WO2022111287A1 - 降解脱氧雪腐镰刀菌烯醇的菌株及应用 - Google Patents

降解脱氧雪腐镰刀菌烯醇的菌株及应用 Download PDF

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WO2022111287A1
WO2022111287A1 PCT/CN2021/129928 CN2021129928W WO2022111287A1 WO 2022111287 A1 WO2022111287 A1 WO 2022111287A1 CN 2021129928 W CN2021129928 W CN 2021129928W WO 2022111287 A1 WO2022111287 A1 WO 2022111287A1
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don
strain
biological
detoxifier
nocardioides
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PCT/CN2021/129928
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French (fr)
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罗会颖
张宏海
姚斌
黄火清
王亚茹
柏映国
苏小运
王苑
涂涛
张�杰
于会民
秦星
王晓璐
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中国农业科学院北京畜牧兽医研究所
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Priority to US18/254,176 priority Critical patent/US20240093142A1/en
Publication of WO2022111287A1 publication Critical patent/WO2022111287A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
    • A23L5/28—Removal of unwanted matter, e.g. deodorisation or detoxification using microorganisms
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09C—RECLAMATION OF CONTAMINATED SOIL
    • B09C1/00—Reclamation of contaminated soil
    • B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/34—Organic compounds containing oxygen
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

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  • the invention relates to the field of microorganisms, in particular to a strain and application for degrading deoxynivalenol.
  • DON Deoxynivalenol
  • Vomitoxin Voltoxin
  • DON can cause vomiting in humans and animals
  • the physical and chemical properties of DON are extremely stable, and it is difficult to remove it by general cooking and processing techniques. During the high-temperature baking process, it can be converted into other compounds with unknown toxicity, such as nor-DON, DON lactone, etc.
  • DON can exist in plants in a "cryptic" form, such as 3- ⁇ -D-glucose-DON. Derivatives of this "covert" form are thought to be a product of the plant's self-defense. Unfortunately, cryptic DON can still be reduced to DON by gut microbes, further increasing the exposure risk of humans and animals.
  • the biodegradation technology of DON has the characteristics of high efficiency, greenness, mild reaction conditions and easy large-scale application.
  • the acquisition of pure culture of DON degrading strains and the revealing of the DON degradation mechanism of the strains will bring important inspiration to the development of DON degradation products.
  • Devosia is a class of DON-degrading strains that have been widely studied. Several species and subspecies of the genus DeVore have been shown to degrade DON into 3-keto-DON and 3-epi-DON. In 2018, the Zhou Ting group in Canada confirmed that two enzymes, DepA and DepB, are responsible for these two steps of degradation, respectively. Since then, multiple teams have also successively confirmed that the independently isolated Dvoria can transform DON, but not all Dvora can achieve the above two-step transformation.
  • Nocardioides Nocardioides
  • Nocardioides Nocardioides
  • Nocardioides were found to have DON degradation function almost at the same time.
  • the public literature only the Tsushima group in Japan reported that Nocardioides could completely transform and utilize DON as a carbon source.
  • Nocardioides produce 3-epi-DON in the process of degrading DON, which will be further transformed.
  • the complete degradation mechanism has not been studied in depth.
  • One of the main reasons is that Nocardioides have DON degradation functions. The strains are very rare, and it is difficult to enrich and isolate them to obtain pure culture.
  • the object of the present invention is to provide a Nocardioid strain for degrading deoxynivalenol.
  • Another object of the present invention is to provide a biological detoxifier prepared by using the above Nocardioid strains.
  • Another object of the present invention is to provide a preparation method for preparing the above-mentioned biological detoxifier.
  • the present invention provides a novel strain of Nocardioides sp., the strain number is ZHH-013, which has been deposited in the China Center for Type Culture Collection on September 30, 2020, and its deposit number is CCTCC No.M 2020565.
  • the function of this strain is similar to that of Nocardioides WSN05-2, but the 16S rDNA homology is less than 97%.
  • the formula of the fermentation medium tryptone 10g, yeast extract powder 5g, sodium chloride 10g, agar 1.5%, add distilled water to 1L, pH 7.0-7.2, sterilize at 121°C for 20min.
  • the biological detoxifying agent according to the present invention comprises as an active ingredient Nocardioides ZHH-013, or intracellular lysates thereof, such as intracellular proteins, the Nocardioids ZHH-013 deposit number is CCTCC No. M 2020565.
