抑黄曲霉毒素的Hitwh-B05菌株有效成分的分离纯化与鉴定
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摘要
黄曲霉毒素是一类由真菌代谢产生的有毒化合物，存在于土壤及花生、玉米等农作物中，不但对环境造成污染，人或动物若食用了受黄曲霉毒素污染的食品或饲料后会导致机体病变甚至死亡。因此，如何快速有效的抑制黄曲霉毒素对农作物、人畜及环境的污染是急需解决的问题，也是国内外学者研究的热点。本研究采用微生物源天然产物中具有抑黄曲霉毒素活性的物质作用于易感染农产品，提高农产品的防霉抑毒能力，从污染源头有效阻止霉菌侵染，减少毒素产生，为减少化学防腐剂对环境和人体的危害起到重要作用。
以本实验室分离获得的具有自主知识产权的枯草芽孢杆菌新菌株Hitwh-B05为主要研究对象，对其代谢产生的抑黄曲霉毒素有效成分的发酵条件进行优化；从多角度建立抑黄曲霉毒素有效成分的分离方法；对分离得到的抑黄曲霉毒素有效成分进行鉴定；初步探讨其抑制黄曲霉毒素产生的机制。本研究在国内率先采用tip culture方法研究枯草芽孢杆菌Hitwh-B05的抑黄曲霉毒素能力，其代谢产生的抑黄曲霉毒素有效成分能够有效抑制寄生曲霉菌丝生长及黄曲霉毒素合成途径中第一个稳定、且呈现橘红色的前体物质—NorsolorinicAcid（NA）的产生，进而抑制了黄曲霉毒素的产生，并且对黄曲霉毒素的抑制具有广谱性。Tip culture方法特点是直观，操作简单，实验周期短，能够进行定量分析，研究结果更准确可靠。与以往使用的产黄曲霉毒素菌株相比，无毒害作用，减少了对实验人员的危害和对环境的污染。
利用统计学方法优化了Hitwh-B05菌株抑黄曲霉毒素有效成分产生的条件，使其产量得到显著提高。抑黄曲霉毒素有效成分产生的适宜条件为温度35℃、培养时间6d、培养基初始pH值为6.00，培养基配方为硫酸锰0.04g/L、酵母粉10g/L、牛肉膏12g/L、葡萄糖40g/L、大豆蛋白胨10g/L、硫酸镁4g/L、磷酸氢二钾4g/L。使用优化培养基获得的Hitwh-B05菌株发酵上清液对NA积累的抑制率是优化前的GY液体培养基的2.98倍，对菌丝体生长的抑制率是优化前的GY液体培养基的5.21倍，菌株代谢产生的脂肽产量是优化前的GY液体培养基的2.16倍。
大孔树脂富集法可作为实际生产应用中快速有效的分离枯草芽孢杆菌Hitwh-B05代谢产生的抑黄曲霉毒素有效成分的方法。筛选出大孔弱极性树脂DM130能够吸附大量抑黄曲霉毒素有效成分且不易吸收色素杂质，有效成分总回收率达到69.43%，色素去除率在96%以上。
通过分子生物学手段鉴定Hitwh-B05菌株含有的脂肽编码基因合成的脂肽包括bacillomycin、bacilysin、ericin、fengycin、mersacidin、mycosubntilin、iturinA、surfactin和sublancin，表明Hitwh-B05菌株在适宜的条件下可产生的脂肽种类比较广泛。MALDI/TOF/MS分析Hitwh-B05菌株发酵上清液分离得到的抑黄曲霉毒素有效成分主要为脂肽类物质iturin A、bacillomycin D、surfactin和fengycin A。结果表明枯草芽孢杆菌Hitwh-B05产生的脂肽种类比较广泛，国内外相关研究报道中，通过MALDI/TOF/MS方法在同一菌株中同时检测到4种不同种类的脂肽鲜有报道，多数仅检测到了1~3种脂肽。
Hitwh-B05菌株分离得到的抑黄曲霉毒素有效成分抗寄生曲霉侵染花生能力显著高于常用的化学合成防腐剂双乙酸钠（SDA），具有较大的应用潜力，为开发具有我国自主知识产权的、并能够有效抑制黄曲霉毒素合成的生物新农药提供理论和技术支持，使我国农产品的利用更加优质可靠。在抑黄曲霉毒素有效成分作用下，寄生曲霉孢子延迟萌发，菌丝体出现较多分节，干瘪、表面粗糙、失水严重，并且断裂成碎片。原因可能是在抑黄曲霉毒素有效成分作用下，寄生曲霉细胞膜中的磷脂双分子层产生孔隙，细胞膜结构被破坏而破裂，细胞无法正常吸收营养物质，导致菌丝体断裂，影响黄曲霉毒素合成。
Aflatoxins were a type of toxic compounds produced by fungus. They werefound in soil, peanuts, corn and other crops, not only causing pollution to theenvironment, but also causing the body disease of human or animal and even death.Therefore, it was a pressing problem about how to fast and effective inhibitaflatoxin pollution on crops, persons, livestock and the environment, as well asresearching hotspot of scholars at home and abroad. In this study, theantiaflatoxigenic effective components in microbial natural products were used toeffect on agricultural products, improving the ability of agricultural productsagainst mold and aflatoxin, which effectively prevented fungal infection andreduced the productin of aflatoxins, and played an important role in order to reducechemical preservative on the environment and human health.
