CN116211936A - 一种基于微生物的抗菌发酵液、其制备方法和在促进皮肤修复或伤口愈合中的应用 - Google Patents
一种基于微生物的抗菌发酵液、其制备方法和在促进皮肤修复或伤口愈合中的应用 Download PDFInfo
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Abstract
本发明公开一种基于微生物的抗菌发酵液、其制备方法和在促进皮肤修复或伤口愈合中的应用,制备包括步骤:配置生产水,过滤、灭菌,接种菌种后静态发酵,过滤得到抗菌发酵液;菌种为棘孢木霉(Trichoderma asperellum),于2022年12月5日保藏于中国典型培养物保藏中心,保藏编号为CCTCCNO:M20221879;生产水包括柴胡、茵陈、茯苓、山萸肉、枸杞、大枣、夜交藤、干姜、玉竹、怀山药、太子参、桔梗、黄芩、生地、决明子、牛膝、丹参中一种或多种中药成分。该抗菌发酵液兼具抑菌、促进成纤维细胞的增殖和迁移、促进伤口愈合等多种功能。
Description
技术领域
本发明涉及微生物发酵工程和医疗领域,具体涉及一种基于微生物的抗菌发酵液、其制备方法和在促进皮肤修复或伤口愈合中的应用。
背景技术
随着科技的不断发展和物质文化水平的提高,人们对生活质量有了更高的要求。日常生活中,公共场所的公共设施表面很难避免细菌贴附,增加细菌感染性疾病风险,细菌性感染所引起的疾病对人们健康构成巨大威胁。在医疗卫生领域,医疗器械表面的细菌会增加患者二次感染的风险,导致疾病治愈难度增加。抗菌剂可杀死细菌或抑制细菌增殖,抗菌剂的有效使用是减少细菌性感染的有效途径。因此,抗菌剂的研发被国内外研究学者广泛关注。
目前,已有的抗菌剂按照形态可分为固体粉体抗菌剂、液体抗菌剂。固体粉末抗菌剂多被作为添加剂添加于材料中,以赋予材料抗菌功能。液体抗菌剂(抗菌液)具有作用速度快、性质稳定、可在常温下使用等优点,可应用于不同环境和场合。液体抗菌剂通常含有抗菌纳米粒子,含纳米粒子的抗菌液的抗菌性能取决于纳米粒子在液体中的分散性和分散稳定性。在日常储存和运输过程中往往会导致纳米粒子的分散性变差,从而降低抗菌效果。不含纳米粒子的抗菌液,表现出较好的抗菌稳定性。
通过微生物发酵可获得天然液体抗菌剂,真菌作为微生物中庞大的家族之一,其本身及其次级代谢产物可表现出抗菌、抗炎、抗肿瘤、抗病毒等一系列生物活性。利用真菌发酵所得抗菌发酵液体无任何化学成分添加,符合绿色环保理念,满足人们对天然绿色产品的需求,具有较好的应用前景。
专利CN 114680106 A公开了一种抗菌液及其制备方法和应用、抗菌制品,所述抗菌液中包含改性聚乙烯醇、全氟烷基季铵盐、纳米抗菌剂、有机助剂多种成分,具有较高的抗菌效果、分散性稳定性和耐水性,但其含有多种化学制剂、成分复杂,功能单一。
专利CN 107320579公开了一种天然抗菌液及其配制方法,该抗菌液的抗菌抑菌效果明显,无毒副作用。但其由多种草本提取物配制而成,成分复杂,且含有乙醇。
专利CN 113057995 A公开了一种用于皮肤消毒的植物抗菌液,该抗菌液包括山苍子或山苍子挥发油、鸡肝散货鸡肝散挥发油、野坝子或野坝子挥发油、芫荽或芫荽挥发油,具有安全有效,能杀灭金黄色葡萄球菌、大肠杆菌、白念珠菌等性能,但其每份所含成分种类较多,且功能单一。
