CN114984114A - 一种液态发酵中药微生态制剂及其在抗猪流行性腹泻病毒方面的应用 - Google Patents
一种液态发酵中药微生态制剂及其在抗猪流行性腹泻病毒方面的应用 Download PDFInfo
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- CN114984114A CN114984114A CN202210630179.3A CN202210630179A CN114984114A CN 114984114 A CN114984114 A CN 114984114A CN 202210630179 A CN202210630179 A CN 202210630179A CN 114984114 A CN114984114 A CN 114984114A
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Abstract
本发明公开了一种液态发酵中药微生态制剂及其在抗猪流行性腹泻病毒方面的应用，属于抗病毒应用领域。本发明中选取丹参、党参、大青叶、黄芪、茯苓、猪苓和甘草七味中药，粉碎后加水浸泡过夜，文火煎煮浓缩后进行高温高压灭菌，待冷却后加入由植物乳杆菌、双歧杆菌、丁酸梭菌和解淀粉芽孢杆菌组成的混合菌液进行发酵，将发酵液进行过滤，药渣进行二次发酵，合并两次滤液，加入山梨酸钾作保护剂，最终得到该液态发酵中药微生态制剂。该液态中药微生态制剂可抑制冠状病毒PEDV对细胞的感染。
Description
技术领域
本发明涉及一种能抑制PEDV感染的液态中药微生态制剂，属于微生态制剂抗病毒领域。
背景技术
PEDV是线性单股正链RNA病毒，属冠状病毒科(Coronaviridae)；PEDV感染猪以后主要临床症状为呕吐、腹泻、脱水性下痢，对各年龄段猪均有感染性，主要对新生仔猪有高致病率与致死率。
临床主要采用注射PEDV疫苗或返饲法来防控PED，但PEDV容易产生变异，经典疫苗对病毒变异株的保护率低，返饲法防控仔猪腹泻又存在较大风险；市面上也尚无商品化的抗猪流行性腹泻病毒制剂，仍旧以注射疫苗为主；近些年，中药与益生菌渐渐应用于畜禽养殖业，实验室研究表明多种中药、活性成分或其中药复方制剂与益生菌具有抵抗病毒的作用，注射疫苗并用中药、中药制剂或益生菌制剂辅助防控已成为疫病防控的主流。
中药微生态制剂作为运用现代微生态学方法将中药与益生菌联合发酵后用于防治动物疾病的一种新型制剂，具有价格低廉、无残留和不产生耐药性等优势。现代研究表明，中药微生态制剂可通过直接杀灭病毒、阻断病毒对宿主细胞的吸附、入侵和抑制病毒的复制等方式发挥抗病毒作用；中药微生态制剂中的有效成分可通过抑制病毒表面关键受体的表达，或激活细胞基因与多种信号通路，表达多种抗病毒蛋白来抑制病毒感染细胞的过程；此外，中药微生态制剂也可以通过增强动物机体免疫力来间接发挥抗病毒作用，如通过增强自然杀伤细胞毒性、增加T淋巴细胞的细胞毒性来刺激免疫系统，以达到抗病毒的目的。同时中药微生态制剂又能调节炎症因子的分泌，减轻炎症反应，缓解机体的患病症状，达到温和治愈的效果。而将中药与益生菌联合发酵后制得的微生态制剂中富含多种生物活性物质及益生菌产物，如氨基酸、维生素、胞外多糖、乳酸等，这些生物活性物质及益生菌产物以多种方式与途径间接发挥抗病毒的作用。
