CN110622968B - 一种大豆疫霉抑制剂及其制备方法 - Google Patents
一种大豆疫霉抑制剂及其制备方法 Download PDFInfo
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- CN110622968B CN110622968B CN201910806605.2A CN201910806605A CN110622968B CN 110622968 B CN110622968 B CN 110622968B CN 201910806605 A CN201910806605 A CN 201910806605A CN 110622968 B CN110622968 B CN 110622968B
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- propyl gallate
- phytophthora sojae
- carbon dioxide
- inhibitor
- phytophthora
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- A01N37/36—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids
- A01N37/38—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids having at least one oxygen or sulfur atom attached to an aromatic ring system
- A01N37/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids having at least one oxygen or sulfur atom attached to an aromatic ring system having at least one carboxylic group or a thio analogue, or a derivative thereof, and one oxygen or sulfur atom attached to the same aromatic ring system
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
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Abstract
本发明提供一种大豆疫霉抑制剂及其制备方法，涉及植物病害生物防治领域，包括没食子酸丙酯，所述没食子酸丙酯作为食品添加剂主要用于食品的抗氧化作用，对大豆疫霉的值EC50％仅为0.458mmol/L，即抑制率为50％时，没食子酸丙酯的浓度仅为0.458mmol/L，安全性较高。其抑制大豆疫霉危害主要来自两方面其一没食子酸丙酯抑制大豆疫霉的生长，其作用机理及靶位点，通过蛋白组学技术揭示大豆疫霉的抑制机理与脂肪转运与代谢、能量代谢和碳水化合物代谢等关键通路蛋白表达的大幅下调紧密相关；其二没食子酸丙酯可以激发植物的防卫反应，提高抗性基因表达量抵抗大豆疫霉的侵染。
Description
技术领域
本发明涉及植物病害生物防治领域，尤其涉及一种大豆疫霉抑制剂及其制备方法。
背景技术
本发明涉及植物病害生物防治领域，尤其涉及一种大豆疫霉抑制剂及其制备方法。
背景技术
大豆疫霉病菌引起的大豆疫病是大豆生产中的毁灭性病害之一,是我国对外公布的一类植物检疫危险性病原真菌。该病菌以土壤传播为主，大豆疫霉病原真菌是典型的土壤习居菌，主要以抗逆性很强的卵孢子在土壤中和土壤中的寄主残体内越冬并长期生存。当田间温度和湿度适宜时,卵孢子打破休眠萌发产生孢子囊,雨后有积水时，孢子囊很快释放出大量游动孢子,游动孢子被雨水冲溅到寄土表面.或受寄主分泌物的吸引。朝根系游动并最终侵染寄主。目前的防治大豆疫霉的方法有如下几种：(1)选用对当地小种具抵抗力的抗病品种。(2)加强田间管理，及时深耕及中耕培土。雨后及时排除积水防止湿气滞留。(3)播种时沟施甲霜灵颗粒剂，使大豆根吸收防止根部侵染。(4)播种前种子重量0.3％的35％甲霜灵粉剂拌种。(5)必要时喷洒或浇灌25％甲霜灵可湿性粉剂800倍液、58％甲霜灵锰锌可湿性粉剂600倍液或64％杀毒矾可湿性粉剂900倍液。可知在大豆疫霉防治中甲霜灵或杀毒矾抑制大豆疫霉的生长作为主要防治手段，其作用机制为抑制RNA聚合酶从而抑制了RNA的生物合成。