发明内容
基于真菌及其次级代谢产物的多样的生物活性优势及现有技术中此类抗菌剂的研究不足,本发明提供一种基于微生物的抗菌发酵液,该抗菌发酵液由天然微生物发酵获得,属于天然来源。通过抗菌试验验证其对革兰氏阴性菌大肠杆菌和革兰氏阳性菌金黄色葡萄球菌均表现出抑菌/杀菌活性。通过体外细胞实验,证明该抗菌发酵液可促进成纤维细胞增殖、迁移,从而促进皮肤组织的再生。此外,动物实验证明该抗菌发酵液可促进伤口愈合。本发明所述基于微生物的抗菌发酵液在抗菌、皮肤修复、促伤口愈合等多领域中表现出较好的应用前景。
为实现上述目的,本发明采用的技术方案是:
一种基于微生物的抗菌发酵液的制备方法,其特征在于,包括步骤:
步骤1,配置生产水、过滤并灭菌处理;
步骤2,在灭菌后的生产水中接种菌种,静态发酵,过滤得到所述抗菌发酵液;
所述菌种为棘孢木霉(Trichoderma asperellum),命名为RPT1,于2022年12月5日保藏于中国典型培养物保藏中心,保藏编号为CCTCCNO:M20221879。
所述生产水包括柴胡、茵陈、茯苓、山萸肉、枸杞、大枣、夜交藤、干姜、玉竹、怀山药、太子参、桔梗、黄芩、生地、决明子、牛膝、丹参中一种或多种中药成分。
优选地,所述生产水为中药液和水的混合液,所述生产水里中药液与水的体积比为1~10:1000;
所述中药液为中药成分通过文武火煎煮后所得;具体地,是将中药成分混合研磨后装袋,置于煎药机内,加水寖泡后,再使用文武火煎煮后得到中药液。
所用水包括山泉水、去离子水、超纯水、蒸馏水中任一种或多种。本发明中的发酵液需选用无污染的水源,山泉水取自山区泉眼产生的天然水,无污染。去离子水和超纯水均已去除了离子杂质,因此采用上述水制备生产水可减少发酵液中的杂质含量,进而减少对皮肤的伤害。
优选地,所述中药成分选自如下四种组成之一:
组成一为柴胡,茵陈,茯苓,山萸肉,枸杞和大枣;
组成二为夜交藤,玉竹,干姜,枸杞和大枣;
组成三为怀山药,太子参,桔梗,黄芩,枸杞和大枣;
组成四为生地,决明子,牛膝,丹参,枸杞和大枣。
柴胡,含有挥发油、皂苷、黄酮、多元醇、多糖、香豆素和微量元素等成分,具有驱散退热、疏肝利胆、疏气解郁等功效。
茵陈,含有香豆素、挥发油和黄酮等成分,具有清热利湿、保肝、降血脂、扩张冠脉、促纤溶、降血压、抗炎、消炎等功效。
茯苓,含有挥发油、萜类化合物、多糖类等成分,具有利尿消肿、保肝、健脾止泻、补益心脾、宁心安神等功效。
山萸肉,含有环烯醚萜、鞣质、黄酮、三萜、芳香酚酸等成分,具有肝肾保护、抗糖尿病、抗氧化、滋补等功效。
夜交藤,含有蒽醌、黄酮等成分,具有养血安神、祛风通络等功效。
玉竹,含有多糖、甾体皂苷、黄酮、挥发油等成分,具有抗肿瘤、抗氧化、抗疲劳、延缓皮肤衰老等药理作用。
干姜,含姜辣烯酮、姜辣醇、姜酮等成分,具有温中逐寒、回阳通脉等功效。
怀山药,含有糖类、糖脂类、脂肪酸类等成分,具有健脾、补肾、润肺止咳等功效。
太子参,含有环肽类、苷类、糖类、氨基酸类和微量元素等成分,
桔梗,含有黄酮、多酚、皂苷、多糖、钙、磷、铁等矿物质成分,具有抗氧化、抗肿瘤和抗炎等功效。
