WO2022156338A1 - 精氨酸作为脱氧雪腐镰刀菌烯醇生成抑制剂的用途 - Google Patents

精氨酸作为脱氧雪腐镰刀菌烯醇生成抑制剂的用途 Download PDF

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WO2022156338A1
WO2022156338A1 PCT/CN2021/131518 CN2021131518W WO2022156338A1 WO 2022156338 A1 WO2022156338 A1 WO 2022156338A1 CN 2021131518 W CN2021131518 W CN 2021131518W WO 2022156338 A1 WO2022156338 A1 WO 2022156338A1
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arginine
don
deoxynivalenol
concentration
fusarium
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PCT/CN2021/131518
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韩铮
范楷
郭文博
聂冬霞
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上海市农业科学院
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
    • A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
    • A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
    • A01N47/44—Guanidine; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/02—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing liquids as carriers, diluents or solvents

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  • the invention relates to the field of biotechnology, in particular to the use of arginine as a deoxynivalenol production inhibitor.
  • Deoxynivalenol also known as DON, is an important mycotoxin produced by Fusarium graminearum and Fusarium flavus, with acute toxicity (diarrhea, vomiting, leukocytosis, etc.) and chronic toxicity (anorexia , weight loss, decreased feed titer, etc.), has been identified as one of the most dangerous food contaminants.
  • JAFAC proposed in 2010 that the tentative daily maximum intake of DON should not exceed 1 ⁇ g/kg bw/day. China is one of the countries most seriously endangered by DON in the world.
  • the present invention provides the use of arginine as a deoxynivalenol production inhibitor
  • arginine when used, it can be formulated into an aqueous solution with a concentration of 10 mM and above, and the preferred concentration of the arginine aqueous solution is 10-500 mM;
  • the preferred arginine aqueous solution concentration is 100-500mM
  • Deoxynivalenol is mainly produced by fungi such as Fusarium graminearum, Fusarium oxysporum, Fusarium flavus, and Fusarium moniliformes;
  • the arginine aqueous solution can be directly sprayed on the surface of agricultural products, or directly added to the substrate.
  • the present invention also provides the application of arginine as a fungal inhibitor, wherein the fungus is Fusarium graminearum, Fusarium oxysporum, Fusarium flavus or Fusarium moniliformes;
  • arginine When arginine is used as a fungal inhibitor, it can be formulated into an aqueous solution with a concentration of 50-500 mM.
  • the present invention finds for the first time that arginine can effectively inhibit the generation of deoxynivalenol in different substrates
  • the arginine of the present invention is a commercial product with wide sources and low price, and the formed inhibitor has low cost and can be applied on a large scale.
  • the arginine of the present invention belongs to the essential amino acid of human body, has good safety to humans and animals, and has no pollution to the environment;
  • the inhibitor of the present invention is easy to use, and can be used by ordinary personnel after simple training, which is beneficial to large-scale promotion;
  • the inhibitor of the present invention has a good effect of inhibiting the generation of deoxynivalenol, and can even inhibit the growth of fungal strains at high concentrations, providing an effective means for the prevention and control of mycotoxins in agricultural products.
  • arginine The activity of arginine to inhibit the biosynthesis of Fusarium deoxynivales is proposed for the first time. Compared with other existing inhibitors, arginine has a more significant inhibitory effect, lower cost, no harm to organisms, and Not easy to develop drug resistance.
  • Arginine purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China
  • Fusarium graminearum strain F4582 purchased from DSMZ, German Collection of Microorganisms
  • Raw materials used in PDA medium, PDB liquid medium, as well as wheat and corn common commercial products.
  • PDA medium 200g of peeled potatoes were boiled for 30min and then the filtrate was taken, added with 20g of glucose and 16g of agar, dilute to 1000mL with distilled water, sterilized by autoclaving at 115°C for 30 minutes, cooled to about 55°C and poured into a plate, 20mL per plate.
  • PDB liquid medium boil 200 g of peeled potatoes for 30 min, take the filtrate, add 20 g of glucose, dilute to 1000 mL with distilled water, and sterilize by autoclaving at 115°C for 30 min.
  • F. graminearum strain F4582 (purchased from DSMZ, Germany) was inoculated into PDA medium, cultivated in the dark at 28°C for 7 days, inoculated into PDB liquid medium, and shaken at 150 r/min at 25°C Continue to cultivate for 5 days.
  • the spore liquid of F. graminearum F4582 was taken, the spore concentration was observed under a microscope, adjusted to 10 5 /mL with sterile water, and used for inoculation in subsequent Examples 1-3.
  • Chromatographic column Agilent Poroshell 120EC-C 18 column (100mm ⁇ 3.0mm, 2.7mm); mobile phase: mobile phase A is 5mmol/L ammonium acetate solution, mobile phase B is methanol; gradient elution program: 0 ⁇ 0.5mim , 10%A; 4min, 90%A; 4.5min, 90%A; 4.7min, 10%A; 6min, 10%A; flow rate 0.4mL/min; injection volume 3 ⁇ L; column temperature 40°C.
  • the electrospray ionization source (ESI) is used for simultaneous scanning in positive and negative ion modes; the atomization gas and auxiliary gas are high-purity air, and the collision gas is high-purity nitrogen; atomization gas: 50Psi; auxiliary gas: 50Psi; atomization temperature: 500.0°C ; Spray voltage: 5500V; Spray voltage curtain gas: 35Psi; Collision gas: 8Psi:
  • the target compound is accurately quantified by multiple reaction monitoring (MRM) mode.
  • MRM multiple reaction monitoring
  • the DON parent ion (m/z) was 297.3, the quantitative product ion (m/z) was 203.0, the collision voltage was 28 eV, the qualitative product ion (m/z) was 175.1, and the collision voltage was 28 eV.
  • arginine solutions with concentrations of 1 mM, 10 mM, 100 mM and 500 mM, respectively, and sterilized by filtration.
  • the conical flask was sealed with sterile air-permeable sealing film, and after shaking, 100 ⁇ L of F. graminearum F4582 spore solution was added and cultured in the dark in a constant temperature and humidity incubator at 28°C for 28 days to detect the yield of DON. Five replicates were set for each concentration.
  • arginine solutions were weighed and dissolved in sterile ultrapure water to prepare arginine solutions with concentrations of 1 mM, 10 mM, 100 mM and 500 mM, respectively, and sterilized by filtration.
  • the conical flask was sealed with sterile air-permeable sealing film, and after shaking, 100 ⁇ L of F. graminearum F4582 spore solution was added and cultured in the dark in a constant temperature and humidity incubator at 28°C for 28 days to detect the yield of DON. Five replicates were set for each concentration.

