CN112226370B - 一种狐粪青霉新菌株及其应用 - Google Patents
一种狐粪青霉新菌株及其应用 Download PDFInfo
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Abstract
本发明涉及一种狐粪青霉(Penicillium vulpinum)新菌株,该菌株已作为专利菌种保藏于中国微生物菌种保藏管理委员会普通微生物中心CGMCC,保藏编号:CGMCC NO:17187。该菌株对真菌具有显著的抑制效果。
Description
技术领域
本发明涉及一种狐粪青霉新菌株及其在抑菌方面的应用,属于微生物领域。
背景技术
真菌感染疾病是一类通过真菌感染动物的皮肤、粘膜、毛发以及手指甲和脚趾甲所导致的一类皮肤性疾病,由真菌感染导致的皮肤病占所有皮肤病的25%左右。由于抗生素的广泛使用,使得皮肤真菌发病率不断升高。
目前致病的皮肤真菌约有20多种,常见的主要包括红色毛癣菌、小孢子菌、念珠菌、毛霉菌等。而红色毛癣菌是浅表性致病菌,一般的体癣、股癣和手足癣的致病均为红色毛癣菌所致。由于真菌耐药性的增加,亟需找到一种新的广谱性抗菌物质。
发明内容
为解决现有技术存在的问题,本发明提供一种狐粪青霉新菌株,该菌株具有易于培养、产量高、培养性状稳定等的特点,其培养物具有显著的抑菌功效。
作为本发明的一个方面,本发明提供一种狐粪青霉新菌株,其分类命名为Penicillium vulpinum,该菌株已作为专利菌种保藏于位于我国北京的中国微生物菌种保藏管理委员会普通微生物中心,保藏编号:CGMCC NO:17187,保藏日期:2019年2月28日。
经中国科学院微生物研究所形态特征鉴定,本发明狐粪青霉菌CGMCC No.17187的主要形态特征为:
在马铃薯葡萄糖培养基及麦芽汁培养基上菌落直径为20-23mm,菌落边缘呈现白色,中部呈现灰绿色,平铺,气生菌丝稀少;质地颗粒状,孢梗束大量产生,长约2-5mm,菌丝体红褐色,背面浅黄褐色,色素不溶于培养基中。分生孢子结构大量产生于孢梗束顶端,帚状枝多见三轮生,叉开。分生孢子梗短,波浪状弯曲,壁光滑,无色,在孢梗束上排列紧密;瓶梗柱状,颈部短,5.0-9.3×2.0-3.0μm;分生孢子椭球形或近球形,光滑,串生,2.5-4.0μm。
本发明的狐粪青霉菌CGMCC NO.17187的ITS序列包含如SEQ ID NO:1所示的核苷酸序列。
作为本发明的第二个方面,本发明提供所述狐粪青霉菌CGMCC NO.17187在制备具有抑制真菌功效的药物或保健品中的应用。所述真菌包括红色毛癣菌、小孢子菌、念珠菌、毛霉菌。
作为本发明的第三个方面,本发明提供一种狐粪青霉菌培养物,所述培养物为狐粪青霉菌CGMCC NO.17187经液体培养获得的培养物。
其中,所述液体培养包括摇瓶培养、种子罐培养、发酵罐培养中的任意一种或几种方法的组合;所述液体培养时间为9-12天。所述液体培养应用的培养基为本领域常规的液体培养基,如PSA培养基、PDA培养基、PDY培养基、PSB培养基、酵母浸出粉-复合氨基酸-蔗糖培养基,酵母浸出粉-白砂糖-大豆水解蛋白培养基,麸皮煮汁-白砂糖培养基等;优选为PDA培养基。
作为本发明的第四个方面,本发明提供一种狐粪青霉菌提取物,所述提取物为狐粪青霉菌CGMCC NO.17187的液体培养物经有机溶剂提取获得提取物。
其中,所述液体培养包括摇瓶培养、种子罐培养、发酵罐培养中的任意一种或几种方法的组合;所述液体培养应用的培养基为本领域常规的液体培养基,如PSA培养基、PDA培养基、PDY培养基、PSB培养基、酵母浸出粉-复合氨基酸-蔗糖培养基,酵母浸出粉-白砂糖-大豆水解蛋白培养基,麸皮煮汁-白砂糖培养基等;优选为PDA培养基。所述有机溶剂选自甲醇、乙醇、乙酸乙酯、氯仿、乙醚、石油醚、丙酮中的任意一种。优选为甲醇或乙酸乙酯。