黄芩,含有黄酮、黄酮苷、锌、铁等微量元素成分,具有抗炎、增强免疫等功效。
生地,含有鞣质类、植物甾醇、香豆素类、糖类、蒽醌类等成分,具有散瘀消肿、清热凉血等功效。
决明子,含有蒽醌类、萘并吡喃酮类、苷类、脂肪酸类、多糖等成分,具有明目、降压调脂、抗菌、抗氧化等功效。
牛膝,含有甾酮、黄酮、皂苷、多糖等成分,具有抗炎、抗肿瘤、抗衰老等功效。
丹参,含有酚酸、参酮和多糖等成分,具有抗炎、抗氧化等功效。
枸杞,含有生物碱、黄酮、多糖等成分,具有保肝、抗氧化、抗炎等功效。
大枣,含有氨基酸、维生素、多糖和钙、铁、钾等微量元素成分,具有增强免疫、抑制癌细胞增值等功效。
本发明所选菌株为棘孢木霉,棘孢木霉可产生酮类、萜类、多肽类等抗菌物质,本发明所选择生产水含有中药成分,所选中药含有多糖和微量元素等营养成分和生物碱、黄酮等抗菌成分,利用多种中药中有效成分的相互补充,可为棘孢木霉生长提供营养成分,并促进棘孢木霉产生具有抗菌活性的酮类和肽类化合物。同时本发明所选生产水中含有生物碱、黄酮等抗菌成分类,发酵过程中可避免细菌污染。从而获得抗菌效果优异,具有促进皮肤修复效果的发酵液。本发明所选组合物中主要功效组分是天然来源,易于获取且安全温和,同时所用中药组分价格低廉。
进一步优选地,所述中药成分选自如下四种组成之一:
组成一为柴胡0.1~1Kg,茵陈0.1~1Kg,茯苓0.1~1Kg,山萸肉0.1~1Kg,枸杞0.1~1Kg,大枣0.1~1Kg;
组成二为夜交藤0.1~1Kg,玉竹0.1~1Kg,干姜0.1~1Kg,枸杞0.1~1Kg,大枣0.1~1Kg;
组成三为怀山药0.1~1Kg,太子参0.1~1Kg,桔梗0.1~1Kg,黄芩0.1~1Kg,枸杞0.1~1Kg,大枣0.1~1Kg;
组成四为生地0.1~1Kg,决明子0.1~1Kg,牛膝0.1~1Kg,丹参0.1~1Kg,枸杞0.1~1Kg,大枣0.1~1Kg。
进一步优选地,所述中药成分选自如下四种组成之一:
组成一为柴胡1Kg,茵陈1Kg,茯苓1Kg,山萸肉1Kg,枸杞1Kg,大枣1Kg;
组成二为夜交藤1Kg,玉竹1Kg,干姜1Kg,枸杞1Kg,大枣1Kg;
组成三为怀山药1Kg,太子参1Kg,桔梗1Kg,黄芩1Kg,枸杞1Kg,大枣1Kg;
组成四为生地1Kg,决明子1Kg,牛膝1Kg,丹参1Kg,枸杞1Kg,大枣1Kg。
步骤2中,所述混合菌种的接种体积浓度为10-20%,接种太多,会影响发酵速度;接种太少,会影响发酵时间。
步骤2中接种混合菌种后再25-37℃下发酵7-21天;
步骤1或2中过滤采用孔径为0.22微米的滤膜过滤,过滤掉发酵液中的真菌孢子。
本发明还提供一种根据所述的方法制得的抗菌发酵液。该抗菌发酵液由天然微生物发酵获得,属于天然来源,通过抗菌试验验证其对革兰氏阴性菌大肠杆菌和革兰氏阳性菌金黄色葡萄球菌均表现出抑菌/杀菌活性。
发明人进一步研究发现所述抗菌发酵液还可促进成纤维细胞的增殖和迁移,同时具有促进皮肤修复或伤口愈合的作用,兼具多重功能,可用作皮肤表面、物体表面杀菌,也可作为活性成分添加于抗菌、护理、护肤等产品中,具有广阔的应用前景。
本发明还提供所述的抗菌发酵液在制备促进皮肤修复或伤口愈合药物中的应用。
