CN111406795A - 一种黄盖小脆柄菇代谢物在制备黄曲霉菌抑菌剂中的应用 - Google Patents
一种黄盖小脆柄菇代谢物在制备黄曲霉菌抑菌剂中的应用 Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B9/00—Preservation of edible seeds, e.g. cereals
- A23B9/16—Preserving with chemicals
- A23B9/24—Preserving with chemicals in the form of liquids or solids
- A23B9/26—Organic compounds; Microorganisms; Enzymes
- A23B9/28—Microorganisms; Enzymes; Antibiotics
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- C12N1/14—Fungi; Culture media therefor
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- C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- 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
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Abstract
本发明公开了一种黄盖小脆柄菇代谢物在制备黄曲霉菌抑菌剂中的应用,所述黄盖小脆柄菇代谢物是将黄盖小脆柄菇发酵液超声破碎,过滤,滤液浓缩后用乙酸乙酯萃取,取乙酸乙酯层浓缩至恒重获得的。本发明提供一种黄盖小脆柄菇代谢物对黄曲霉菌抑菌活性以及用于储粮抑菌剂的应用。传统抑制霉菌制剂为制霉菌素等抗生素,残留危害较大,天然的抑菌剂将是未来市场的趋势,该抑菌剂产量大,工艺简单,更加安全,环保。
Description
(一)技术领域
本发明涉及一种黄曲霉抑菌剂,特别涉及一种黄盖小脆柄菇代谢物在制备黄曲霉菌抑菌剂中的应用。
(二)背景技术
植物内生菌是特指一类在其整个生活史或者生活史的部分阶段寄生于健康植物的部分细胞间隙、器官或者组织中,植物与其内生菌是共生体,而不使宿主植物出现任何反常症状或者病症的微生物。近年来,研究人员从银杏中分离出了具有多种生物活性的内生菌,其中部分银杏内生菌通过次生代谢途径可以产生与银杏次生代谢产物相似的产物。根据文献表明,从银杏中分离出的内生菌次级代谢产物有:黄酮类物质,银杏内酯类化合物,生长素类物质,抑菌物质,其他类物质如色素类物质、银杏多糖等,上述物质均有较好的药理作用。
以银杏果外种皮为研究材料分离该组织中的内生菌的报道极少,而该组织作为白果的一部分,其中可能存在着与白果种子相同的活性成分,而因组织不同其中所含有的内生菌种类也会大相径庭,可能分离得到在银杏其他组织中未被分离报道的微生物种类,而微生物来源的活性成分也是现今研究的热点;研究银杏果外种皮内生菌及其次级代谢产物可为银杏类活性物质的开发寻找新的途径;对内生菌及其代谢产物在抑制黄曲霉菌的研究及在粮储中应用具有十分重要的意义。
(三)发明内容
本发明目的是提供一种黄盖小脆柄菇代谢物在制备黄曲霉菌抑菌剂中的应用,能够抑制黄曲霉菌的生长,并且在稻谷储藏过程中控制黄曲霉毒素的产生。
本发明采用的技术方案是:
本发明提供一种黄盖小脆柄菇代谢物在制备黄曲霉菌抑菌剂中的应用,所述黄盖小脆柄菇代谢物是将黄盖小脆柄菇发酵液超声处理,使细胞破碎,过滤,滤液浓缩后用乙酸乙酯萃取,取乙酸乙酯层浓缩至恒重获得的。
进一步,所述黄盖小脆柄菇代谢物按如下方法制备:将黄盖小脆柄菇接种于含200mL发酵培养基的500mL锥形瓶中,28℃、180r/min培养7d,获得发酵液;将发酵液超声破碎后抽滤,取滤液经孔径为0.45μm微孔滤膜过滤后,微滤液利用旋转蒸发仪浓缩至原体积的1/3,用1倍体积乙酸乙酯萃取至乙酸乙酯相肉眼观察无明显颜色变化,合并乙酸乙酯萃取相,再次利用旋转蒸发仪浓缩至恒重,获得黄盖小脆柄菇代谢物;所述发酵培养基组成:硝酸钠3g/L,磷酸氢二钾1g/L,硫酸镁(MgSO4·7H2O)0.5g/L,氯化钾0.5g/L,硫酸亚铁0.01g/L,蔗糖30g/L,溶剂为蒸馏水,pH7.0~7.2。