而农药使用不当可对植物产生药害，引起人畜中毒，杀伤有益微生物，导致病原物产生抗药性，农药的高残留还可造成环境污染。
发明内容
本发明的目的在于提供一种大豆疫霉抑制剂及其制备方法，以解决上述技术问题。
本发明为解决上述技术问题，采用以下技术方案来实现：一种大豆疫霉抑制剂，包括没食子酸丙酯和癸醇，癸醇作为没食子酸丙酯的助溶剂，其用量比例为：0.1mg-0.3mg的癸醇：1-2mmol的没食子酸丙酯。
所述没食子酸丙酯采用超临界二氧化碳萃取方法，包括如下步骤：
(1)精确称取颗粒大小在2-5mm大小的赤芍粉1.3-1.8kg，将其加入17-22％的温度为6-8℃的纯水中，在5℃条件下放置50-70分钟，超临界二氧化碳萃取中，超临界二氧化碳提取压力为33-37MPa，提取时间为54-57分钟，二氧化碳流率为4.5-5.7L/min，即可提取出没食子酸丙酯粗提物；
(2)没食子酸丙酯纯化：将(3)中没食子酸丙酯粗提物按照1:5的质量比溶于85-90℃的热水中，用截留分子量10kDa的超滤膜超滤去大分子杂质；然后用旋转蒸发仪中浓缩至没食子酸丙酯含量大于20-50mg/L；
(3)结晶纯化与干燥：将(4)中的没食子酸丙酯转移到50ml的离心管中，4-6℃下放置12小时后有沉淀出现在离心管底部，在10000rpm/min下离心5分钟，去上清，液氮速冻沉淀后，在-60℃、真空度为45的真空条件下冷冻干燥，待干燥至粉末，即获得没食子酸丙酯。
所述没食子酸丙酯还可由没食子酸和丙醇在单宁酶的催化下制备，包括如下制备步骤：
(1)没食子酸提取工艺：采用超临界二氧化碳萃取方法，称取颗粒大小在2-5mm大小的蓝莓叶子或绿茶叶粉1.3-1.8kg，将其加入17-22％的温度为2-8℃的纯水中，在5℃条件下放置50-70分钟，超临界二氧化碳萃取中，超临界二氧化碳提取压力为23-27MPa，提取时间为32-50分钟，即可提取出没食子酸；
(2)单宁酶制备工艺：黑曲霉在液体土豆培养基中培养3-5天，过滤收集发酵液，用硫酸铵沉淀法沉淀，复溶提取粗蛋白，用截留分子量10kDa的超滤膜超滤去硫酸铵并浓缩蛋白，即得到单宁酶；
(3)没食子酸丙酯合成方法：将8.9mmol没食子酸溶于8.2％-13.6％丙醇溶液中，加入0.3-0.6mol的单宁酶在二氧化碳培养箱中，在20℃恒温条件下催化反应10-15小时，在-60℃、真空度为45的真空条件下冷冻干燥，待干燥至粉末，即获得没食子酸丙酯。
大豆疫霉抑制剂的制备方法为没食子酸丙酯与癸醇混合：将0.1mg-0.3mg的癸醇，1-2mmol的没食子酸丙酯混合，完全溶解后依次添加60-70℃的热水和20-25℃常温水各500mL，即可以得到大豆疫霉抑制剂。
本发明的有益效果是：
作为食品添加剂没食子酸丙酯具有以下优点：安全性高；无农药残留；可生物合成，耗能低对环境友好；激发大豆抗性基因显著上调表达抵抗大豆疫霉的侵染；对大豆疫霉的防治效果好，抑制50％的大豆疫霉生长是的浓度仅需0.458mmol/L，没食子酸丙酯抑制50％的大豆疫霉生长所用抑制剂剂量更小，较常用农药的防治效果更好，没食子酸丙酯还可以激发植物的防卫反应，提高抗性基因表达量抵抗大豆疫霉的侵染。
附图说明：
图1是本发明质谱鉴定到肽段的长度分布柱状图；
图2是本发明质谱鉴定到的蛋白分子量与覆盖度的关系示意图；
图3是本发明质谱数据的质量精度分布图；
图4是本发明差异表达蛋白KOG功能分类分布图；
图5是本发明差异表达蛋白KEGG通路富集分析图；
图6是本发明脂肪酸代谢途径示意图；
图7是本发明淀粉和糖代谢途径示意；
图8是本发明PG处理后与未处理的大豆相对基因表达量的柱状图。
具体实施方式