与现有技术相比,本发明具有以下有益效果:
本发明的抗菌发酵液采用混合菌种在中药成分中发酵而成,无任何化学成分添加,属于天然、绿色产品。依据试验数据来看,本发明所制备的发酵液,不仅能够抑制革兰氏阴性菌大肠杆菌和革兰氏阳性菌金黄色葡萄球菌的生长,还可促进成纤维细胞的增殖和迁移,同时又可促进伤口愈合,兼具多重功能,可用作皮肤表面、物体表面杀菌,也可作为活性成分添加于抗菌、护理、护肤等产品中,具有广阔的应用前景。
附图说明
图1为实施例1发酵液和对照组对大肠杆菌的抑菌结果图。
图2为实施例1发酵液和对照组金黄色葡萄球菌的抑菌结果图。
图3为对比例1发酵液对大肠杆菌和金黄色葡萄球菌的抑菌结果图。
图4为实施例1发酵液和对照组对物体表面大肠杆菌的杀菌作用图。
图5为实施例1发酵液和对照组对物体表面金黄色葡萄球菌的杀菌作用图。
图6为实施例1发酵液和对照组对成纤维细胞增值作用图。
图7为实施例1发酵液和对照组对成纤维细胞迁移作用图。
图8为实施例1发酵液和对照组对小鼠伤口愈合作用图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例和对比例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅用以解释本发明,并不用于限定本发明。在不脱离真菌发酵原理的前提下,还可以作出诸多修改和变化,这些改进和变化均应涵盖在本发明的保护范围内。
以下具体实施方式中棘孢木霉菌采用如下方法进行分离和鉴定,其他原料均购于市场,未经处理直接使用。
棘孢木霉菌的分离和检定
(1)真菌分离纯化
从东北小兴安岭的白色云母石分离得到所述棘孢木霉菌,将取自东北小兴岭的白色云母石使用无菌水冲洗2~3次,置于液体培养基中,28℃振荡培养24小时,取100μL菌液均匀涂布于于固体培养基平板上,25~28℃条件下倒置培养3~7天后,观察菌落形态,并根据不同菌落形态对真菌进行分离纯化。
其中,液体培养基为PDB培养基,配方为:马铃薯浸粉5g/L,葡萄糖20g/L,氯霉素0.1g/L,余量为去离子水。
固体培养基为PDA培养基,配方为:马铃薯浸粉5g/L,葡萄糖20g/L,氯霉素0.1g/L,琼脂粉15g/L,余量为去离子水。
(2)所述棘孢木霉菌的鉴定
菌落形态特征:所述棘孢木霉菌株在PDA培养基上生长较快,生长3~5天左右便可长满整个培养板,菌落表面产生短绒毛状菌丝,颜色为白色,生长5~8天后,菌落变为绿色,分生孢子梗细长,分生孢子为绿色、球形和近球形。
分子生物学鉴定
利用PCR技术,测定所述菌株的18S sDNA/ITS序列,并通过NCBI(https://wwwjicbi.nlm.nih.gov/)上的BLAST程序进行对比,结果显示所述菌为棘孢木霉(Trichoderma asperellum),命名为RPT1,于2022年12月5日保藏于中国典型培养物保藏中心,地址:中国.武汉.武汉大学,保藏编号为CCTCCNO:M20221879;
实施例1
一种抗菌发酵液的制备过程:
1.称取柴胡、茵陈、茯苓、山萸肉、枸杞、大枣,各1Kg;
2.将上述中药研磨至50目细粉,并装袋置于煎药机内,加水100L,寖泡3~5小时后,使用文武火煎煮各1小时后,滤出得到中药液;