进一步,所述黄盖小脆柄菇优选为黄盖小脆柄菇(Psathyrella candolleana)Gb.PY-F1,保藏于中国典型培养物保藏中心,保藏编号为:CCTCC M 2019125,保藏日期为2019年3月6日,保藏地址:中国武汉武汉大学,邮编:430072,已在专利申请CN110024696A中公开。
进一步,所述超声破碎条件为:在405W条件下,每工作3s,间歇4s,进行300次循环。
进一步,所述黄盖小脆柄菇发酵前先接种于PDA培养基中,置于28℃恒温培养箱中培养7d;再用无菌打孔器将黄盖小脆柄菇沿着菌落边缘打直径为5mm的菌饼,接种于发酵培养基;PDA培养基组成:马铃薯200g/L,葡萄糖20g/L,琼脂15~20g/L,溶剂为蒸馏水,自然pH。
进一步,所述抑菌剂是将黄盖小脆柄菇代谢物溶于丙酮中,即为抑菌剂溶液;所述丙酮体积用量以黄盖小脆柄菇代谢物重量计为10-20mL/g。
进一步,所述黄曲霉菌为黄曲霉菌(Aspergillus flavus)CGMCC 3.4408。
进一步,所述抑菌剂为储粮抑菌剂,所述储粮优选稻谷,所述储粮抑菌剂为0.1-0.5g/mL的黄盖小脆柄菇代谢物乙醇溶液,施用量为0.5升/吨。
与现有技术相比,本发明有益效果主要体现在:本发明提供一种黄盖小脆柄菇代谢物对黄曲霉菌(CGMCC 3.4408)抑菌活性以及用于储粮抑菌剂的应用。传统抑制霉菌制剂为制霉菌素等抗生素,残留危害较大,天然的抑菌剂将是未来市场的趋势,该抑菌剂产量大,工艺简单,更加安全,环保。
(四)具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
实施例1黄盖小脆柄菇CCTCC M 2019125代谢物
1、将保存在4℃冰箱中的黄盖小脆柄菇CCTCC M 2019125接种于PDA培养基中,置于28℃恒温培养箱中培养7d;PDA培养基组成:马铃薯200g,葡萄糖20g,琼脂15~20g,蒸馏水1000mL,自然pH。
2、在超净工作台中,用无菌打孔器将步骤1黄盖小脆柄菇CCTCC M 2019125沿着菌落边缘打直径为5mm的菌饼,接种于含200mL发酵培养基的500mL锥形瓶中,28℃,180r/min培养7d,获得发酵液;将发酵液在405W条件下,每工作3s,间歇4s,进行300次循环超声破碎后抽滤,取滤液经孔径为0.45μm微孔滤膜过滤后,微滤液利用旋转蒸发仪浓缩至原体积的1/3,用1倍体积乙酸乙酯萃取至乙酸乙酯相肉眼观察无明显颜色变化,合并乙酸乙酯萃取相,再次利用旋转蒸发仪浓缩至恒重,获得黄盖小脆柄菇CCTCC M 2019125代谢物3.2g。
所述发酵培养基组成:硝酸钠3g/L,磷酸氢二钾1g/L,硫酸镁(MgSO4·7H2O)0.5g/L,氯化钾0.5g/L,硫酸亚铁0.01g/L,蔗糖30g/L,溶剂为蒸馏水,pH7.0~7.2;利用高压蒸汽灭菌锅对其进行灭菌,条件为121℃,20分钟。
实施例2黄曲霉菌孢子悬液
将黄曲霉菌(Aspergillus flavus)CGMCC 3.4408(购自中国普通微生物菌种保藏管理中心)接种在PDA斜面培养基,28℃培养10d后,用接种针挑取斜面上的孢子,加入10mL含体积浓度0.05%吐温-80的0.2mol/L、pH6无菌磷酸盐缓冲液,通过纱布过滤以除去菌丝残体,并用0.2mol/L、pH6磷酸盐缓冲液将孢子浓度调整为1×107CFU/mL,即为黄曲霉菌孢子悬液。
实施例3黄盖小脆柄菇CCTCC M 2019125代谢物对黄曲霉菌的抑菌试验
1、取实施例1方法制备的黄盖小脆柄菇CCTCC M 2019125代谢物1.0g溶于10mL丙酮中,即为抑菌剂溶液。取1mL抑菌剂溶液加入平皿中,倒入15mL PDA培养基混匀,待凝固后在每个平板上用接种棒溶出3个直径为2.5mm的小洞,每个小洞分别加入0.01mL的实施例2制备的黄曲霉菌孢子悬液,28℃培养5天,观察抑菌圈直径大小,结果见表1。同样条件下,用丙酮代替抑菌剂溶液作为空白对照。
表1抑菌圈直径(mm)
2、将黄盖小脆柄菇CCTCC M 2019125代谢物1.0g用4mL无水乙醇溶解,获得抑菌剂乙醇溶液。按照0.5升/吨的施用量,均匀喷洒在稻谷表面,混合均匀,于20℃储存。一年之后,使用黄曲霉毒素酶联免疫试剂盒(深圳市博奥通科生物制品有限公司)检测稻谷中黄曲霉毒素含量,未检出黄曲霉毒素,未加抑菌剂的对照组检测黄曲霉毒素含量8.7μg/kg。
