腸道pH 2.5-8.0環境下，CK1均有高於65%的吸附力；在4-42℃的測試條件下，CK1均有75%的吸附力。是故CK1做為黴菌毒素吸附劑，具有穩定的吸附效力。
在in vitro試驗中已經知道CK1有成為益生菌的潛力，因此我們進一步利用in vivo的BALB/c小鼠動物試驗，評估CK1的益生菌功能。在免疫功能評估結果顯示，餵食CK1小鼠的血液中，吞噬細胞活性、脾臟細胞增殖反應及自然殺手細胞活性較對照組顯著提升。在改善胃腸道功能研究中發現，餵食CK1小鼠腸道中有害菌Clostridium perfringens的量顯著降低。在研究腸道菌相與腸腦軸的影響結果顯示，餵食高劑量1010 CFU/ kg BW/day組別的小鼠，其糞便中的菌相以NGS方法分析顯示與其它組別不同，且此劑量的小鼠大腦中的血清素含量顯著增加，暗示CK1能夠改變腸道菌相進而影響腸腦軸的功能。
總結以上各項結果顯示，CK1做為飼料用添加劑，不僅能夠做為黴菌毒素去除劑，又能夠發揮益生菌免疫調節的功能以及改善胃腸道的功能，成為具有多重功效的飼料添加劑。另外，因為CK1可以提昇腦內血清素的含量，未來具有防禦疾病與舒緩緊迫的潛力。

關鍵字

地衣芽孢桿菌 ； 黴菌毒素去除劑 ； 益生菌 ； 免疫調節 ； 胃腸道功能 ； 腸腦軸

並列摘要

According to the latest survey from the Food and Agriculture Organization of the United Nations, up to 50 percent of the grain were contaminated by mycotoxins. Mycotoxins have adverse effects on humans, animals, and crops that result in illnesses and economic losses. The supplementation of mycotoxin detoxifiers to the contaminated feed could be the most promising approach to reduce the loss. Our previous study showed that Bacillus licheniformis CK1 (CK1) decreased more than 98% of zearalenone in ZEN-contaminated corn meal medium after 36 hours of incubation, thus suggesting that CK1 might be the solution for ZEN contamination in feed. In addition, CK1 was non-hemolytic, non-enterotoxin producing, and was resistant to gastric acid and bile salts. Furthermore, CK1 could adhere to human epithelial cell line (Caco-2) and reduce the adhesion of pathogens, such as enterohemorrhagic Escherichia coli and Salmonella typhimurium to surfaces of Caco-2 cells. Therefore, all these data showed that CK1 can be qualified as a food probiotic.
Although CK1 is a candidate for mycotoxin detoxifier, we still need more evidence to assess its market potential. By comparing CK1 to a commercial inorganic adsorbent (Omniguard®) and a well understood microbial original adsorbent (Lactobacillus rhamnosus GG, LGG), CK1 exhibited approximately 30% and 25% higher performance in removing ZEN than LGG and Omniguard®, respectively. Furthermore, to reveal the detoxification mechanism of CK1, viable and non-viable (acid treated and heat treated) CK1 were used in a ZEN removing assay. The process of toxin removal by CK1 is rapid since approximately 75% of ZEN was removed from the liquid media within the 10-min centrifugation. Even after 24 hours of incubation, the removal rate is slightly increased (from 0.5% to 2.2%) in all samples of CK1 without any significant difference. Under these conditions, the ZEN removal mechanism of CK1 was concluded to be through adsorption but not through biotransformation.
Since CK1 had exhibited probiotic characteristics in in vitro experiments, we also evaluated its other potential functions in vivo. In BALB/c mouse model, we evaluated the non-specific immune functions, gastrointestinal functions, and gut-brain axis effects of CK1. In immune functions study, the results showed that granulocyte phagocytosis, splenocyte proliferation and natural killer (NK) cell activity were increased after oral administration of CK1. In gastrointestinal functions study, levels of harmful bacteria Clostridium perfringens in the intestine were significantly decreased. Moreover, serotonin levels in the brain were significantly increased which imply that CK1 could influence gut-brain axis.
In conclusion, our data showed that CK1 had the best ZEN removing ability compared to two other commercial adsorbents. Moreover, CK1 possesses immunomodulatory functions, improves gastrointestinal function, and may involves in affects gut-brain axis, thus, making it a super multi-functional probiotic. In the future, CK1 can be applied in preventing diseases as well as in reducing stress.

並列關鍵字

Bacillus licheniformis ； zearalenone ； adsorbent ； probiotic ； immunomodulation ； gut-brain axis ； gastrointestinal function

參考文獻

易秉蓉。2010。以地衣芽孢桿菌去除黴菌毒素之研究。國立臺灣大學動物科學技術學系碩士論文。

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