玉米赤黴烯酮之生物性脫毒法於動物飼糧之研究

玉米赤黴烯酮（zearalenone, ZEN）是一個具雌激素生物活性的黴菌毒素，它會對造成動物的生殖毒性，造成畜產經濟嚴重的損失。本研究之目的為發展適合使用在飼料工業上的ZEN生物性脫毒法。研究分為兩個部分：第一部分研究為驗證瘤胃原蟲（rumen protozoa）的ZEN脫毒能力與其脫毒的方式；第二部分研究則著重在篩選具有ZEN脫毒潛力的芽孢桿菌（Bacillus spp.），並利用發酵過程驗證其在飼料工業的應用性。最後將挑選出的芽孢桿菌菌株，與先前篩選出具ZEN脫毒能力的植物乳桿菌（Lactobacillus plantarum）（LAB B2 strain）組合，發展兩階段的液態飼料發酵程序。瘤胃原蟲由餵飼60%精料飼料的瘤胃開窗牛採集，經由體外試驗證實了瘤胃原蟲的確具有ZEN降解能力，並擁有相當好的ZEN吸附特性。於高濃度ZEN（5 mg･L-1）存在下，瘤胃原蟲的ZEN吸附率超過60%以上，且ZEN吸附率不受環境pH值之影響。由MCF-7細胞增生試驗結果顯示，瘤胃原蟲可以顯著降低ZEN造成的細胞增生效應（p<0.05）。經由掃描式電子顯微鏡觀察結果，以2.5%褐藻膠製成的瘤胃原蟲微膠囊（encapsulated rumen protozoa）可以抵抗冷凍乾燥與胃蛋白酶體外消化作用的傷害。於BALB/c小鼠28天餵食試驗中，餵飼ZEN處理組（30 mg･kg BW･day-1）會造成BALB/c小鼠白血球數（white blood cell）、淋巴球數（lymphocyte）、單核球數（monocyte）與顆粒性白血球數（granulocyte）顯著減少（p<0.05）。ZEN處理同時增加小鼠血漿尿素氮（blood urine nitrogen）與減少總膽固醇（total cholesterol）（p<0.05）。此外，ZEN也會造成生殖器官的相對重量增加（p<0.05）。添加1.3%凍乾的瘤胃原蟲微膠囊（freeze-dried encapsulated rumen protozoa），則可以顯著減緩前述ZEN對BALB/c小鼠所產生的負面影響。第二部分研究由發酵大豆、土壤與污水樣品中分離出14株具有較高ZEN脫毒能力之分離株。經16S rRNA基因序列分析結果，14株分離株均為Bacillus spp.。初步排除會產生腸毒素的菌株後，5株檢測不會產生腸毒素的芽孢桿菌ZEN脫毒菌株（ZEN-detoxifying Bacillus, ZDB）作為後續研究使用。所有的ZDB菌株均具有可降解ZEN的酯酶活性，同時也具有ZEN吸附能力。於高濃度ZEN （5 mg･kg-1）污染玉米粉發酵試驗中，ZDB B2 strain的ZEN脫毒能力最佳，脫毒率約56%。由gyrB基因序列分析顯示，ZDB B2 strain為枯草芽孢桿菌（Bacillus subtilis subsp. subtilis）。當ZDB B2 strain接種於含ZEN之TSB中生長時，顯著增加其菌落數、乳酸、乙酸、總揮發性脂肪酸和氨態氮濃度（p<0.05），ZDB B2 strain以ZEN污染玉米粉作為發酵基質時，發酵產物擁有更好之發酵特性：乳酸濃度超過110 mmol･L-1、乙酸濃度少於20 mmol･L-1與pH值低於4.5。此外，ZDB B2 strain同時具黃麴毒素B1（aflatoxin B1）、脫氧雪腐鐮刀烯醇（deoxynivalenol）、伏馬鐮孢毒素B1（fumonisin B1）、和T-2毒素（T-2 toxin）的脫毒能力。第二部分研究後段接著針對已分離出具ZEN脫毒作用的LAB B2 strain進行研究，確認其ZEN降解機制。LAB B2 strain是從蝙蝠糞便所分離出具ZEN脫毒能力的植物乳桿菌。由脂酶（lipase）活性分析中，LAB B2 strain對於C2或C4短鏈酯類分解的活性優於C8、C10或C16的長鏈酯類。由ZEN移除試驗結果顯示，有無添加脂酶活性抑制劑（tetrahydrolipstatin）不影響LAB B2 strain培養液上清液的ZEN移除率。後續初步建立LAB B2 strain液態飼料發酵之條件，基質與水量比例1：3，菌株接種濃度為109 CFU･mL-1，可以獲得良好菌數之維持與較高之乳酸產量。最後將ZDB B2 strain與LAB B2 strain搭配進行兩階段液態飼料發酵試驗，發酵結果顯示，ZDB B2 strain有助於支持第二階段接種菌株LAB B2 strain於ZEN污染玉米之生長。本研究證實瘤胃原蟲能有效進行ZEN之脫毒，降低ZEN對BALB/c小鼠造成的毒性。ZDB B2 strain有很強的酯酶活性，對高濃度ZEN污染玉米粉有最佳的脫毒效果，是一株適合於飼料在餵飼動物前，以發酵進行ZEN脫毒之菌株。