实施例4黄盖小脆柄菇CCTCC M 2019125代谢物对黄曲霉菌的抑菌试验
1、取实施例1方法制备的黄盖小脆柄菇CCTCC M 2019125代谢物1.0g溶于15mL丙酮中,即为抑菌剂溶液。取1mL抑菌剂溶液加入平皿中,倒入15mL PDA培养基混匀,待凝固后在每个平板上用接种棒溶出3个直径为2.5mm的小洞,每个小洞分别加入0.01mL的实施例2制备的黄曲霉菌孢子悬液,28℃培养5天,观察抑菌圈直径大小,结果见表2。同样条件下,用丙酮代替抑菌剂溶液作为空白对照。
表2抑菌圈直径(mm)
2、将黄盖小脆柄菇CCTCC M 2019125代谢物1.0g用5mL无水乙醇溶解,获得抑菌剂乙醇溶液。按照0.5升/吨的施用量,均匀喷洒在稻谷表面,混合均匀,于20℃储存。一年之后,使用黄曲霉毒素酶联免疫试剂盒(深圳市博奥通科生物制品有限公司)检测稻谷中黄曲霉毒素含量,未检出黄曲霉毒素,未加抑菌剂的对照组检测黄曲霉毒素含量8.7μg/kg。
实施例5黄盖小脆柄菇CCTCC M 2019125代谢物对黄曲霉菌的抑菌试验
1、取实施例1方法制备的黄盖小脆柄菇CCTCC M 2019125代谢物1.0g溶于20mL丙酮中,即为抑菌剂溶液。取1mL抑菌剂溶液加入平皿中,倒入15mL PDA培养基混匀,待凝固后在每个平板上用接种棒溶出3个直径为2.5mm的小洞,每个小洞分别加入0.01mL的实施例2制备的黄曲霉菌孢子悬液,28℃培养5天,观察抑菌圈直径大小,结果见表3。同样条件下,用丙酮代替抑菌剂溶液作为空白对照。
表3抑菌圈直径(mm)
2、将黄盖小脆柄菇CCTCC M 2019125代谢物1.0g用6mL无水乙醇溶解,获得抑菌剂乙醇溶液。按照0.5升/吨的施用量,均匀喷洒在稻谷表面,混合均匀,于20℃储存。一年之后,使用黄曲霉毒素酶联免疫试剂盒(深圳市博奥通科生物制品有限公司)检测稻谷中黄曲霉毒素含量,未检出黄曲霉毒素,未加抑菌剂的对照组检测黄曲霉毒素含量8.7μg/kg。
Claims (10)
1.一种黄盖小脆柄菇代谢物在制备黄曲霉菌抑菌剂中的应用。
2.如权利要求1所述的应用,其特征在于所述黄盖小脆柄菇代谢物是将黄盖小脆柄菇发酵液超声破碎,过滤,滤液浓缩后用乙酸乙酯萃取,取乙酸乙酯层浓缩至恒重获得的。
3.如权利要求1所述的应用,其特征在于所述黄盖小脆柄菇代谢物按如下方法制备:将黄盖小脆柄菇接种于发酵培养基中,28℃、180r/min培养7d,获得发酵液;将发酵液超声破碎后抽滤,取滤液经孔径为0.45μm微孔滤膜过滤后,微滤液浓缩至原体积的1/3,用乙酸乙酯萃取至乙酸乙酯相肉眼观察无明显颜色变化,合并乙酸乙酯萃取相,再次浓缩至恒重,获得黄盖小脆柄菇代谢物;所述发酵培养基组成:硝酸钠3g/L,磷酸氢二钾1g/L,硫酸镁0.5g/L,氯化钾0.5g/L,硫酸亚铁0.01g/L,蔗糖30g/L,溶剂为蒸馏水,pH7.0~7.2。
4.如权利要求1所述的应用,其特征在于所述黄盖小脆柄菇为黄盖小脆柄菇(Psathyrella candolleana)CCTCC M 2019125。
5.如权利要求1所述的应用,其特征在于所述超声破碎条件为:在405W条件下,每工作3s,间歇4s,进行300次循环。
6.如权利要求1所述的应用,其特征在于所述黄盖小脆柄菇发酵前先接种于PDA培养基中,置于28℃恒温培养箱中培养7d;再用无菌打孔器将黄盖小脆柄菇沿着菌落边缘打直径为5mm的菌饼,接种于发酵培养基;PDA培养基组成:马铃薯200g/L,葡萄糖20g/L,琼脂15~20g/L,溶剂为蒸馏水,自然pH。
7.如权利要求1所述的应用,其特征在于所述抑菌剂是将黄盖小脆柄菇代谢物溶于丙酮中,即为抑菌剂溶液;所述丙酮体积用量以黄盖小脆柄菇代谢物重量计为10-20mL/g。
8.如权利要求1所述的应用,其特征在于所述黄曲霉菌为黄曲霉菌(Aspergillusflavus)CGMCC 3.4408。
9.如权利要求1所述的应用,其特征在于所述抑菌剂为储粮抑菌剂。
10.如权利要求9所述的应用,其特征在于所述储粮为稻谷,所述储粮抑菌剂为0.1-0.5g/mL的黄盖小脆柄菇代谢物乙醇溶液。
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