  • the biological detoxifier can be in liquid dosage form or solid dosage form, and is prepared by the preparation method disclosed in the prior art.
  • the present invention provides a method for preparing the above-mentioned biological detoxifier, the method comprising:
  • Nocardioides ZHH-013 with the deposit number CCTCC No.M 2020565 was activated, and multi-stage expansion was carried out.
  • the fermentation broth was collected to prepare a liquid biological detoxifier.
  • the fermentation broth is concentrated by methods such as natural sedimentation, centrifugation, filtration, etc. that do not affect the activity of the bacteria to obtain a bacterial suspension with a higher concentration; more preferably, the bacteria
  • the suspension can also be added with a nutrient solution or a mixed solution of a nutrient solution and a protective agent to prepare a liquid biological detoxifier.
  • the liquid-type biological detoxifier can also be prepared into a solid-type biological detoxifier by using the existing methods in the art, for example, adding an adsorbent, a protective agent, and the like.
  • the method further comprises the following steps:
  • the obtained bacterial suspension or bacterial cell with a higher concentration is broken by existing methods such as homogenization, ultrasonication, etc. After removing impurities such as bacterial cell fragments, a protein solution with a higher concentration is obtained by concentration, and a liquid type is obtained.
  • Biological detoxifier The liquid-type biological detoxifier can be prepared into a solid-type biological detoxifier by using existing methods in the art, such as adding adsorbents, protective agents, and the like.
  • the above-mentioned biological detoxifier can be packaged by conventional packaging technology in the art, and stored according to specific environmental conditions.
  • the present invention separates and obtains a Nocardioid strain ZHH-013 with DON degradation function from soil in China, and the DON degradation function of this bacterial strain is the same as the reported Nocardioid strain isolated in Japan
  • the DON-degrading functions of the 16S rDNA are similar, but the 16S rDNA homology is less than 97%.
  • the bacterial strain provided by the invention can use the toxic compound DON as the sole carbon source to grow, convert DON into its own chemical composition, the reaction process is irreversible, the reaction conditions are mild, and secondary pollution will not be caused;
  • bacterial strains provided by the invention and various DON biological detoxification preparations prepared therefrom can be used for the application of degrading DON in feed and food raw materials, primary processed products, deep processed products and related processing by-products;
  • strains provided by the present invention and various DON biological detoxification preparations prepared therefrom can be applied to various ecosystems such as soil or water bodies polluted by DON to achieve the purpose of degrading DON and restoring ecosystems.
  • Figure 2 shows the degradation effect of Nocardioides strain ZHH-013 of the present invention on DON in corn steep liquor.
  • Soil samples were collected from Zhangjiakou City, Hebei province, and the shaking flask enrichment method was used. First, the samples were made into bacterial suspensions in sterile water, and were inoculated into LB liquid medium with 10% inoculum. The final concentration of DON in the medium was 50 ⁇ g/ml, after culturing for 7 days and then inoculation with 10% inoculum, the DON concentration in the medium remained unchanged. After five consecutive inoculations, the DON content was detected, and the DON-LB medium containing the same DON concentration without bacteria was used as a negative control.
  • the bacterial suspension with degraded DON was spread on the LB agar plate at a suitable dilution according to the dilution coating method, and after culturing at 30 °C for 72 h, single colonies with good separation degree and different colony shapes were picked, and the concentration of DON was 50 ⁇ g/g/
  • the detoxification test was carried out in LB medium of ml, and the DON content was determined according to Example 1. After repeated screening, a strain capable of degrading DON was finally obtained, numbered ZHH-013. Pick a single colony of ZHH-013 in LB liquid medium, and when cultured to the mid-log phase, mix it with an equal volume of 50% glycerol and store it at -80°C.
  • the degrading strains were classified and identified by morphological, physiological and biochemical classification and 16S rDNA sequence analysis.
  • the ZHH-013 strain of the present invention was cultured on LB agar medium at 30°C. On the 7th day, circular white colonies were seen, with a diameter of 1 mm, neat edges, glossy surfaces, and transparent circles around colonies. When cultured on TSB agar medium at 30°C, round white colonies were seen on the 7th day, with neat edges and a glossy surface, and a transparent circle could be seen around the colonies. On the 14th day, the diameter of the colonies could reach 3 mm. Gram stain was positive.