目前中药微生态制剂在抗病毒领域的研究报告还相对较少，但中药微生态制剂由中药与益生菌复配发酵制备而成，兼具了中药与益生菌的优点与疗效，在抗病毒领域展现出巨大的潜力。
发明内容
本发明的目的是通过液态发酵，提高制剂中中药活性成分含量；本发明的另一目的是针对引起猪流行性腹泻的PEDV，提供一种能抗冠状病毒PEDV感染的液态发酵中药微生态制剂及其在抗猪流行性腹泻病毒方面的应用。
本发明的目的可通过如下技术方案实现：一种液态发酵中药微生态制剂，按以下方法制备：选取七味中药，粉碎后加入中药总质量10~14倍的水浸泡过夜；然后文火煎煮浓缩至原体积的1/5~1/6倍，高温高压灭菌后，加入浓缩液1/5~2/5倍总体积的益生菌混合菌液进行发酵，37℃发酵48小时后，将发酵液进行过滤，滤液备用，将药渣加入8~12倍的水再次煎煮浓缩至原体积1/4~1/5，加入浓缩液1/10~2/10倍体积的混合菌液，进行二次发酵，发酵后再次过滤，合并两次滤液，加入0.5~1.0g/L山梨酸钾作保护剂，最终得到该液态中药微生态制剂；
所述七味中药是丹参、党参、大青叶、甘草、茯苓、猪苓、黄芪，所述中药由丹参3～5份、党参2～4份、大青叶2～4份、甘草2～4份、猪苓1～3份、茯苓1～3份、黄芪1～3份组成，均为质量份；
所述益生菌混合菌液中，植物乳杆菌MⅢ 2~4份、双歧杆菌2~4份、解淀粉芽孢杆菌1~3份、丁酸梭菌1~3份，皆为体积份。
所述解淀粉芽孢杆菌为解淀粉芽孢杆菌L2，该菌株分离自健康鸡的盲肠，具有蛋白酶和淀粉酶活性，保藏在中国典型培养物保藏中心，保藏编号为CCTCC NO：M 2020570。
上述的液态发酵中药微生态制剂可用于预防或治疗冠状病毒PEDV所引起的猪流行性腹泻。
上述液态中药微生态制剂应用在防治和治疗猪冠状病毒方面。
本发明所选取中药，丹参可提高淋巴细胞的增殖能力，增加抗体生成细胞的数量，并能增强巨噬细胞的吞噬能力和NK细胞活性，丹参中的酚酸类化合物均呈现出较强抗炎活性。党参、茯苓、猪苓等所含的多糖也可以提高机体的免疫力。黄芪常用于流感、病毒性心肌炎、乙型肝炎、带状疱疹等病毒感染性疾病的治疗。甘草提取物甘草素能有效抑制PEDV的入胞和复制，从而有效地抑制PEDV的感染。党参和黄芪均为补气类中药，党参偏于阴而补中，黄芪偏于阳而实表，两药结合，奏扶正补气之功。而甘草在整个体系中作为调和剂，起抗炎解毒的作用。将这七味中药互相配伍，能使其发挥整体综合效能。
本发明选用的益生菌，据报道，乳酸杆菌与双歧杆菌是应用于发酵食品与微生态制剂中最多的益生菌，它们可通过多种机制刺激机体免疫系统，例如增强自然杀伤细胞毒性、刺激促炎细胞因子的产生以及增加T淋巴细(CD3+CD16+CD56+)的细胞毒性，以增强免疫。双歧杆菌具有营养、抑菌、提高免疫力、保护肝脏及改善胃肠道功能等生理活性。而丁酸梭菌作为专性厌氧菌，具有良好的肠道定植能力，可以促进乳酸杆菌、双歧杆菌等益生菌的增殖。丁酸梭菌在肠道代谢过程中会产生短链脂肪酸，它対肠道中的致病菌具有抑制生长或杀灭的作用，产生的丁酸有利于肠道屏障的维持和修复。口服补充丁酸梭状芽孢杆菌可提高丁酸水平，降低促炎细胞因子水平，减轻烧伤小鼠的肠道损伤。