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  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Pest Control & Pesticides (AREA)
  • Plant Pathology (AREA)
  • Engineering & Computer Science (AREA)
  • Dentistry (AREA)
  • Agronomy & Crop Science (AREA)
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Abstract

本发明公开了精氨酸作为脱氧雪腐镰刀菌烯醇生成抑制剂的用途，其中精氨酸使用时配制成浓度为10mM及以上浓度的水溶液。本发明采用精氨酸作为脱氧雪腐镰刀菌烯醇生成抑制剂，健康、无毒，在较低的含量下即可有效抑制镰刀菌等真菌在食用农产品中产生脱氧雪腐镰刀菌烯醇。

Description

精氨酸作为脱氧雪腐镰刀菌烯醇生成抑制剂的用途 技术领域
本发明涉及生物技术领域，具体的说涉及精氨酸作为脱氧雪腐镰刀菌烯醇生成抑制剂的用途。
背景技术
脱氧雪腐镰刀菌烯醇(DON)，又称呕吐毒素，为禾谷镰刀菌和黄色镰刀菌产生的一种重要真菌毒素，具有急性毒性(腹泻、呕吐、白细胞增多等)和慢性毒性(厌食、体重减轻、饲料效价下降等)，已被确定为最危险的食品污染物之一。JAFAC于2010年提出，DON的暂定日内最大摄入量均不得超过1μg/kg bw/day。中国是世界上受DON危害最严重的国家之一，2007-2008年，中国13个省市292份小麦粉和347份玉米制品(玉米面、玉米渣、玉米片等)中DON的污染研究发现，小麦粉中DON的检出率为100％，平均值为178.4μg/kg，玉米制品的阳性率为97.4％，平均值为272.2μg/kg。2010-2012年连续3年对江苏省21个县180份小麦的调查结果发现，DON的检出率高达74.4％，浓度为14.5-41157.1μg/kg。2010年，浙江省的63份及吉林省的6份玉米样品的调查结果发现，61份含有DON，检出率为88.4％，浓度为2.7-311.2μg/kg。由此可见，DON污染严重威胁我国食品安全。
因此，寻找能够抑制DON产生的活性物质，对于保障农产品质量安全、促进国家经济发展具有非常重要的意义。
发明内容
本发明提供了精氨酸作为脱氧雪腐镰刀菌烯醇生成抑制剂的用途；
具体的说，精氨酸使用时可配制成浓度为10mM及以上浓度的水溶液，优选的精氨酸水溶液浓度为10-500mM；
进一步优选的，精氨酸水溶液用于农产品如小麦、玉米、花生、燕麦、黑麦等作为脱氧雪腐镰刀菌烯醇生成抑制剂时，优选的精氨酸水溶液浓度为100-500mM；
脱氧雪腐镰刀菌烯醇主要由禾谷镰刀菌、尖孢镰刀菌、黄色镰刀菌、串珠镰刀菌等真菌产生；
精氨酸作为脱氧雪腐镰刀菌烯醇生成抑制剂时，可以将精氨酸水溶液直接喷洒到农产品表面、或者直接加入基质等。
本发明还提供了精氨酸作为真菌抑制剂的应用，其中所述的真菌为禾谷镰刀菌、尖孢镰刀菌、黄色镰刀菌或串珠镰刀菌；
精氨酸作为真菌抑制剂使用时，可配制成浓度为50-500mM的水溶液。
本发明提供的精氨酸作为脱氧雪腐镰刀菌烯醇生成抑制剂的用途，其具备以下优点：
(1)本发明首次发现了精氨酸在不同基质中可以有效抑制脱氧雪腐镰刀菌烯醇的生成；
(2)本发明的精氨酸属于商品化产品来源广、价格低廉，形成的抑制剂成本低，能够大规模应用。