本发明的有益效果:本发明狐粪青霉CGMCC NO.17187菌株的液体培养物对于红色毛癣菌、小孢子菌等真菌具有显著的抑制作用,其抑菌率能达到100%,抑菌效果显著优于市售抑菌产品达克宁和酮康唑,与盐酸特比萘芬相当。
附图说明
图1为狐粪青霉CGMCC NO.17187菌株9天发酵液甲醇和乙酸乙酯提取物对红色毛癣菌的抑制作用。
图2为狐粪青霉CGMCC NO.17187菌株9天发酵液甲醇和乙酸乙酯提取物对石膏样小孢子菌的抑制作用。
图3为狐粪青霉CGMCC NO.17187菌株12天发酵液甲醇和乙酸乙酯提取物对红色毛癣菌的抑制作用。
图4为狐粪青霉CGMCC NO.17187菌株12天发酵液甲醇和乙酸乙酯提取物对石膏样小孢子菌的抑制作用。
图5为狐粪青霉CGMCC NO.17187菌株9天发酵液甲醇、乙酸乙酯提取物与达克宁和盐酸特比奈芬溶液的抑菌比较实验。
生物材料保藏信息
本发明狐粪青霉(Penicillium vulpinum)新菌株,已作为专利菌种保藏于位于我国北京的中国微生物菌种保藏管理委员会普通微生物中心CGMCC,保藏编号:CGMCC NO:17187,保藏日期:2019年2月28日。
具体实施方式
实施例1狐粪青霉菌CGMCC No.17187的主要形态特征
将狐粪青霉CGMCC No.17187菌株接于马铃薯葡萄糖培养基上,24℃培养十天,菌落直径为20-23mm,菌落边缘呈现白色,中部呈现灰绿色,平铺,气生菌丝稀少;质地颗粒状,孢梗束大量产生,长约2-5mm,菌丝体红褐色,背面浅黄褐色,色素不溶于培养基中。分生孢子结构大量产生于孢梗束顶端,帚状枝多见三轮生,叉开。分生孢子梗短,波浪状弯曲,壁光滑,无色,在孢梗束上排列紧密;瓶梗柱状,颈部短,5.0-9.3×2.0-3.0μm;分生孢子椭球形或近球形,光滑,串生,2.5-4.0μm。
实施例2狐粪青霉菌CGMCC No.17187的液体培养
菌株的活化:将保藏的狐粪青霉划线接种于PDA斜面培养基上,置于26±2℃培养箱中培养3-5天;
种子液配制:配制200ml/瓶的PDA液体培养基,将生长好的斜面直接接入液体摇瓶,置于26±2℃,180rpm摇床中培养两天,制备种子液;
液体扩大培养:将制备好的种子液分别取40ml加入到新的200ml PDA培养基中,置于26±2℃,180rpm摇床中继续培养,扩大培养7-10天,取出,即得液体培养物(即发酵液)。
所述PDA培养基配方:200g土豆煮汁,加入20g葡萄糖,定容至1L,再加入20g琼脂粉。
实施例3狐粪青霉CGMCC No.17187菌株甲醇提取物的制备
取实施例2制备的发酵液,水浴加热至体积达到原来的1/5,加入等体积的甲醇,常温静置提取2h,或者60℃温浴提取1h。取上清液进行蒸发,产物加入原液3%量的甲醇溶解(100ml发酵液加入3ml甲醇),得甲醇提取物。
实施例4狐粪青霉CGMCC No.17187菌株乙酸乙酯提取物的制备
取实施例2制备的发酵液,水浴加热至体积达到原来的1/5,分别加入等体积的乙酸乙酯,常温静置提取2h,或者60℃温浴提取1h。取上清液进行蒸发,产物加入原液3%量的乙酸乙酯溶解(100ml发酵液加入3ml乙酸乙酯),得乙酸乙酯提取物。
实施例5狐粪青霉CGMCC No.17187菌株9天发酵液甲醇和乙酸乙酯提取物对红色毛癣菌的抑制作用
取发酵9天的狐粪青霉发酵液(按实施例2方法制备),并制备甲醇提取液和乙酸乙酯提取液(分别按实施例3、4方法制备),获得提取液后采用打孔法对红色毛癣菌进行抑菌实验。实验设置五个组,分别为:空白对照组(不加入任何试剂)、甲醇溶液组(加入甲醇溶液)、酮康唑组(加入2mg/ml的酮康唑溶液)、发酵液甲醇提取物组和发酵液乙酸乙酯提取物组。
实验结果:涂有甲醇溶液的平板红色毛癣菌会生长,且生长状况与空白对照相比无异,说明甲醇对红色毛癣菌的生长无明显的抑制作用。2mg/ml的酮康唑溶液可以抑制红色毛癣菌的生长速度,使其生长缓慢。而涂有甲醇和乙酸乙酯提取液的平板上,红色毛癣菌基本没有生长,说明采用本发明狐粪青霉菌的甲醇和乙酸乙酯提取液能抑制红色毛癣菌的生长,且效果明显。在统计的十天内,狐粪青霉发酵液对红色毛癣菌的抑制效果达到100%。实验结果见图1。