The Bacillus subtilis Hitwh-B05strain used in this study was isolated in thislaboratory and was a new Bacillus subtilis strain with independent intellectualproperty rights. The fermentation conditions of antiaflatoxigenic effctivecomponents were opmized. The separation methods were established from variousangles for the antiaflatoxigenic effective components. The antiaflatoxigeniceffective components separated were identified. The inhibitive mechanism wasdicussed. The tip culture method used in this study was the first time in China todetect the antiaflatoxigenic abilities of effective components produced byHitwh-B05. Its supernatant could inhibit the growth of Aspergillus parasiticus andthe production of Norsolorinic Acid (NA)—the first stable intermediate product ofaflatoxin, and then inhibited the production of aflatoxins. The inhibition had broadspectrum to aflatoxins. The merit of this method was direct-viewing, easy operating,short experiment period, could be controlled by quantificationally analyzing and theresults were more accurate and reliable. The Aspergillus parasiticus mutant strainused in this study was nonhazardous and reducing the harm of experiment personneland environmental pollution compared with the aflatoxigenic strain.
The activities of antiaflatoxigenic effective components were obviouslyincreased through opmizing fermention conditions. The felicity conditions ofantiaflatoxigenic effective components produced were as follows:35℃,6d,pH6.00,MnSO40.04g/L, yeast powder10g/L, beef extract12g/L, glucose40g/L, soyapeptone10g/L, MgSO44g/L and K2HPO44g/L. Comparing the effects of optimalmedium and GY under the same condition, we found that the inhibitory rate of NAaccumulation using optimal medium was2.98-fold to using GY medium, and5.21-fold to the inhibitory rate of mycelia growth. The lipopeptides were2.16-fold to GY liquid medium.
Macroporous resin enrichment method was the best method of separated theantiaflatoxigenic effective components from Bacillus subtilis Hitwh-B05in actualproduction application. Macroporous resins DM130could absorb amount ofantiaflatoxigenic effective components, but was not that easy to absorb pigmentimpurities. The recovery rate of peptide could reach69.43%, while the averageremove rate of pigment could reach above96%.
By the detection of molecular biological techniques, lipopeptides bacillomycin,bacilysin, ericin, fengycin, mersacidin, mycosubntilin, iturin A, surfactin andsublancin were found in the fermentation broth of Hitwh-B05, which indicated thatthe lipopeptides produced by Hitwh-B05were with wide ranges. Analized byMALDI/TOF/MS showed that the type of antiaflatoxigenic effective componentswere lipopeptides: iturin A, bacillomycin D, surfactin and fengycin A. The types oflipopeptides were more than other majority Bacillus subtilis had been reported.There were fewer reports at home and abroad about four different types oflipopipties detected in one strain, and most of them were only detected one to threetypes of lipopeptides.
The mould proof and inhibitory ability of active substances separated fromHitwh-B05on the inhibition of Aspergillus parasiticus infected peanuts was muchhigher than the commonly used chemical synthetic preservatives sodium diacetate,the substances had great applictional potential, applying theoretical and techinicalsupports for the developing of the new biological pesticides with China'sindependent intellectual property rights and inhibitory ability to aflatoxin synthesis,making the use of agricultural products in China more reliable and superior. TheAspergillus parasiticus spores germination time was delayed under the treatment ofantiaflatoxigenic effective components. The mycelia were dry, surface was rough,fluid loss seriously and some had broken into pieces. This demonstrated that underthe treatment of antiaflatoxigenic effective components, the plasma membrane ofthe Aspergillus parasiticus cells form pores and the struture of plasma membranewas damaged, leading to the disability of absorbing the nutrient substances normally,and influenced the synthesis of aflatoxin.