3.将上述中药液与山泉水以1:1000体积比混合,孔径为0.22微米滤膜过滤灭菌,经得到生产水;
4.将棘孢木霉菌种接种到已灭菌的生产水中,并反复吹打,以确保真菌菌液均匀分散于生产水中,接种体积浓度为10~20%;
5.再将接种棘孢木霉菌种的生产水静态发酵,发酵温度为25~28℃,发酵时间为21天;
6.将发酵所得发酵液用孔径为0.22微米滤膜过滤,即可得到抗菌发酵液。
实施例2
同上述实施例1,其区别在于:步骤(1)中称取柴胡、茵陈、茯苓、山萸肉、枸杞、大枣,各0.1Kg;步骤(3)中药液与山泉水的体积比为10:1000;
实施例3
同上述实施例1,其区别在于:步骤(1)中称取夜交藤,玉竹,干姜,枸杞,大枣;各1Kg;
实施例4
同上述实施例1,其区别在于:步骤(1)中称取怀山药、太子参、桔梗、黄芩、枸杞、大枣,各1Kg;
实施例5
同上述实施例1,步骤(1)中称取生地、决明子、牛膝、丹参、枸杞、大枣,各1Kg;
实施例6
同上述实施例1,其区别在于:第5步中发酵温度为28~30℃,发酵时间为21天。
实施例7
同上述实施例1,其区别在于:第5步中发酵温度为30~35℃,发酵时间为14天。
实施例8
同上述实施例1,其区别在于:发酵温度为37℃,发酵时间为7天。
对比例1
按照实施例1的方法,区别在于,将山泉水替换为人工海水,制得抗菌发酵液。所述人工海水配方为:氯化钠24.53g/L,氯化镁5.2g/L,硫酸钠4.09g/L,氯化钙1.16g/L,氯化钾0.695g/L,碳酸氢钠0.201g/L,溴化钾0.101g/L,硼酸0.027g/L,氯化锶0.025g/L,氟化钠0.003g/L,余量为去离子水,溶液pH值为8.2(使用氢氧化钠调节pH值)。
应用例以下测试以实施例1和对比例1制备的抗菌液进行性能测试,结果如下:
1、抗菌性能测试
悬液抗菌测试,测试菌种为大肠杆菌和金黄色葡萄球菌,方法如下:
先将已灭菌的磷酸盐缓冲液(对照组)和实施例1和对比例1所制备抗菌发酵液(实验组)分别置于超净台中,并在紫外灯下照射30min杀灭容器表面的细菌。取处于对数生长期的大肠杆菌和金黄色葡萄球菌的菌液,并将菌液光密度(Optical Density,OD)值调整到0.1。取OD值为0.1的上述菌液100μL,分别用磷酸盐缓冲液和抗菌发酵液稀释至10mL。放入37℃的烘箱中培育0和3小时后涂板,其抗菌性能如图1-2所示,可以看到,图1中为大肠杆菌的对比情况,a、c为对照组,b、d为实验组,经实验3小时后,实验组具有明显抑菌的效果。同样图2为金黄色葡萄球菌的抑菌情况,a、c为对照组,b、d为实验组,经实验3小时后,实验组具有明显抑菌的效果。说明本产品对大肠杆菌和金黄色葡萄球菌均具有优异的抗菌活性。图3为对比例1所得发酵液与细菌作用3小时结束后对大肠杆菌和金黄色葡萄球菌的抑菌情况,其中a为大肠杆菌,b为金黄色葡萄球菌,与图1和图2对比说明采用对比例1所得发酵液的发酵效果较差,可能是人工海水中所添加的无机盐不利于真菌发酵和抗菌活性物质的产生。
表面杀菌测试,测试菌种为大肠杆菌和金黄色葡萄球菌,方法如下:
