Study on Fungistasis of Marine Bacteria BMF04 and Removal Effect on Toxin
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摘要: 为了明确海洋细菌BMF04菌株的抑菌作用和毒素去除作用及其降解毒素机理。采用平板对峙法测定BMF04菌株及无菌发酵液对小麦赤霉病菌（Fusarium graminearum）、小麦雪腐镰刀菌（Fusarium nivale (Fr) Ces.）的抑制作用；采用含毒平板法和HPLC法测定该菌株、无菌发酵液、细胞壁悬浮液、胞内液对玉米赤霉烯酮（ZEN）毒素的去除作用和高温以及蛋白酶处理对BMF04菌株去除ZEN毒素作用的影响，明确该菌株去除毒素的作用机理。结果表明，BMF04菌株及无菌发酵液对小麦赤霉病菌和小麦雪腐镰刀菌具有较强的抑菌作用；BMF04菌株对ZEN毒素具有较强的去除作用，当培养液中ZEN毒素浓度为15 µg/mL时，菌株活菌、无菌发酵液、细胞壁悬浮液和胞内液均对ZEN毒素具有较强的去除作用，去除率分别98.92%±0.07%、98.70%±0.19%、97.85%±0.07%和98.54%±0.10%，菌液在121 ℃条件下高温处理30 min，灭活菌液和灭活无菌发酵液对ZEN毒素的去除率明显下降，仅为60.32%±0.21%、2.09%±1.15%，胰蛋白酶、胃蛋白酶和蛋白酶K处理后，去除率分别下降到3.52%±0.77%、0.50%±0.39% 和0.18%±0.12%，说明高温灭活和蛋白酶对BMF04菌株发酵液去除ZEN毒素作用具有显著影响；菌株去除ZEN毒素既有胞外蛋白质降解作用也有细胞壁的吸附作用。研究结果为微生物去除污染粮食、饲料中的ZEN毒素提供了新的菌种资源。
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关键词:
- 海洋细菌 /
- 玉米赤霉烯酮(ZEN) /
- 抑菌带宽度 /
- 去除率 /
- 生物降解
Abstract: In order to clarify the inhibitory effects, toxin removal capacity and toxin degradation mechanism of marine bacteria BMF04. Firstly, the inhibitory effects of BMF04 strain and its aseptic fermentation broth on Fusarium graminearum and Fusarium nivale (Fr) Ces. were determined by the plate-stand method; Secondly, the removal of ZEN by live strain, aseptic fermentation broth, cell wall suspension and intracellular fluid, as well as the effect of hightemperature and protease treatment on the removal of ZEN by BMF04 strain were determined by toxicity plate method and HPLC method, to clarify the mechanism of its action of removing toxin. The results showed that BMF04 had good inhibitory effect on F. graminearum and F. nivale (Fr) Ces.. The strain BMF04 strain had a strong ability to remove Zen toxin, and when the concentration was 15 µg/mL, the live strain, the aseptic fermentation liquid, the cell wall suspension and the intracellular liquid had strong removal effect on ZEN, the removal rates were 98.92%±0.07%, 98.70%±0.19%, 97.85%±0.07% and 98.54%±0.10%, respectively. After high temperature inactivation, the removal rate of ZEN was reduced to 60.32%±0.21% and 2.09%±1.15%. After treatment with trypsin, pepsin and proteinase K, the removal rates decreased to 3.52%±0.77%, 0.50%±0.39% and 0.18%± 0.12%, indicating that high temperature inactivation and protease had significant effects on the removal of ZEN from BMF04 fermentation broth. Therefore, the removal of ZEN by the strain had both the degradation of extracellular protein and the adsorption of cell wall. The results of this study provided a new strain resource for the biological removal of ZEN toxin from contaminated food and feed, and also provided a theoretical basis for the biodegradation of ZEN toxin.-
Key words:
- marine bacteria /
- zearalenone (ZEN) /
- inhibition zone width /
- degradation rate /
- biodegradation
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图 1 BMF04菌株及无菌发酵液对小麦赤霉病菌和小麦雪腐镰刀菌菌丝的抑制作用
Figure 1. Inhibitory effects of strain BMF04 and its aseptic fermentation broth on F. graminearum and F. nivale (Fr) Ces
注：A和C为菌株及无菌发酵液对小麦赤霉病菌的抑制作用；B和D为菌株及无菌发酵液对小麦雪腐镰刀菌的抑制作用。
图 2 不同菌株在含ZEN毒素平板上的生长状态
Figure 2. The growth state of different strains on ZEN plate
注：大写字母A、B、C分别为BMF04菌株、大肠杆菌（E. coli）、GM-1-2菌株。
表 1 BMF04菌株对不同浓度ZEN毒素的去除率
Table 1. Removal rate of BMF04 strain to different concentration of ZEN
ZEN初始浓度
（µg/mL）CK 处理 去除率（%） ZEN浓度（µg/mL） ZEN浓度（µg/mL） 4 3.89±0.07 0.00±0.00 100.00±0.00 10 9.88±0.06 0.00±0.00 100.00±0.00 15 14.91±0.30 0.14±0.01 98.95±1.27 
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表 2 不同处理BMF04菌株ZEN毒素的峰面积和浓度
Table 2. Peak area and concentration of ZEN by strain BMF04 with different treatments
处理 ZEN浓度（µg/mL） 去除率（%） CK 14.86±0.08a 0.00±0.00d 活菌菌悬液 0.16±0.01d 98.92±0.07a 灭活菌悬液 5.90±0.03b 60.32±0.21b 细胞壁悬液 0.32±0.01c 97.85±0.07a 胞内液 0.22±0.02c 98.54±0.10a 无菌发酵液 0.19±0.03cd 98.70±0.19a 灭活的无菌发酵液 14.55±0.17a 2.09±1.15c 注：不同字母表示差异显著（P<0.05），表3同。
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表 3 不同蛋白酶处理BMF04菌株无菌发酵液后ZEN毒素的峰面积、浓度和去除率
Table 3. Peak area and concentration of ZEN culture medium after different protease treatment of aseptic fermentation broth
不同处理 ZEN浓度（µg/mL） 去除率（%） CK1 0.53±0.03c 86.54±0.72a CK2 3.97±0.02a 0.00±0.00c 胰蛋白酶 3.83±0.03b 3.52±0.77b 胃蛋白酶 3.95 ±0.02a 0.50±0.39c 蛋白酶K 3.96±0.00a 0.18±0.12c
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