以本实验室分离获得的具有自主知识产权的枯草芽孢杆菌新菌株Hitwh-B05为主要研究对象，对其代谢产生的抑黄曲霉毒素有效成分的发酵条件进行优化；从多角度建立抑黄曲霉毒素有效成分的分离方法；对分离得到的抑黄曲霉毒素有效成分进行鉴定；初步探讨其抑制黄曲霉毒素产生的机制。本研究在国内率先采用tip culture方法研究枯草芽孢杆菌Hitwh-B05的抑黄曲霉毒素能力，其代谢产生的抑黄曲霉毒素有效成分能够有效抑制寄生曲霉菌丝生长及黄曲霉毒素合成途径中第一个稳定、且呈现橘红色的前体物质—NorsolorinicAcid（NA）的产生，进而抑制了黄曲霉毒素的产生，并且对黄曲霉毒素的抑制具有广谱性。Tip culture方法特点是直观，操作简单，实验周期短，能够进行定量分析，研究结果更准确可靠。与以往使用的产黄曲霉毒素菌株相比，无毒害作用，减少了对实验人员的危害和对环境的污染。
利用统计学方法优化了Hitwh-B05菌株抑黄曲霉毒素有效成分产生的条件，使其产量得到显著提高。抑黄曲霉毒素有效成分产生的适宜条件为温度35℃、培养时间6d、培养基初始pH值为6.00，培养基配方为硫酸锰0.04g/L、酵母粉10g/L、牛肉膏12g/L、葡萄糖40g/L、大豆蛋白胨10g/L、硫酸镁4g/L、磷酸氢二钾4g/L。使用优化培养基获得的Hitwh-B05菌株发酵上清液对NA积累的抑制率是优化前的GY液体培养基的2.98倍，对菌丝体生长的抑制率是优化前的GY液体培养基的5.21倍，菌株代谢产生的脂肽产量是优化前的GY液体培养基的2.16倍。
大孔树脂富集法可作为实际生产应用中快速有效的分离枯草芽孢杆菌Hitwh-B05代谢产生的抑黄曲霉毒素有效成分的方法。筛选出大孔弱极性树脂DM130能够吸附大量抑黄曲霉毒素有效成分且不易吸收色素杂质，有效成分总回收率达到69.43%，色素去除率在96%以上。
通过分子生物学手段鉴定Hitwh-B05菌株含有的脂肽编码基因合成的脂肽包括bacillomycin、bacilysin、ericin、fengycin、mersacidin、mycosubntilin、iturinA、surfactin和sublancin，表明Hitwh-B05菌株在适宜的条件下可产生的脂肽种类比较广泛。MALDI/TOF/MS分析Hitwh-B05菌株发酵上清液分离得到的抑黄曲霉毒素有效成分主要为脂肽类物质iturin A、bacillomycin D、surfactin和fengycin A。结果表明枯草芽孢杆菌Hitwh-B05产生的脂肽种类比较广泛，国内外相关研究报道中，通过MALDI/TOF/MS方法在同一菌株中同时检测到4种不同种类的脂肽鲜有报道，多数仅检测到了1~3种脂肽。
Hitwh-B05菌株分离得到的抑黄曲霉毒素有效成分抗寄生曲霉侵染花生能力显著高于常用的化学合成防腐剂双乙酸钠（SDA），具有较大的应用潜力，为开发具有我国自主知识产权的、并能够有效抑制黄曲霉毒素合成的生物新农药提供理论和技术支持，使我国农产品的利用更加优质可靠。在抑黄曲霉毒素有效成分作用下，寄生曲霉孢子延迟萌发，菌丝体出现较多分节，干瘪、表面粗糙、失水严重，并且断裂成碎片。原因可能是在抑黄曲霉毒素有效成分作用下，寄生曲霉细胞膜中的磷脂双分子层产生孔隙，细胞膜结构被破坏而破裂，细胞无法正常吸收营养物质，导致菌丝体断裂，影响黄曲霉毒素合成。
Aflatoxins were a type of toxic compounds produced by fungus. They werefound in soil, peanuts, corn and other crops, not only causing pollution to theenvironment, but also causing the body disease of human or animal and even death.Therefore, it was a pressing problem about how to fast and effective inhibitaflatoxin pollution on crops, persons, livestock and the environment, as well asresearching hotspot of scholars at home and abroad. In this study, theantiaflatoxigenic effective components in microbial natural products were used toeffect on agricultural products, improving the ability of agricultural productsagainst mold and aflatoxin, which effectively prevented fungal infection andreduced the productin of aflatoxins, and played an important role in order to reducechemical preservative on the environment and human health.