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解，下面结合具体实施例，进一步阐述本发明，但下述实施例仅仅为本发明的优选实施例，并非全部。基于实施方式中的实施例，本领域技术人员在没有做出创造性劳动的前提下所获得其它实施例，都属于本发明的保护范围。下述实施例中的实验方法，如无特殊说明，均为常规方法，下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1
一种大豆疫霉抑制剂，其特征在于：包括没食子酸丙酯和癸醇，癸醇作为没食子酸丙酯的助溶剂，其用量比例为：0.1mg-0.3mg的癸醇：1-2mmol的没食子酸丙酯。
如图1-7所示，通过蛋白质组学挖掘没食子酸丙酯抑制大豆疫霉的机理：通过对未处理的大豆疫霉和没食子酸丙酯处理的大豆疫霉(在液体V8中添加没食子酸使其最终浓度为0.5mmol/L)培养3-5天后收集菌丝，加液氮充分研磨至粉末。各组样品分别加入粉末4倍体积酚抽提缓冲液(含1％蛋白酶抑制剂)，超声裂解。加入等体积的Tris平衡酚，4℃，5500g离心10min，取上清并加入5倍体积的0.1M乙酸铵/甲醇沉淀过夜，蛋白沉淀分别用甲醇和丙酮进行洗涤得蛋白质溶液。蛋白溶液中加入二硫苏糖醇使其终浓度为5mM，56℃还原30min。之后加入碘代乙酰胺使其终浓度为11mM，室温避光孵育15min。最后将样品的尿素浓度稀释至低于2M。以1:50的质量比例(胰酶：蛋白)加入胰酶，37℃酶解12h。再以1:100的质量比例(胰酶：蛋白)加入胰酶，继续酶解4h。胰酶酶解的肽段用Strata X C18(Phenomenex)除盐后真空冷冻干燥。以0.5M TEAB溶解肽段，根据TMT试剂盒操作说明标记肽段。
简单的操作如下：标记试剂解冻后用乙腈溶解，与肽段混合后室温孵育2h，标记后的肽段混合后除盐，真空冷冻干燥。肽段用高pH反向HPLC分级，色谱柱为Agilent300Extend C18(5μm粒径，4.6mm内径，250mm长)。操作如下：肽段分级梯度为8％-32％乙腈、pH 9，60min时间分离60个组分，随后肽段合并为18个组分，合并后的组分经真空冷冻干燥后进行后续操作。
肽段用液相色谱流动相A相(0.1％(v/v)甲酸水溶液)溶解后使用EASY-nLC 1000超高效液相***进行分离。流动相A为含0.1％甲酸和2％乙腈的水溶液；流动相B为含0.1％甲酸和90％乙腈的水溶液。液相梯度设置：0～24min，9％-25％B；24～32min，25％-36％B；32～36min，36％-80％B；36～40min，80％B，流速维持在350nL/min。
肽段经由超高效液相***分离后被注入NSI离子源中进行电离然后进QExactiveTM Plus质谱进行分析。离子源电压设置为2.0kV，肽段母离子及其二级碎片都使用高分辨的Orbitrap进行检测和分析。一级质谱扫描范围设置为350-1800m/z，扫描分辨率设置为70,000；二级质谱扫描范围则固定起点为100m/z，二级扫描分辨率设置为17,500。数据采集模式使用数据依赖型扫描(DDA)程序，即在一级扫描后选择信号强度最高的前20肽段母离子依次进入HCD碰撞池使用31％的碎裂能量进行碎裂，同样依次进行二级质谱分析。为了提高质谱的有效利用率，自动增益控制(AGC)设置为5E4，信号阈值设置为10000ions/s，最大注入时间设置为200ms，串联质谱扫描的动态排除时间设置为30秒避免母离子的重复扫描。
数据库搜索及蛋白质注释：二级质谱数据使用Maxquant(v1.5.2.8)进行检索，检索参数设置：数据库为NCBI Phytophthora_sojae(26489条序列)，添加了反库以计算随机匹配造成的假阳性率(FDR)，并且在数据库中加入了常见的污染库，用于消除鉴定结果中污染蛋白的影响；酶切方式设置为Trypsin/P；漏切位点数设为2；肽段最小长度设置为7个氨基酸残基；肽段最大修饰数设为5；First search和Main search的一级母离子质量误差容忍度分别设为20ppm和5ppm，二级碎片离子的质量误差容忍度为0.02Da。将半胱氨酸烷基化设置为固定修饰，可变修饰为甲硫氨酸的氧化，蛋白N端的乙酰化。定量方法设置为TMT-6plex，蛋白鉴定、PSM鉴定的FDR都设置为1％。