  • LB medium tryptone 10g, yeast extract powder 5g, sodium chloride 10g, agar 1.5%, add distilled water to 1L, pH 7.0-7.2, sterilize at 121°C for 20min.
  • the TSB medium was purchased from BD Company under the brand name Difco.
  • Example 2 strains are used to degrade deoxynivalenol
  • DON stock solution Accurately weigh 5.0 mg of DON standard substance, dissolve it in 1 mL of sterile water, and prepare a DON stock solution with a final concentration of 5 mg/mL. Sterilize using a 1 mL syringe filter. DON stock solution is stored at -20°C and is effective within 3 months.
  • Sample preparation Take 500 ⁇ L of the sample solution, add an equal volume of pure methanol, shake and mix, centrifuge (12000 rpm, 4° C., 10 min), and draw 500 ⁇ L of the supernatant for HPLC detection.
  • the DON content was determined by HPLC, and the determination conditions were as follows: Column: Agilent 5 TC-C18(2) reversed-phase column (250 ⁇ 4.6 mm, 5 ⁇ m); Mobile phase: methanol: water (15:85); wash Desorption method: isocratic elution; flow rate: 1 mL/min; injection volume: 20 ⁇ L; column temperature: 30 °C; detection wavelength of UV detector: 220 nm; retention time of DON: 14.9 min.
  • a is the control with only inoculated bacterial liquid
  • e is the control with only DON added
  • b and c are the two treatment groups after overnight culture, DON was completely degraded compared with the control.
  • a is the treatment group with 10% corn steep liquor aqueous solution
  • b is the treatment group without 10% corn steep liquor aqueous solution
  • c is the control group with neither corn steep liquor nor degrading strains added
  • a single clone of Nocardioides sp. ZHH-013 was picked from the plate, inoculated into 1 mL of M9D medium, and cultured at 30°C for 7 days, and then transferred once every 7 days according to the transfer method. Pick up 3 times. Collect the fermentation broth after 7 days of each cultivation, take 150 ⁇ L and spread it on LB agar plate for cultivation, and measure the DON content of the remaining fermentation broth according to the above method.