先将硅片置于超净台中正反面分别灭菌30min,取处于对数生长期的大肠杆菌和金黄色葡萄球菌菌液,将菌液OD值调整为0.1,取500μL菌液将硅片浸没后,置于37℃恒温烘箱中,静置贴附24小时,取出,并用磷酸盐缓冲液,轻轻柔细2遍,以洗去硅片表面未贴附细菌。随后分别加入磷酸盐缓冲液(对照组)和实施例1抗菌发酵液(实验组)各1mL,作用12小时后,使用荧光染料PI(碘化丙啶)避光染色15分钟后,用磷酸盐缓冲液清洗2遍,并置于激光共聚焦下观察,PI可进入死细菌而对核染色,死细菌在荧光下显示红色。
试验结果见图4-5,图4中为大肠杆菌的对比情况,a、c为对照组,b、d为实验组,图5为金黄色葡萄球菌的杀菌对比情况,a、c为对照组,b、d为实验组,从图4和5可以看出,本发明所述抗菌发酵液可杀死物体表面细菌。
2、对成纤维细胞的增殖作用
将处于对数生长期的L929小鼠成纤维细胞以1×103个/孔的密度接种于96孔板,于37℃,5%CO2细胞培养箱中培养24小时后,将细胞培养液吸弃,并于对照组中加入100μL含DMEM完全培养基(含10%FBS和1%双抗的DMEM培养基),实验组中加入含有10%抗菌发酵液的DMEM完全培养基,分别培养1,3,5,7天后,采用CCK-8方法检测发酵抗菌液对细胞增殖的影响。
试验结果如图6所示,a为对照组,b为实验组,从图中可以看出含10%抗菌发酵液的培养液对成纤维细胞具有一定的促增殖作用。
3、抗菌发酵液对细胞划痕愈合作用
通过成纤维细胞划痕愈合实验探究抗菌发酵液对成纤维细胞迁移的影响,以表征抗菌发酵液对伤口愈合的潜在作用。
将处于生长对数器的生长期的L929小鼠成纤维细胞以105/孔的密度接种于6孔板中,用马克笔在6孔板的盖子中央垂直划十字标记线,并置于37℃,5%CO2细胞培养箱中培养。当细胞融合到80%时,将培养移去,然后用20微升移液枪头沿十字标记线划线。用磷酸盐缓冲液轻柔3次,以洗去分离的细胞。分别加入DMEM完全培养基(对照组)、含10%抗菌发酵液的DMEM完全培养基(实验组),每组设3个复孔。培养0、12、24、48、72小时后置于倒置显微镜下观察拍照。
试验结果如图7所示,a为对照组,b为实验组,由图可以看出,与空白对照组相比,实验组促进了成纤维细胞迁移。
4、动物伤口愈合作用
选取6只5周龄ICR雌性小鼠,平均分为2组,包括空白组和实验组。取ICR小鼠,每只小鼠进行称重,并用记号笔在尾部做好标记。根据小鼠体重吸取水合氯醛0.1ml/10g,排气泡,腹腔注射麻醉。待小鼠用剃毛器剃去小鼠背部毛,然后用打孔器在小鼠背部打孔。往装有棉球的50mL离心管内加入约0.5mL异氟烷,数次振荡,然后将小鼠头部置于离心管口作气体维持麻醉,随后用针线将小动物皮肤防缩支架缝合到创面上,缝线松紧度适中,使支架能固定。然后拍摄创面照片(0天),注意标记。向创面分别滴加生理盐水(空白组)和抗菌发酵液(实验组),然后用3M敷料贴附,覆盖整个创面,最后盖上支架盖子,将小鼠放回笼子,等待苏醒,在1天、3天、5天更换敷料并拍摄照片。
图8为小鼠培养5天后的伤口照片,a为对照组,b为实验组,可见抗菌发酵液表现出促进伤口愈合的作用。
发明人将实施例2-8制得的发酵液进行抗菌液性能测试,其效果与实施例1的抗菌液相当。
Claims (10)