实施例6狐粪青霉CGMCC No.17187菌株9天发酵液甲醇和乙酸乙酯提取物对石膏样小孢子菌的抑制作用
取发酵9天的狐粪青霉发酵液(按实施例2方法制备),并制备甲醇提取液和乙酸乙酯提取液(分别按实施例3、4方法制备),获得提取液后采用打孔法对石膏样小孢子菌进行抑菌实验。分组同实施例5。
实验结果显示石膏样小孢子菌在涂有甲醇提取液和乙酸乙酯提取液的平板并没有生长,即使在持续了12天的平板上也没有进行生长。2mg/ml的酮康唑溶液抑制了石膏样小孢子菌的生长速度。狐粪青霉9天发酵液对石膏样小孢子菌的抑制效果达到100%。实验结果见图2。
实施例7狐粪青霉CGMCC No.17187菌株12天发酵液甲醇和乙酸乙酯提取物对红色毛癣菌的抑制作用
取发酵12天的狐粪青霉发酵液(按实施例2方法制备),并制备甲醇提取液和乙酸乙酯提取液(分别按实施例3、4方法制备),获得提取液后采用打孔法对红色毛癣菌进行抑菌实验。分组同实施例5。
实验结果:酮康唑溶液可以抑制红色毛癣菌的生长速度,使其生长缓慢。而涂有甲醇和乙酸乙酯提取液的平板上,在生长前10天的时候红色毛癣菌基本没有生长,直到第12天的时候菌丝体才开始生长,说明采用本发明狐粪青霉菌的甲醇和乙酸乙酯提取液能抑制红色毛癣菌的生长,且效果明显。其抑制效果在5天内达到100%。实验结果图见图3
实施例8狐粪青霉CGMCC No.17187菌株12天发酵液甲醇和乙酸乙酯提取物对石膏样小孢子菌的抑制作用
取发酵12天的狐粪青霉发酵液(按实施例2方法制备),并制备甲醇提取液和乙酸乙酯提取液(分别按实施例3、4方法制备),获得提取液后采用打孔法对石膏样小孢子菌进行抑菌实验。分组同实施例5。
实验结果显示石膏样小孢子菌在涂有甲醇提取液的平板上抑菌天数只持续了四天,而到第五天的时候菌丝体开始生长。在涂有乙酸乙酯提取液的平板直到第七天才开始生长,说明12天发酵液提取物的抑菌效果与9天发酵液提取物的效果相比较差。
狐粪青霉12天发酵液乙酸乙酯提取液对石膏样小孢子菌的抑制效果在5天内达到100%,超过五天后,抑制率下降,但仍然保持在90%以上。而甲醇提取物的抑制与乙酸乙酯提取物相比,其抑菌能力下降,从第5天开始,甲醇提取液的抑制作用开始下降,但其抑制作用仍然维持在70%以上。实验结果见图4。
实施例9狐粪青霉菌CGMCC No.17187发酵液甲醇和乙酸乙酯提取物与达克宁和盐酸特比奈芬溶液的抑菌比较实验
为了进一步确定狐粪青霉提取物所具有的抑菌效果,我们购买了市场上流通的两种抑菌药品,主要包括达克宁和盐酸特比萘芬溶液,以及纯品酮康唑进行实验。
取发酵9天的狐粪青霉发酵液(按实施例2方法制备),并制备甲醇提取液和乙酸乙酯提取液(分别按实施例3、4方法制备),获得提取液后采用打孔法对石膏样小孢子菌进行抑菌实验。
实验结果发现,在分别涂上酮康唑溶液、达克宁软膏以及盐酸特比萘芬溶液的平板上,生长7天后0.2%和1%的酮康唑平板上开始有菌丝体生长,同时达克宁平板上也有菌丝体可以生长。而涂有盐酸特比萘芬溶液和发酵9天狐粪青霉乙酸乙酯提取液的平板上石膏样小孢子菌并没有生长,说明发酵9天狐粪青霉乙酸乙酯的提取液其抑菌效果与盐酸特比萘芬溶液的抑菌效果相当,均达到了100%,显著优于酮康唑和达克宁。实验结果见图5,统计结果见表1。
表1 不同溶液对石膏样小孢子菌的抑菌效果比较
不同的溶液 | 抑菌率 |
0.2%酮康唑溶液 | 85% |
1%酮康唑溶液 | 95% |
达克宁 | 84% |
盐酸特比萘芬 | 100% |
9天狐粪青霉乙酸乙酯提取液 | 100% |
序列表
<110> 浙江泛亚生物医药股份有限公司
<120> 一种狐粪青霉新菌株及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 411
<212> DNA
<213> 狐粪青霉菌(Penicillium vulpinum)