(3)本发明的精氨酸属于人体必需氨基酸，对人畜安全性好，对环境无污染；
(4)本发明的抑制剂使用简单，普通人员简单培训即可使用，有益于大规模推广；
(5)本发明的抑制剂抑制脱氧雪腐镰刀菌烯醇生成的效果好，高浓度时甚至可以抑制真菌菌株生长，为农产品中真菌毒素的防控提供了有效手段。
(6)精氨酸抑制脱氧雪腐镰刀菌生物合成的活性为首次提出，与现有的其它抑制剂相比，精氨酸抑制效果更为显著、成本更低、对生物体无危害，且不易产生抗药性。
附图说明
图1不同浓度精氨酸对PDA培养基中DON生成的抑制作用
图2不同浓度精氨酸对PDA培养基中DON产毒真菌菌株生长的抑制作用
图3不同浓度精氨酸对小麦中DON生成的抑制作用
图4不同浓度精氨酸对玉米中DON生成的抑制作用
具体实施方式
原料来源：
精氨酸：购自国药集团化学试剂有限公司，上海，中国
禾谷镰刀菌菌株F4582：购至德国微生物菌种保藏中心DSMZ
PDA培养基、PDB液体培养基中所用原料，以及小麦、玉米：普通市售产品。
下列实施例中用到的实验方法：
1、禾谷镰刀菌菌株F4582菌株的培养方法
PDA培养基：去皮马铃薯200g煮沸30min后取滤液，加入葡萄糖20g、琼脂16g，用蒸馏水定容至1000mL，115℃高压灭菌30分钟，降温到55℃左右倒平板，每平板20mL。
PDB液体培养基：去皮马铃薯200g煮沸30min后取滤液，加入葡萄糖20g，用蒸馏水定容至1000mL，115℃高压灭菌30分钟。
菌株活化培养：将禾谷镰刀菌菌株F4582(购至德国微生物菌种保藏中心DSMZ)接种于PDA培养基，28℃黑暗培养7天后，接种至PDB液体培养基中，25℃下150r/min震荡继续培养5天。取禾谷镰刀菌F4582孢子液，显微镜观察孢子浓度，用无菌水调整至10 5个/mL，用于后续实施例1-3中接种使用。
2、DON的提取方法
将PDA培养基、小麦、玉米分别于50℃烘箱干燥，粉碎混匀后，准确称取2g粉碎样品于50mL离心管，加入10mL乙腈/水(84/16，v/v)，旋涡震荡1min，浸泡5min后，超声提取1小时。4000r/min离心10min后，取5mL上清液，在40℃下氮气吹干，1mL的5mmol/L乙酸铵水溶液/甲醇(80:20，v:v)溶解残渣，涡旋30s，超声1min，涡旋30s，充分溶解后，适量稀释，过0.22μm滤膜，UPLC-MS/MS测定。
3、DON的UPLC-MS/MS检测条件和方法
色谱柱：Agilent Poroshell 120EC-C 18色谱柱(100mm×3.0mm，2.7mm)；流动相：流动相A为5mmol/L乙酸铵溶液，流动相B为甲醇；梯度洗脱程序：0～0.5mim，10％A；4min，90％A；4.5min，90％A；4.7min，10％A；6min，10％A；流速0.4mL/min；进样量3μL；柱温40℃。
采用电喷雾电离源(ESI)正负离子模式同时扫描；雾化气、辅助气均为高纯空气，碰撞气为高纯氮气；雾化气：50Psi；辅助气：50Psi；雾化温度：500.0℃；喷雾电压：5500V；喷雾电压气帘气：35Psi；碰撞气：8Psi：通过多反应监测(multiple reaction monitoring，MRM)模式对目标化合物准确定量。DON母离子(m/z)为297.3，定量子离子(m/z)为203.0，碰撞电压为28eV，定性子离子(m/z)为175.1，碰撞电压为28eV。
实施例1 PDA培养基中精氨酸对DON合成的抑制作用