(1)Gene Ontology分析Gene Ontology(GO)对蛋白质组学层面的注释来源于UniProt-GOA数据库。如果蛋白来源于Uniprot数据库，首先用UniProt ID去匹配GO ID，并依据GO ID从UniProt-GOA数据库中调取相应的信息。如果UniProt-GOA数据库中没有所查询的蛋白信息，那么会使用一款基于蛋白序列的算法软件，InterProScan，去预测该蛋白的GO功能。之后按照细胞成分、分子功能或生理进程对此蛋白进行分类。使用KEGG通路数据库对蛋白通路进行注释：首先，使用KEGG在线服务工具KAAS对提交的蛋白进行注释，之后通过KEGG mapper将注释过的蛋白匹配入数据库中相应的通路中。
如图1所示为样品蛋白质肽段长度分布结果，样品经胰酶酶解和TMT标记后，经反向HPLC分级，用Q ExactiveTM Plus液相色谱-质谱联用分析鉴定蛋白质，小于5个氨基酸的肽段产生的碎片离子过少，造成序列鉴定不准确；而当肽段大于20个氨基酸时，由于质量和电荷数较高导致它们不适合HCD碎裂方式。本实验结果显示，大部分肽段分布在7-20个氨基酸范围内，该结果显示肽段长度分布符合基于胰蛋白酶酶解和HCD碎裂的一般规律，质谱鉴定到的肽段长度的分布符合质控要求。
如图2所示，蛋白质组分的分子量和覆盖度之间为反比关系，随着蛋白质组分分子量的增加水解时会产生较多的酶解肽段；换而言之相同的覆盖率肽段数目会随之蛋白质组分分子量的增加而增加，大多数蛋白质组分蛋白质分子量在50kD左右，其覆盖度在70％左右。
如图3所示，对照组和处理组蛋白质组分谱图一级质量误差≤6ppm，该结果显示质谱结果符合轨道阱质谱的高精度特性，即质谱仪运转正常，能满足蛋白组分的定性定量分析要求。谱图匹配肽段得分较高，也反映了蛋白质组分鉴定质量较好，可靠性高可以满足两组样品蛋白质组分分析的要求。
如图4所示，通过对真核生物KOG数据库比对分析和统计，差异蛋白同源组蛋白集群(Clusters of Orthologous Groups of proteins，COG)分为20类功能。
下调表达的蛋白主要分布在脂肪转运与代谢(Lipid transport andmetabolism)、碳水化合物转运与代谢(Carbohydrate transport and metabolism)、信号转导机制(Signal transduction mechanisms)、次级代谢合成、转运和分解代谢(Secondary metabolites biosynthesis,transport and catabolism)和能量的产生与转化(Energy production and conversion)等16类KOG功能，这些功能涉及的差异蛋白数量分别为17、16、12、11和10。涉及脂肪转运与代谢、能量代谢和碳水化合物运输与代谢等蛋白表达大幅下调与大豆疫霉生长受到抑制的原因。
如图5所示为差异蛋白KEGG富集分析，KEGG分析显示，共有17个上调表达差异蛋白被富集到“psoj03010 Ribosome(核糖体，6个差异表达蛋白)”、“psoj00010 Glycolysis/Gluconeogenesis(糖酵解糖质新生，4个差异表达蛋白)”和“psoj01110 Biosynthesis ofsecondary metabolites(次生代谢产物的生成，7个差异表达蛋白)”。从富集通路可知它们可能与细胞器核糖体功能翻译蛋白，糖类利用途径糖酵解和糖质新生相关，而更多的差异蛋白可能参与了大豆疫霉的次级代谢过程。另外，“psoj03010 Ribosome”、“psoj00010Glycolysis/Gluconeogenesis”和“psoj01110 Biosynthesis of secondary metabolites的-log10(p value)分别为3.01、1.73和1.4。