  • the M9D medium formula is: M9 medium (Difco), DON 50mg, pH 7.0, add distilled water to 1L.
  • the transfer method is as follows: transfer to 1 mL of M9D medium with 0.1% inoculum, and culture at 30°C for 7 days.

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Abstract

本发明涉及微生物、饲料、食品及生态修复领域，具体涉及降解脱氧雪腐镰刀菌烯醇(DON)的菌株及应用。该菌株的保藏编号为CCTCC No.M 2020565。该菌株能够以有毒化合物DON为唯一碳源生长，将DON转化为自身化学组成成分，反应过程不可逆，反应条件温和，不会引起二次污染。本发明提供的菌株可用于制备DON的生物脱毒制剂。本发明提供的菌株可用于降解饲料和食品原料、初加工产品、深加工产品及相关加工副产物中的DON。本发明提供的菌株可应用于被DON污染的土壤或水体等各种生态系统，达到降解DON和生态修复的目的。

Description

降解脱氧雪腐镰刀菌烯醇的菌株及应用 技术领域
本发明涉及微生物领域，具体涉及降解脱氧雪腐镰刀菌烯醇的菌株及应用。
背景技术
脱氧雪腐镰刀菌烯醇(Deoxynivalenol,DON)，因DON能引起人和动物呕吐，又称呕吐毒素(Vomitoxin)，它是一种常见的食源性真菌毒素，广泛存在于小麦、大麦、燕麦和玉米等食品原料中。DON理化性质极其稳定，一般的烹饪和加工技术很难将其脱除，在高温烘培过程中，能够转化为其他毒性未知的化合物，如nor-DON，DON内酯等。此外，DON能够以“隐蔽”的形式存在于植物中，如3-β-D-葡萄糖-DON。这种“隐蔽”形式的衍生物被认为是植物自我防御的产物。不幸的是，隐蔽型DON经肠道微生物作用后仍能够还原为DON，人和动物的暴露风险会进一步增加。
DON的生物降解技术具有其高效、绿色、反应条件温和以及易于规模化应用等特点，近年来引起了广泛地关注，但可供研究与应用的生物资源却十分匮乏。DON降解菌株纯培养的获得及菌株对DON降解机制的揭示将为DON降解产品的开发带来重要启发。
德沃氏菌(Devosia)是被广泛研究的一类DON降解菌株。德沃氏属中多个种和亚种被证实能够将DON降解为3-keto-DON和3-epi-DON。2018年，加拿大的Zhou Ting组证实两个酶DepA和DepB分别负责这两步降解过程。此后多个团队也相继证实独自分离的德沃氏菌能够转化DON，但并不是所有德沃氏菌都能实现上述两步转化。
类诺卡氏菌(Nocardioides)与德沃氏菌几乎同期被发现具有DON降解功能。公开文献资料中，仅有日本的Tsushima组报道称类诺卡氏菌能够将DON作为碳源完全转化利用。类诺卡氏菌降解DON的过程中会产生3-epi-DON，后者会进一步被转化，然而完整的降解机制未被深入研究，主要原因之一是具有DON降解功能的类诺卡氏菌菌株的十分稀有，难以富集分离获得纯培养。
发明内容
为解决上述技术问题，本发明的目的是提供一株用于降解脱氧雪腐镰刀菌烯 醇的类诺卡氏菌菌株。
本发明的再一目的是提供利用上述类诺卡氏菌菌株制成的生物脱毒剂。
本发明的另一目的是提供制备上述生物脱毒剂的制备方法。
本发明提供了一株新型类诺卡氏菌(Nocardioides sp.)，菌株编号为ZHH-013，已于2020年9月30日保藏于中国典型培养物保藏中心，其保藏编号为CCTCC No.M 2020565。该菌株功能与类诺卡氏菌WSN05-2相似，但16S rDNA同源性小于97％，通过比对亲缘模式菌株的形态学、分子生物学和生理生化特性，确定其为类诺卡氏属(Nocardioides genus)中的一个新种。
所述发酵培养基的配方：胰蛋白胨10g、酵母浸粉5g、氯化钠10g，琼脂1.5％，加蒸馏水至1L，pH7.0-7.2，121℃灭菌20min。
根据本发明的生物脱毒剂包括作为活性成分的类诺卡氏菌ZHH-013，或其细胞内溶物，例如胞内蛋白，所述类诺卡氏菌ZHH-013保藏编号为CCTCC No.M 2020565。该生物脱毒剂可以是液体剂型也可以是固体剂型，并通过现有技术中已公开的制备方法来制备。
本发明提供了上述生物脱毒剂的制备方法，该方法包括：
将保藏编号为CCTCC No.M 2020565的类诺卡氏菌ZHH-013活化，多级扩培，当菌体处于稳定期时，收集发酵液，制备成液体型生物脱毒剂。
