赭曲霉毒素A降解菌的分离筛选及生物学特性研究

Isolation,Screening and Biological Characterization of Ochratoxin A Degrading Bacteria

试验旨在筛选出对赭曲霉毒素A(Ochratoxin A,OTA)有降解能力的菌株,以用于赭曲霉毒素A污染的饲料谷物降解。研究采集霉变饲料、土壤等样品,通过在LB培养基中添加OTA制成改良固体培养基进行菌株初筛,以初筛菌株对OTA标准品的降解率为指标,采用ELISA法进行复筛;通过形态学、生理生化试验、16S rRNA序列分析等方法对筛选菌株进行鉴定,并对筛选菌株进行耐酸、耐胆盐、模拟人工胃液和肠液耐受性、抑菌性及产酶特性等生物学特性进行研究。结果表明:通过筛选获得1株对OTA降解率为86.31%的菌株MM28,鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。其在pH2.0的酸性液中存活率为52.13%,0.3%胆盐中存活率可达到65.94%,人工模拟胃液和肠液中存活率分别为50.54%和55.68%。菌株MM28发酵上清液对鼠伤寒沙门氏菌(Salmonella typhimurium)、大肠杆菌(Escherichia coli)和金黄色葡萄球菌(Staphylococcus aureus)均有抑制作用。产酶水解圈试验表明,该菌具有产纤维素酶、蛋白酶、淀粉酶及木聚糖酶的活性。综上,筛选获得的高效降解OTA的解淀粉芽孢杆菌有一定的抗逆性、抑菌性及产酶性,有望开发成为降解OTA的益生菌用于饲料工业中。

The experiment aimed to screen strains with degradation ability to ochratoxin A(OTA)for the degradation of ochratoxin A contaminated feed grains.Samples of moldy feed and soil were collected,and the strains were initially screened by adding OTA to LB medium to make modified solid medium,and the degradation rate of the initially screened strains to OTA standards was used as an index,and the ELISA method was used for re-screening.The screening strains were identified by morphology,physiolog⁃ical and biochemical tests,16S rRNA sequence analysis and other methods.The screening strains were also investigated for their biological properties such as acid tolerance,bile salt tolerance,simulated artifi⁃cial gastric and intestinal fluid tolerance,bacterial inhibition and enzyme production characteristics.The results showed that one strain MM28 with 86.31%degradation rate of OTA was obtained through screen⁃ing,which was identified as Bacillus amyloliquefaciens.Its survival rate was 52.13%in acidic solution at pH 2.0,65.94%at 0.3%bile salt concentration,and 50.54%and 55.68%in artificial simulated gastric and intestinal fluids,respectively.The fer⁃mentation supernatant of strain MM28 had inhibi⁃tory effects on Salmonella typhimurium,Escherich⁃ia coli and Staphylococcus aureus.The enzyme-producing hydrolysis circle test showed that it also had cellulase,protease,amylase and xylanase produc⁃ing activities.In conclusion,a strain of Bacillus amyloliquefaciens with high OTA degradation has certain stress resistance,bacteriostasis and enzyme production,and is expected to be developed into OTA degra⁃dation probiotics for feed industry.