下调表达的蛋白中有159个(占下调表达总蛋白的75.7％)分别富集在“psoj00350Tyrosine metabolism(酪氨酸，8个差异表达蛋白)”、“psoj00360Phenylalanine metabolism(苯丙氨酸代谢，36个差异表达蛋白)”、“psoj00130Ubiquinone and other terpenoid-quinone biosynthesis(泛醌和其他萜烯类醌的生物合成，6个差异表达蛋白)”、“psoj01110 Biosynthesis of secondarymetabolites(次级代谢生物合成，22个差异表达蛋白)”、“psoj00071 Fatty aciddegradation(脂肪酸降解，6个差异表达蛋白)”、“psoj00500 Starch and sucrosemetabolism(淀粉和糖代谢，4个差异表达蛋白)”、“psoj00010 Glycolysis/Gluconeogenesis(糖酵解糖质新生，6个差异表达蛋白)”和“psoj00460 Cyanoamino acidmetabolism(氰基氨基酸代谢，3个差异表达蛋白)”等主要八个代谢通路。我们沉默大豆疫霉的琥珀酸脱氢酶亚基A、B和延胡索酸还原酶均会使得大豆疫霉生长速率下降。
如图6所示，其中，蛋白XP_009534955.1、XP_009522539.1和XP_009527170.1参与调控长链脂酰辅酶A合成、乙酰辅酶A氧化和乙酰辅酶A脱氢氧化，三个关键蛋白的下调导致大豆疫霉脂肪代谢紊乱，以及相关细胞结构如细胞膜等完整性受损，图中阴影标识为蛋白下降通路。
另外如图7所示，蛋白XP_009526054.1、XP_009526052.1、XP_009526051.1和XP_009517993.1分别为糖苷水解酶、糖苷水解酶家族、糖苷水解酶和糖基转移酶，参与淀粉、糖和能量代谢过程，该通路关键蛋白翻译量下降会给大豆疫霉的生长带来负面影响，图中阴影标识为蛋白下降通路。
实施例2
所述没食子酸丙酯由没食子酸和丙醇在单宁酶的催化下制备，包括如下制备步骤：
(1)没食子酸提取工艺：采用超临界二氧化碳萃取方法，称取颗粒大小在2-5mm大小的蓝莓叶子或绿茶叶粉1.3-1.8kg，将其加入17-22％的温度为2-8℃的纯水中，在5℃条件下放置50-70分钟，超临界二氧化碳萃取中，超临界二氧化碳提取压力为23-27MPa，提取时间为32-50分钟，即可提取出没食子酸；
(2)单宁酶制备工艺：黑曲霉在液体土豆培养基中培养3-5天，过滤收集发酵液，用硫酸铵沉淀法沉淀，复溶提取粗蛋白，用截留分子量10kDa的超滤膜超滤去硫酸铵并浓缩蛋白，即得到单宁酶；
(3)没食子酸丙酯合成方法：将8.9mmol没食子酸溶于8.2％-13.6％丙醇溶液中，加入0.3-0.6mol的单宁酶在二氧化碳培养箱中，在20℃恒温条件下催化反应10-15小时，在-60℃、真空度为45的真空条件下冷冻干燥，待干燥至粉末，即获得没食子酸丙酯。
没食子酸丙酯还可采用超临界二氧化碳萃取方法，包括如下步骤：
(1)精确称取颗粒大小在2-5mm大小的赤芍粉1.3-1.8kg，将其加入17-22％的温度为6-8℃的纯水中，在5℃条件下放置50-70分钟，超临界二氧化碳萃取中，超临界二氧化碳提取压力为33-37MPa，提取时间为54-57分钟，二氧化碳流率为4.5-5.7L/min，即可提取出没食子酸丙酯粗提物；
(2)没食子酸丙酯纯化：将(3)中没食子酸丙酯粗提物按照1:5的质量比溶于85-90℃的热水中，用截留分子量10kDa的超滤膜超滤去大分子杂质；然后用旋转蒸发仪中浓缩至没食子酸丙酯含量大于20-50mg/L；
(3)结晶纯化与干燥：将(4)中的没食子酸丙酯转移到50ml的离心管中，4-6℃下放置12小时后有沉淀出现在离心管底部，在10000rpm/min下离心5分钟，去上清，液氮速冻沉淀后，在-60℃、真空度为45的真空条件下冷冻干燥，待干燥至粉末，即获得没食子酸丙酯。如表1所示，
表1.没食子酸丙酯提取工艺优化实验表
提取工艺是利用Box-Behnken试验模型，经实验，分析数据得回归方程：
Y＝10.7+0.89A+1.43B+0.94C+0.05AB+0.33AC+0.6BC-0.44A2-1.01B2-0.84C2；表1中A、B、C、Y分别代表提取压力，提取时间、CO2流率和没食子酸提取得率。根据方程模型以没食子酸提取得率最大值为指标，计算并验证得到超临界二氧化碳提取的最佳工艺为：提取压力为33-37MPa，提取时间为54-57分钟，二氧化碳流率为4.5-5.7L/min。
实施例3