根据本发明的生物脱毒剂的制备方法，其中，该发酵液经自然沉降、离心、过滤等不影响菌体活性的方法浓缩后得到浓度较高的菌悬液；更优选地，所述菌悬液还可以加入营养液或营养液和保护剂的混合液，制备成液体型生物脱毒剂。进一步地，也可以利用本领域现有方法制备将该液体型生物脱毒剂制成固体型生物脱毒剂，例如加入吸附剂、保护剂等。
根据本发明的生物脱毒剂的制备方法，其中，所述方法还包括以下步骤：
将得到的浓度较高的菌悬液或菌体细胞，利用均质、超声等现有的方法进行破碎，除去菌体细胞碎片等杂质后通过浓缩获得浓度较高的蛋白液，制得液体型生物脱毒剂。可以利用本领域现有方法制备将该液体型生物脱毒剂制成固体型生物脱毒剂，例如加入吸附剂、保护剂等。
上述生物脱毒剂可采用本领域的常规包装技术包装，依据具体环境条件保存。
本发明的优点在于：
(1)本发明从中国境内的土壤中分离获得一株具有DON降解功能的类诺卡氏菌菌株ZHH-013，该菌株的DON降解功能与已报道的日本境内分离的类诺 卡氏菌菌株的DON降解功能相似，但16S rDNA同源性小于97％。
(2)通过形态学、生理生化和16S rDNA序列分析等分类法对本发明提供的菌株进行分类鉴定，通过比对亲缘模式菌株的形态学和生理生化特性资料，确定其为类诺卡氏属的新种；
(3)本发明提供的菌株能够以有毒化合物DON为唯一碳源生长，将DON转化为自身化学组成成分，反应过程不可逆，反应条件温和，不会引起二次污染；
(4)本发明提供的菌株可用于制备各种DON的生物脱毒制剂；
(5)本发明提供的菌株及其制备的各种DON生物脱毒制剂可用于在饲料和食品原料、初加工产品、深加工产品及相关加工副产物中降解DON的应用；
(6)本发明提供的菌株及其制备的各种DON生物脱毒制剂可应用于被DON污染的土壤或水体等各种生态系统，达到降解DON和生态体修复的目的。
附图说明
图1为本发明的类诺卡氏菌菌株ZHH-013及其内溶物降解DON的色谱图(RT DON＝14.9)；
图2显示本发明的类诺卡氏菌菌株ZHH-013对玉米浆中DON的降解效果。
类诺卡氏菌(Nocardioides sp.)ZHH-013，保藏编号CCTCC No：M 2020565，保藏于中国典型培养物保藏中心，保藏日期：2020-09-30，保藏地址：中国，武汉，武汉大学。
具体实施方式
实施例1脱氧雪腐镰刀菌烯醇降解菌株的分离和鉴定
从河北省张家口市采集土样，采用摇瓶富集方法，首先将样品在无菌水中制成菌悬液，以10％的接种量接种于LB液体培养基中，培养基中DON终浓度为50μg/ml，培养7天后再以10％接种量移种，培养基中DON浓度不变。经过连续5次移种后，检测DON含量，以未接菌、含相同DON浓度的DON-LB培养基为阴性对照。将具有降解DON的菌悬液按稀释涂布法以合适的稀释度涂布在LB琼脂平板，30℃培养72h后，挑取分离程度良好且菌落形态不同的单菌落，在DON浓度为50μg/ml的LB培养基中进行脱毒试验，按实施例1测定DON含量，经过反复的筛选最终获得一株能够降解DON的菌株，编号为ZHH-013。挑取ZHH-013单菌落于LB液体培养基中，培养至对数期中期时，用50％的甘油 与培养物等体积混合后置于-80℃保存。
采用形态学、生理生化和16S rDNA序列分析等分类法对降解菌株进行分类鉴定。将本发明的ZHH-013菌株在LB琼脂培养基上于30℃培养，第7天可见圆形白色菌落，直径为1mm，边缘齐整，表面有光泽，菌落周围可见透明圈。当在30℃下于TSB琼脂培养基上培养时，第7天可见圆形白色菌落，边缘齐整，表面有光泽，菌落周围可见透明圈，第14天，菌落直径可达3mm。革兰氏染色结果为阳性。
其中LB培养基的配方：胰蛋白胨10g、酵母浸粉5g、氯化钠10g，琼脂1.5％，加蒸馏水至1L，pH 7.0-7.2，121℃灭菌20min。
其中TSB培养基购于BD公司，品牌为Difco。
所有结果表明，本发明ZHH-013菌株应归属为类诺卡氏菌(Nocardioides sp.)，这一分类是基于直接的实验室比较和对已发表的类似物种的描述检索。
实施例2菌株用于降解脱氧雪腐镰刀菌烯醇
1、DON的检测方法
DON母液的制备：准确称取DON标准品5.0mg，溶于1mL无菌水中，配制终浓度为5mg/mL的DON母液。使用1mL的针筒过滤器除菌。DON母液储存在-20℃，3个月内有效。
样品制备：取500μL样品液，加入等体积的纯甲醇，震荡混匀后，离心(12000rpm，4℃，10min)，吸取500μL上清液，用于HPLC检测。使用HPLC法对DON含量进行测定，测定条件如下：色谱柱：Agilent 5 TC-C18(2)反相色谱柱(250×4.6mm，5μm)；流动相：甲醇:水(15:85)；洗脱方式：等度洗脱；流速：1mL/min；进样量：20μL；柱温：30℃；紫外检测器检测波长：220nm；DON的保留时间：14.9min。