将上述七味中药与四种益生菌进行复配发酵，在发酵过程中，益生菌产生了大量的代谢酶，这些酶通过降解植物细胞壁促进对中药活性成分的释放，并对皂苷类化合物等口服生物利用度较低的中药活性成分进行分解转化，使其成为更易被机体吸收利用的成分。而中药则为益生菌提供营养物质，促进益生菌的增殖。发酵中药将中药与益生菌联合发酵后制得的微生态制剂中富含多种生物活性物质及益生菌产物，如氨基酸、维生素、胞外多糖、乳酸等。中药与益生菌协同增效，表现出更优良的抗炎、促免疫、抗病毒性能。
本发明有益效果是：将中药与益生菌进行液态发酵后，中药总皂苷的含量得到显著增加；该中药微生态制剂中所含益生菌具有良好的抗逆、定植、抑制有害菌、调节肠道菌群、促肠道屏障修复的作用；该液态中药微生态制剂能抑制冠状病毒PEDV在Vero-CCL81细胞中的复制，并具有一定的直接灭活冠状病毒PEDV的作用；本发明潜在的临床应用效果：该中药微生态制剂具有较好的抗冠状病毒PEDV活性，为该中药微生态制剂临床防治猪流行性腹泻提供了一定的依据。
附图说明
图1是抑制冠状病毒PEDV在细胞内的复制实验中各组病毒GFP荧光强度对比图。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了解释本发明，而不是为了限制本发明的范围。
实施例1：液态中药微生态制剂的制备
1.益生菌种子液制备
挑取植物乳杆菌MIII、双歧杆菌、解淀粉芽孢杆菌L2、丁酸梭菌单菌落至对应培养基中，平板划线后培养24h，进行菌种活化；挑取活化后的单菌落至对应的5mL液体培养基中，37℃培养24h，得一级种子液；吸取100μL一级种子液至5mL液体培养基中，37℃培养至对数生长期，得二级种子液；植物乳杆菌MⅢ、双歧杆菌、丁酸梭菌和解淀粉芽孢杆菌L2生长至对数培养基需分别培养14 h、14 h、20 h和8 h，植物乳杆菌MⅢ的活菌数为1.93×109CFU/mL、双歧杆菌的活菌数为2.54×109CFU/mL、丁酸梭菌的活菌数为1×107CFU/mL，解淀粉芽孢杆菌L2的活菌数为1.1×109CFU/mL。
2. 液态中药微生态制剂的制备
称取总质量120g的中药，其中丹参、党参、大青叶、黄芪、茯苓、猪苓和甘草的质量比=3∶2∶2∶2∶1∶1∶1，加入1440mL的二次水浸泡过夜，文火煎煮浓缩至240mL，按植物乳杆菌MIII∶双歧杆菌∶解淀粉芽孢杆菌L2∶丁酸梭菌=2∶2∶1∶1的体积比接种72mL总体积的混合菌液，37℃发酵48h后，收集滤液；向药渣中加入1200mL的二次水，文火煎煮浓缩至240mL，冷却后加入24mL总体积的混合菌液，37℃发酵48h后，过滤，收集滤液，将两次滤液合并后加入1mg/L的山梨酸钾，最终得到480mL的液态中药微生态制剂，存于4℃冰箱，备用；液态中药微生态制剂的浓度（按中药质量计算）为1mg/4mL。
实施例2：液态中药微生态制剂总皂苷的含量检测
按实施例1的中药配方将中药混合粉碎为中药混合物（丹参、党参、大青叶、黄芪、茯苓、猪苓和甘草的质量比=3∶2∶2∶2∶1∶1∶1），检测此中药混合物、实施例1制备的液态发酵中药微生态制剂中的总皂苷含量，总皂苷检测方法如下：
1.标准曲线的制作：
准确称取黄芪甲苷5mg置于25mL容量瓶中，加入甲醇，定容至刻度线，溶解摇匀，即得0.2mg/mL黄芪甲苷溶液；分别吸取0.1、0.2、0.4、0.6和0.8mL黄芪甲苷标准品加入干燥的试管中，70 ℃水浴蒸发多余甲醇，加入8%香草醛-乙醇溶液0.5mL，72%硫酸5mL，小心振摇混匀后置于60℃恒温水浴中加热10min，取出后冰水冷却10min，摇匀，以相应试剂为空白对照，在540nm处测吸光度；
