以地衣芽孢桿菌去除黴菌毒素之研究

Abstract

黴菌毒素（mycotoxin）是真菌類微生物所產生之二級代謝物，當穀物受到黴菌毒素的汙染，經由飼糧或食物進入動物或人體內，便會對動物產生負面影響，降低生產量及免疫力，亦直接或間接威脅人類的健康，同時也造成經濟損失；因此，去除飼糧或食物中的黴菌毒素成為重要的研究方向。 本論文之目的為評估一能產生胞外聚木糖酶之CK1菌株去除黃麴毒素B1（aflatoxin B1, AFB1）與玉米赤黴毒素（zearalenone, ZEN）之能力，並鑑定CK1菌株生長之相關特性與測量其胞外酵素活性，進而分析該菌株做為益生菌之潛力。經由API 50 CHB套組與16s rDNA序列分析鑑定CK1為地衣芽孢桿菌（Bacillus licheniformis）其相似性達99.9%。酵素活性部分，B. licheniformis CK1較標準菌株（B. licheniformis ATCC 14580）呈現較高的胞外聚木糖酶、羧甲基纖維素酶與蛋白酶活性。將B. licheniformis CK1與 ATCC 14580置於含有2 ppm的AFB1或ZEN之液態培養基中，測量菌株在含有黴菌毒素培養液中的生長，並以高效液相層析儀分析其黴菌毒素含量。結果顯示B. licheniformis CK1與ATCC 14580皆能生長於含有黴菌毒素之培養液，此外B. licheniformis CK1能分別降低86%與92% AFB1及ZEN的含量，顯示該菌株去除黴菌毒素之效率皆高於B. licheniformis ATCC 14580。玉米粉培養基試驗中，兩菌株接種於含有18 ppm ZEN之玉米粉，經過48小時之培養，兩菌株菌數從107 CFU/mL增加至109 CFU/mL，而B. licheniformis CK1與ATCC 14580分別降低玉米粉培養基中20%及13%之ZEN。 B. licheniformis CK1經由分析顯示該菌株不產生內毒素與溶血性；此外，該菌對於clindamycin與erythromycin有抵抗性。B. licheniformis CK1在pH 3.0或0.3% oxgall的環境下菌數不受影響，而在pH 2.0的環境培養3小時後，菌數由109降至105 CFU�mL。抗病原菌能力分析顯示B. licheniformis CK1能抑制Escherichia coli與Listeria monocytogenes。 綜上所述，B. licheniformis CK1具有產生胞外聚木糖酶、羧甲基纖維素酶與蛋白酶，及去除黃麴毒素B1與玉米赤黴毒素之能力，並展現多項益生菌之特性，是故B. licheniformis CK1具有使用在飼糧或食物中去除黴菌毒素之生物製劑之潛力。

Mycotoxins are naturally occurried chemical contaminants. They are produced by certain fungi which pose a threat to human and animal health when ingested. Exposuring to mycotoxins can produce both acute and chronic toxicities ranging from death to deleterious effects. A xylanase-producing bacterial strain CK1 was isolated and used in the detoxification of mycotoxins study. First, according to the molecular identification and API CHB 50 system, bacterial CK strain was identified as Bacillus licheniformis. Thus, in order to compare the ability to detoxification with a control group, B. licheniformis ATCC 14580 was used as a standard strain. These two strains displayed two different colony morphologies but similar figures in microscopic investigation. Enzyme activities assay revealed that CK1 displayed outstanding enzyme activities as compared with the standard strain. In detoxification of mycotoxins study, both CK1 and the standard strain possess the ability to remove aflatoxin B1 (AFB1) and zearalenone (ZEN) in contaminated broth. CK1 removed 86% and 99% of AFB1 and ZEN respectively, whereas the standard strain removed 73% and 60% respectively of these toxins. In feed stock test, CK1 and the standard strain could grow in corn medium containing 18 ppm ZEN; however, both strains only removed 20% and 13% of ZEN in the medium. CK1 was further evaluated its potential application as probiotic. This strain was resistant to clindamycin, neither carry enterotoxin genes nor possess hemolysis property. CK1 could survived in pH 2 condition with 105 CFU/mL and the numbers of CK1 was steady in buffer of pH 3.0 or 0.3% oxgall, which also had anti-pathogenic activity against Escherichia coli and Listeria monocytogenes. In conclusion, this study demonstrated that B. licheniformis CK1 has the properties of fibrolytic enzymes and protease production, detoxification of mycotoxins and probiotic characteristics. These findings suggest that B. licheniformis CK1 has the potential for bio-detoxifying of mycotoxins in feed or food.