1.一种基于微生物的抗菌发酵液的制备方法,其特征在于,包括步骤:
步骤1,配置生产水、过滤并灭菌处理;
步骤2,在灭菌后的生产水中接种菌种,静态发酵,过滤得到所述抗菌发酵液;
所述菌种为棘孢木霉(Trichoderma asperellum),命名为RPT1,于2022年12月5日保藏于中国典型培养物保藏中心,保藏编号为CCTCCNO:M20221879;
所述生产水包括柴胡、茵陈、茯苓、山萸肉、枸杞、大枣、夜交藤、干姜、玉竹、怀山药、太子参、桔梗、黄芩、生地、决明子、牛膝、丹参中一种或多种中药成分。
2.根据权利要求1所述的基于微生物的抗菌发酵液的制备方法,其特征在于,所述生产水为中药液和水的混合液,所述生产水里中药液与水的体积比1~10:1000。
3.根据权利要求2所述的基于微生物的抗菌发酵液的制备方法,其特征在于,所述中药液为中药成分通过文武火煎煮后所得;
所用水包括山泉水、去离子水、超纯水、蒸馏水中任一种或多种。
4.根据权利要求1所述的基于微生物的抗菌发酵液的制备方法,其特征在于,所述中药成分选自如下四种组成之一:
组成一为柴胡,茵陈,茯苓,山萸肉,枸杞和大枣;
组成二为夜交藤,玉竹,干姜,枸杞和大枣;
组成三为怀山药,太子参,桔梗,黄芩,枸杞和大枣;
组成四为生地,决明子,牛膝,丹参,枸杞和大枣。
5.根据权利要求4所述的基于微生物的抗菌发酵液的制备方法,其特征在于,所述中药成分选自如下四种组成之一:
组成一为柴胡0.1~1Kg,茵陈0.1~1Kg,茯苓0.1~1Kg,山萸肉0.1~1Kg,枸杞0.1~1Kg,大枣0.1~1Kg;
组成二为夜交藤0.1~1Kg,玉竹0.1~1Kg,干姜0.1~1Kg,枸杞0.1~1Kg,大枣0.1~1Kg;
组成三为怀山药0.1~1Kg,太子参0.1~1Kg,桔梗0.1~1Kg,黄芩0.1~1Kg,枸杞0.1~1Kg,大枣0.1~1Kg;
组成四为生地0.1~1Kg,决明子0.1~1Kg,牛膝0.1~1Kg,丹参0.1~1Kg,枸杞0.1~1Kg,大枣0.1~1Kg。
6.根据权利要求4所述的基于微生物的抗菌发酵液的制备方法,其特征在于,所述中药成分选自如下四种组成之一:
组成一为柴胡1Kg,茵陈1Kg,茯苓1Kg,山萸肉1Kg,枸杞1Kg,大枣1Kg;
组成二为夜交藤1Kg,玉竹1Kg,干姜1Kg,枸杞1Kg,大枣1Kg;
组成三为怀山药1Kg,太子参1Kg,桔梗1Kg,黄芩1Kg,枸杞1Kg,大枣1Kg;
组成四为生地1Kg,决明子1Kg,牛膝1Kg,丹参1Kg,枸杞1Kg,大枣1Kg。
7.根据权利要求1所述的基于微生物的抗菌发酵液的制备方法,其特征在于,步骤2中,所述混合菌种的接种体积浓度为10-20%;
步骤2中接种混合菌种后再25-37℃下发酵7-21天;
步骤1或2中过滤采用孔径为0.22微米的滤膜过滤。
8.一种根据权利要求1-7任一项所述的方法制得的抗菌发酵液。
9.根据权利要求8所述的抗菌发酵液在促进皮肤修复或伤口愈合中的应用。
10.根据权利要求9所述的抗菌发酵液在制备促进皮肤修复或伤口愈合药物中的应用。
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