The Bacillus subtilis Hitwh-B05strain used in this study was isolated in thislaboratory and was a new Bacillus subtilis strain with independent intellectualproperty rights. The fermentation conditions of antiaflatoxigenic effctivecomponents were opmized. The separation methods were established from variousangles for the antiaflatoxigenic effective components. The antiaflatoxigeniceffective components separated were identified. The inhibitive mechanism wasdicussed. The tip culture method used in this study was the first time in China todetect the antiaflatoxigenic abilities of effective components produced byHitwh-B05. Its supernatant could inhibit the growth of Aspergillus parasiticus andthe production of Norsolorinic Acid (NA)—the first stable intermediate product ofaflatoxin, and then inhibited the production of aflatoxins. The inhibition had broadspectrum to aflatoxins. The merit of this method was direct-viewing, easy operating,short experiment period, could be controlled by quantificationally analyzing and theresults were more accurate and reliable. The Aspergillus parasiticus mutant strainused in this study was nonhazardous and reducing the harm of experiment personneland environmental pollution compared with the aflatoxigenic strain.
The activities of antiaflatoxigenic effective components were obviouslyincreased through opmizing fermention conditions. The felicity conditions ofantiaflatoxigenic effective components produced were as follows:35℃,6d,pH6.00,MnSO40.04g/L, yeast powder10g/L, beef extract12g/L, glucose40g/L, soyapeptone10g/L, MgSO44g/L and K2HPO44g/L. Comparing the effects of optimalmedium and GY under the same condition, we found that the inhibitory rate of NAaccumulation using optimal medium was2.98-fold to using GY medium, and5.21-fold to the inhibitory rate of mycelia growth. The lipopeptides were2.16-fold to GY liquid medium.
Macroporous resin enrichment method was the best method of separated theantiaflatoxigenic effective components from Bacillus subtilis Hitwh-B05in actualproduction application. Macroporous resins DM130could absorb amount ofantiaflatoxigenic effective components, but was not that easy to absorb pigmentimpurities. The recovery rate of peptide could reach69.43%, while the averageremove rate of pigment could reach above96%.
By the detection of molecular biological techniques, lipopeptides bacillomycin,bacilysin, ericin, fengycin, mersacidin, mycosubntilin, iturin A, surfactin andsublancin were found in the fermentation broth of Hitwh-B05, which indicated thatthe lipopeptides produced by Hitwh-B05were with wide ranges. Analized byMALDI/TOF/MS showed that the type of antiaflatoxigenic effective componentswere lipopeptides: iturin A, bacillomycin D, surfactin and fengycin A. The types oflipopeptides were more than other majority Bacillus subtilis had been reported.There were fewer reports at home and abroad about four different types oflipopipties detected in one strain, and most of them were only detected one to threetypes of lipopeptides.
The mould proof and inhibitory ability of active substances separated fromHitwh-B05on the inhibition of Aspergillus parasiticus infected peanuts was muchhigher than the commonly used chemical synthetic preservatives sodium diacetate,the substances had great applictional potential, applying theoretical and techinicalsupports for the developing of the new biological pesticides with China'sindependent intellectual property rights and inhibitory ability to aflatoxin synthesis,making the use of agricultural products in China more reliable and superior. TheAspergillus parasiticus spores germination time was delayed under the treatment ofantiaflatoxigenic effective components. The mycelia were dry, surface was rough,fluid loss seriously and some had broken into pieces. This demonstrated that underthe treatment of antiaflatoxigenic effective components, the plasma membrane ofthe Aspergillus parasiticus cells form pores and the struture of plasma membranewas damaged, leading to the disability of absorbing the nutrient substances normally,and influenced the synthesis of aflatoxin.
引文
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