溶剂的选择及抑制溶液的配制：以大豆疫霉抑制率为指标，从乙醇、丙醇、丁醇、戊醇、己醇、丙酮、二甲亚砜、乙酸乙酯、石油醚和癸醇等有机溶剂中，优选出用癸醇作为没食子酸丙酯的助溶试剂，其用量比例为：0.1mg-0.3mg的癸醇：1-2mmol的没食子酸丙酯，完全溶解后依次添加60-70℃的热水和室温水各500mL水，即可以用于大豆疫霉的防治。如表2所示
表2.溶剂种类及用量优化实验表
没食子酸丙酯还可以激发植物的防卫反应，提高抗性基因表达量抵抗大豆疫霉的侵染。按照takara试剂和提取mRNA反转录cDNA并按照qRT-PCR体系进行(25μL):2μL c DNA模板,12.5μL的SYBR green,8.5μL dd H2O和1μL引物.PCR程序为95℃、30s；39循环(95℃for 5s,60℃for30s)。
如图8所示，实时荧光定量PCR结果显示可以诱导大豆抗性基因表达量显著升高。PR3基因在处理后的12小时内表达量持续升高(最高达149倍)，对抵抗大豆疫霉侵染发挥主要作用，与未处理的大豆样品相同时间基因表达量之比，为便于比较并设未处理组表达量为1。“**”和“*”分别表示处理组，基因表达量较对照组在P<0.01和P<0.05下有显著性差异。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的仅为本发明的优选例，并不用来限制本发明，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (4)
1.一种大豆疫霉抑制剂，其特征在于：包括没食子酸丙酯和癸醇，癸醇作为没食子酸丙酯的助溶剂，其用量比例为：0.1mg-0.3mg的癸醇：1-2mmol的没食子酸丙酯。
2.根据权利要求1所述的一种大豆疫霉抑制剂，其特征在于：所述没食子酸丙酯采用超临界二氧化碳萃取方法，包括如下步骤：
(1)精确称取颗粒大小在2-5mm大小的赤芍粉1.3-1.8kg，将其加入17-22％的温度为6-8℃的纯水中，在5℃条件下放置50-70分钟，超临界二氧化碳萃取中，超临界二氧化碳提取压力为33-37MPa，提取时间为54-57分钟，二氧化碳流率为4.5-5.7L/min，即可提取出没食子酸丙酯粗提物；
(2)没食子酸丙酯纯化：将(3)中没食子酸丙酯粗提物按照1:5的质量比溶于85-90℃的热水中，用截留分子量10kDa的超滤膜超滤去大分子杂质；然后用旋转蒸发仪中浓缩至没食子酸丙酯含量大于20-50mg/L；
(3)结晶纯化与干燥：将(4)中的没食子酸丙酯转移到50ml的离心管中，4-6℃下放置12小时后有沉淀出现在离心管底部，在10000rpm/min下离心5分钟，去上清，液氮速冻沉淀后，在-60℃、真空度为45的真空条件下冷冻干燥，待干燥至粉末，即获得没食子酸丙酯。
3.根据权利要求1所述的一种大豆疫霉抑制剂，其特征在于：所述没食子酸丙酯还可由没食子酸和丙醇在单宁酶的催化下制备，包括如下制备步骤：
(1)没食子酸提取工艺：采用超临界二氧化碳萃取方法，称取颗粒大小在2-5mm大小的蓝莓叶子或绿茶叶粉1.3-1.8kg，将其加入17-22％的温度为2-8℃的纯水中，在5℃条件下放置50-70分钟，超临界二氧化碳萃取中，超临界二氧化碳提取压力为23-27MPa，提取时间为32-50分钟，即可提取出没食子酸；
(2)单宁酶制备工艺：黑曲霉在液体土豆培养基中培养3-5天，过滤收集发酵液，用硫酸铵沉淀法沉淀，复溶提取粗蛋白，用截留分子量10kDa的超滤膜超滤去硫酸铵并浓缩蛋白，即得到单宁酶；
(3)没食子酸丙酯合成方法：将8.9mmol没食子酸溶于8.2％-13.6％丙醇溶液中，加入0.3-0.6mol的单宁酶在二氧化碳培养箱中，在20℃恒温条件下催化反应10-15小时，在-60℃、真空度为45的真空条件下冷冻干燥，待干燥至粉末，即获得没食子酸丙酯。
4.一种大豆疫霉抑制剂，其特征在于：大豆疫霉抑制剂的制备方法为没食子酸丙酯与癸醇混合：将0.1mg-0.3mg的癸醇，1-2mmol的没食子酸丙酯混合，完全溶解后依次添加60-70℃的热水和20-25℃常温水各500mL，即可以得到大豆疫霉抑制剂。
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