2、从平板上挑取类诺卡氏菌(Nocardioides sp.)ZHH-013的单克隆，接种至3mL LB培养基，30℃下培养7d后，以1％接种量，转接至3mL LB培养基中，其中DON的终浓度为50μg/mL，30℃下震荡培养3d，按上述方法测定DON的含量。以不添加DON的试验组为阴性对照。色谱分析结果如图1所示，a为仅接种菌液的对照，e为仅添加DON的对照，b和c为两个处理组过夜培养后，与对照相比DON完全降解。
3、粗酶液对DON的降解
按上述步骤2制备菌体，并使用PBS缓冲液(pH 6.9)重悬菌体，调节OD 600 至1.0左右。使用超声破碎仪破碎菌体后，离心(12000rpm，4℃，10min)后取490μL上清液，加入10μL DON母液，终浓度50μg/mL，过夜培养后，按上述方法测定DON的含量。色谱分析结果如图1的d所示，过夜培养后，DON完全降解。
4、菌株对玉米浆中DON的降解
从平板上挑取类诺卡氏菌(Nocardioides sp.)ZHH-013的单克隆，接种至3mL LB培养基，30℃下培养7d后，以1％接种量，转接至300mL LB培养基中30℃下震荡培养5d，收集菌体。使用无菌水充分洗涤菌体至少3次，完全除净培养基。使用PBS缓冲液(pH 6.9)充分重悬菌体，调节OD 600至1.0左右。分别加入10％的10％玉米浆水溶液，以不加10％玉米浆水溶液的样本为对照。培养过夜后，按上述方法测定DON的含量。色谱分析结果如图2所示，a为添加10％玉米浆水溶液的处理组，b为不添加10％玉米浆水溶液的处理组，c为既不添加玉米浆且不添加降解菌株的对照组，与对照组c相比，处理组a过夜培养后DON降解率大于80％，处理组b中的DON含量也显著下降，因此，加与不加玉米浆，DON都可以被降解，菌株的降解功能稳定，不会因底物组分变化而变化。
5、菌株以DON为唯一碳源生长
从平板上挑取类诺卡氏菌(Nocardioides sp.)ZHH-013的单克隆，接种至1mL M9D培养基，30℃下培养7d后，按转接方法每培养7d转接1次，共转接3次。收集每次培养后7d后的发酵液，取150μL涂布于LB琼脂平板培养，剩余发酵液按上述方法测定DON的含量。其中M9D培养基配方为：M9培养基(Difco)，DON 50mg,pH 7.0，加蒸馏水至1L。其中转接方法为：以0.1％接种量，转接至1mL M9D培养基，30℃下培养7d。
该实验中，通过连续转接3次，意味着每次转接菌液会被稀释1000倍。如果不能利用DON为碳源，那么DON不能被降解，连续的3次实验都不能被降解；LB琼脂平板上的菌落数量规律是：稀释次数越多，数量越少。如果能利用DON作为碳源，则菌群数量每次培养后都会增加，那么3次实验DON均可被降解；LB琼脂平板上的菌落数量较多，且相当。
实验结果表明，收集的3次培养发酵液中DON降解率均大于80％，且对应的LB琼脂平板上均长满类诺卡氏菌(Nocardioides sp.)ZHH-013的菌苔，这说明类诺卡氏菌(Nocardioides sp.)ZHH-013可以以DON为唯一碳源生长。
显然，本发明的上述实施例仅仅是为清楚地说明本发明所作的举例，而并非 是对本发明的实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无法对所有的实施方式予以穷举。凡是属于本发明的技术方案所引伸出的显而易见的变化或变动仍处于本发明的保护范围之列。

Claims (8)

  1. 类诺卡氏菌(Nocardioides sp.)菌株ZHH-013，其特征在于，其保藏编号为CCTCC No.M 2020565。
  2. 一种生物脱毒剂，其特征在于，所述生物脱毒剂的活性组分包括权利要求1所述的类诺卡氏菌(Nocardioides sp.)菌株ZHH-013，或其细胞内溶物。
  3. 根据权利要求2所述的生物脱毒剂，其特征在于，所述生物脱毒剂的剂型为液体或固体。
  4. 一种生物脱毒剂的制备方法，其特征在于，所述方法包括以下步骤：
    活化类诺卡氏菌(Nocardioides sp.)菌株ZHH-013；
    多级扩大培养；以及
    收集发酵液，并制成生物脱毒剂。
  5. 根据权利要求4所述的生物脱毒剂的制备方法，其特征在于，将类诺卡氏菌(Nocardioides sp.)菌株ZHH-013的发酵液制成液体剂型或固体剂型的生物脱毒剂。
  6. 根据权利要求5所述的生物脱毒剂的制备方法，其特征在于，将所述液体剂型的生物脱毒剂进一步制成固体剂型。
  7. 权利要求1所述的类诺卡氏菌(Nocardioides sp.)菌株ZHH-013在饲料和食品原料、初加工产品、深加工产品及相关加工副产物中降解DON的应用。
  8. 权利要求2所述的生物脱毒剂在被DON污染的生态系统中降解DON的应用。
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