關鍵字

玉米赤黴烯酮；生物性脫毒；瘤胃原蟲；芽孢桿菌；植物乳桿菌；發酵

並列摘要

Zearalenone (ZEN) is an estrogenic mycotoxin which can cause loss in animal production. The aim of this study was to develop biological ZEN detoxification for application in the feed industry. This study was divided into two parts. The first part was to verify the ZEN detoxification capability and detoxification mechanisms of rumen protozoa. The second part was to screen Bacillus strains for their potential ZEN detoxification capability and use the fermentation process to validate their application potential in the feed industry. Finally, the selected Bacillus strain was combined with Lactobacillus plantarum B2 (LAB B2 strain) which also has the ZEN detoxification capability to perform a two-stage liquid feed fermentation process. The rumen protozoa were collected from the cannulated cow fed 60% concentrate diet, and their ZEN degradation and ZEN adsorption capability were confirmed. The rumen protozoa showed an excellent properties of ZEN adsorption. The ZEN adsorption rate of rumen protozoa was more than 60% at high ZEN concentration (5 mg･L-1) and it was not affected by the pH of the environment. The MCF-7 cell proliferation assay showed rumen protozoa significantly reduced the proliferative effect of ZEN (p<0.05). The scanning electron microscope photographs presented that with the use of alginate 2.5%, the encapsulated rumen protozoa could resist the freeze-drying process and in vitro pepsin digestion. In BALB/c mice 28-day feeding toxicity study, the complete blood count data indicated ZEN (30 mg･kg BW･day-1) could decrease the numbers of white blood cell, lymphocyte, monocyte and granulocyte (p<0.05). ZEN increased the level of blood urine nitrogen but decreased the level of total cholesterol in mice (p<0.05). ZEN increased the relative weight of the reproductive organs (p<0.05). The addition of 1.3% freeze-dried encapsulated rumen protozoa could reduce the ZEN harmful effect on BALB/c mice significantly. In the second part of this study, there were 14 isolates had markedly greater ZEN detoxification capability than other isolates, which were isolated from fermented soybean products, soil and sewage. The 16S rRNA gene sequence analysis indicated that these 14 isolates shared the highest identity similarity with Bacillus spp. After checking the potential enterotoxin producers, five non-enterotoxin-producing ZEN-detoxifying Bacillus (ZDB) strains were identified and kept for further study. All of the tested ZDB strains demonstrated esterase activity which is linked to their ZEN degradation capability. They were also able to adsorb ZEN. In the high-level ZEN-contaminated maize (5 mg･kg-1) fermentation test, ZDB B2 strain exhibited the highest detoxification rate, removing 56% of the ZEN. The gyrB gene sequencing identified the ZDB B2 strain as Bacillus subtilis subsp. subtilis. When ZDB B2 strain grew in TSB medium with ZEN, it had higher bacterial numbers, lactic acid, acetic acid, total volatile fatty acids, and ammonia nitrogen. The ZEN-contaminated maize fermented by ZDB B2 strain had better fermentation characteristics (pH < 4.5; lactic acid > 110 mmol･L-1; acetic acid < 20 mmol･L-1) than ZEN-free maize. Furthermore, ZDB B2 strain also had detoxification capabilities toward aflatoxins B1, deoxynivalenol, fumonisin B1, and T-2 toxin. The second part of the study also investigated the ZEN degradation mechanism of LAB B2 strain which was isolated in previous study with ZEN detoxification capability. LAB B2 strain was isolated from bat excreta. In the lipolytic activity assay, LAB B2 strain showed higher specific activity against short-chain esters (C2 and C4) than longer-chain esters (C8, C10 and C16). ZEN removal assay indicated the ZEN removal rate in the supernatant of LAB B2 strain remained the same with or without lipase inhibitor tetrahydrolipstatin (THL). And then, the liquid feed fermentation conditions (solid/liquid ratio: 1/3; bacterial inoculation dose: 109 CFU･mL-1 ) of LAB B2 strain was established. At last, ZDB B2 strain and LAB B2 strain were combined to perform the two-stage liquid fermentation process. The results of the fermentation test showed that ZDB B2 strain had beneficial effect on the growth of LAB B2 strain which was inoculated at the second stage. Overall, rumen protozoa could detoxify ZEN effectively, and reduced the extent of ZEN-induced reproductive toxicity. The ZDB B2 strain can be a suitable candidate for ZEN detoxification by fermentation process before feeding because it demonstrated strong esterase activity and exhibited the highest detoxification capability in maize with a high level of ZEN (5 mg･kg-1 maize).