<400> 1
gtaccaattg acaacttact aacttgatta caggcagacc atttctggcg agcacggtct 60
cgatggtgat ggacagtaag ttaatattga tggattatcc agtggaccac aagtctgatg 120
tcttgctagg tacaatggta cctccgacct ccagctcgag cgtatgaacg tctacttcaa 180
cgaagtgagt acaatgactg ggacccggat aatcgtgcat caactgatca gatccttttc 240
tttgataatc taggccaacg gtgacaagta cgttccccgt gccgttctcg tcgatttgga 300
gcccggtacc atggacgctg tccgctccgg tcccttcggc aagcttttcc gccccgacaa 360
cttcgtcttc ggtcagtccg gtgccggtaa caactgggcc aagggtcact a 411
Claims (7)
1.一种狐粪青霉(Penicillium vulpinum)菌株,该菌株已作为专利菌种保藏于中国微生物菌种保藏管理委员会普通微生物中心CGMCC,保藏编号:CGMCC NO:17187。
2.如权利要求1所述的狐粪青霉菌株,其特征在于,所述菌株的ITS序列包含如SEQ IDNO:1所示的核苷酸序列。
3.如权利要求1所述的狐粪青霉菌株在制备具有抑制真菌功效的药物中的应用。
4.如权利要求3所述的应用,其特征在于,所述真菌包括红色毛癣菌、小孢子菌、念珠菌或毛霉菌。
5.一种狐粪青霉菌培养物,其特征在于,所述培养物为狐粪青霉菌CGMCC NO.17187经液体培养获得的培养物。
6.如权利要求5所述的培养物,其特征在于,所述液体培养包括摇瓶培养、种子罐培养、发酵罐培养中的任意一种或几种方法的组合。
7.如权利要求5所述的培养物,其特征在于,所述液体培养时间为9-12天。
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RU2612150C2 (ru) * | 2015-06-09 | 2017-03-02 | Федеральное бюджетное учреждение науки Государственный научный центр прикладной микробиологии и биотехнологии (ФБУН ГНЦ ПМБ) | Штамм микромицета Penicillium vulpinum F-1523, обладающий антибактериальной активностью в отношении возбудителя сибирской язвы Bacillus anthracis |
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RU2612150C2 (ru) * | 2015-06-09 | 2017-03-02 | Федеральное бюджетное учреждение науки Государственный научный центр прикладной микробиологии и биотехнологии (ФБУН ГНЦ ПМБ) | Штамм микромицета Penicillium vulpinum F-1523, обладающий антибактериальной активностью в отношении возбудителя сибирской язвы Bacillus anthracis |
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