称取适量精氨酸溶于10mL无菌超纯水，过滤除菌后加入90mL灭菌后的PDA培养基，使得最终添加浓度分别达到0、0.1、1、10、50、100mM和500mM。混匀倒板后，接种100μL禾谷镰刀菌F4582孢子液于28℃恒温恒湿培养箱黑暗培养培养9天，检测DON的产量。每个浓度设置平行5份。
结果见图1，与对照组相比(精氨酸浓度0mM)，所有浓度精氨酸均能抑制DON 的生物合成(P<0.05)，抑制效果随着精氨酸浓度的升高而增强。精氨酸浓度0.1、1和10mM时DON的产量分别下降24.5％、52.2％和68.6％，精氨酸浓度达到50、100和500mM时几乎完全抑制DON的产生。
与对照组相比(精氨酸浓度0mM)，在低浓度时，精氨酸抑制禾谷镰刀菌F4582生长的效果不明显，但是当浓度达到50mM时，精氨酸可以显著抑制禾谷镰刀菌F4582的生长，当浓度达到500mM时，精氨酸几乎可以完全抑制禾谷镰刀菌F4582的生长(图2)。
实施例2 小麦中精氨酸对DON合成的抑制作用
称取适量精氨酸溶于无菌超纯水，配制浓度分别为1mM、10mM、100mM和500mM的精氨酸溶液，过滤除菌。准确称取50g小麦于250mL无菌锥形瓶中，120℃高压灭菌30分钟后分别加入不同浓度的50mL精氨酸溶液，对照组加入50mL无菌超纯水。用无菌透气封口膜密封锥形瓶，摇匀后加入100μL禾谷镰刀菌F4582孢子液于28℃恒温恒湿培养箱黑暗培养培养28天，检测DON的产量。每个浓度设置平行5份。
结果显示，与对照组相比，低浓度精氨酸对小麦中DON的产生无明显影响，而高浓度精氨酸显著抑制DON的合成(P<0.01)(图3)。100mM精氨酸作用下，DON的产量下降66.6％，500mM精氨酸则几乎完全抑制小麦中DON的产生。
实施例3 玉米中精氨酸对DON合成的抑制作用
称取适量精氨酸溶于无菌超纯水，配制浓度分别为1mM、10mM、100mM和500mM的精氨酸溶液，过滤除菌。准确称取50g玉米于250mL无菌锥形瓶中，120℃高压灭菌30分钟后分别加入不同浓度的50mL精氨酸溶液，对照组加入50mL无菌超纯水。用无菌透气封口膜密封锥形瓶，摇匀后加入100μL禾谷镰刀菌F4582孢子液于28℃恒温恒湿培养箱黑暗培养培养28天，检测DON的产量。每个浓度设置平行5份。
结果显示，与对照组相比，低浓度精氨酸对玉米中DON的产生无明显影响，而高浓度精氨酸显著抑制DON的合成(P<0.01)(图4)。100mM精氨酸作用下，DON的产量下降48.1％，500mM精氨酸则几乎完全抑制玉米中DON的产生。

Claims (5)

  1. 精氨酸作为脱氧雪腐镰刀菌烯醇生成抑制剂的用途。
  2. 根据权利要求1所述的精氨酸作为脱氧雪腐镰刀菌烯醇生成抑制剂的用途，其中精氨酸使用时配制成浓度为10mM及以上浓度的水溶液。
  3. 根据权利要求2所述的精氨酸作为脱氧雪腐镰刀菌烯醇生成抑制剂的用途，其中精氨酸水溶液浓度为10-500mM。
  4. 精氨酸作为真菌抑制剂的应用，其中所述的真菌为禾谷镰刀菌、尖孢镰刀菌、黄色镰刀菌或串珠镰刀菌。
  5. 根据权利要求4所述的精氨酸作为真菌抑制剂的应用，其中精氨酸使用时，配制成浓度为50-500mM的水溶液。
PCT/CN2021/131518 2021-01-19 2021-11-18 精氨酸作为脱氧雪腐镰刀菌烯醇生成抑制剂的用途 WO2022156338A1 (zh)

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