2.总皂苷的提取与含量测定：
精密称取待测样品4. 0 g 至250 mL 锥形瓶中，加20 倍量70% 乙醇于720 W 超声提取2 次，每次20 min，合并提取液，50 ℃减压浓缩至干，加甲醇溶解并定容至10 mL；吸取提取液40μL，置于试管中，70 ℃水浴蒸干，加入0.5mL 的8%香草醛－乙醇溶液和5mL的72%硫酸试液，摇匀后置于60 ℃水浴中加热10min，取出后冰水冷却10min，采用紫外分光光度计于540 nm 测定吸光度。利用标准曲线计算总皂苷含量。
中药混合物、实施例1制备的液态发酵中药微生态制剂的总皂苷含量，见下表1：
表1中结果显示，实施例1制备的液态发酵中药微生态制剂中所含的总皂苷含量大约是未加入益生菌进行发酵的中药混合物的1.5倍，说明微生物有效促进了中药活性物质的转化和释放。
实施例3：液态发酵中药微生态制剂抑制PEDV在细胞内的复制
试验材料：PEDV-DR13毒株，携带绿色荧光蛋白基因，可在蓝光下激发绿色荧光，由河南农业大学李永涛老师馈赠；Vero-CCL81细胞，用含有10% FBS，100 U /mL青霉素和100μg/mL 链霉素的DMEM高糖培养基进行培养，由河南农业大学李永涛老师馈赠；液态发酵中药微生态制剂：按照实施案例1的方法制备。
将Vero-CCL81细胞接种于6孔板，37℃ 5% CO2培养箱培养至细胞贴壁生长为单层。取DR13病毒液（MOI=0.01）接种于Vero-CCL81细胞，37℃ 5% CO2孵育2小时后，弃去原液，PBS清洗2次；用无血清DMEM将液态中药微生态制剂稀释至特定浓度。分别添加31.2mg/mL、7.81mg/mL、0.976mg/mL三个剂量的液态中药微生态制剂至细胞中，病毒对照组则添加2mL无血清DMEM，37℃ 5%CO2培养48小时后，倒置荧光显微镜下观察各组病毒GFP荧光强度；参见图1的结果显示，液态发酵中药微生态制剂能明显抑制PEDV在细胞中的复制。
实施例4：液态中药微生态制剂具有直接灭活PEDV的活性
试验材料：病毒PEDV-DR13毒株，同实施例3；细胞Vero-CCL81，同实施例3；液态发酵中药微生态制剂，其制备方法同实施例1。
将Vero-CCL81细胞接种于6孔板，37℃ 5% CO2培养箱培养至细胞贴壁生长为单层。用无血清DMEM将液态中药微生态制剂稀释至31.2mg/mL、7.81mg/mL、0.976mg/mL；取DR13病毒液（MOI=0.01mL）1mL与上述不同浓度药液37℃共孵育2小时，病毒组添加1mL无血清DMEM作为对照；弃去6孔板中的原细胞培养液，PBS清洗2次后，分组加入病毒-药物混合液，37℃ 5% CO2培养箱孵育2小时，进行接毒；接毒完成后，弃去原液，PBS清洗2次，37℃ 5%CO2继续培养48小时，倒置荧光显微镜下观察各组病毒GFP荧光强度；结果显示，液态中药微生态制剂具有直接灭活PEDV的活性。
Claims (5)
1.一种液态发酵中药微生态制剂，其特征在于，按以下方法制备：选取七味中药，粉碎后加入中药总质量10~14倍的水浸泡过夜；然后文火煎煮浓缩至原体积的1/5~1/6倍，高温高压灭菌后，加入浓缩液1/5~2/5倍总体积的益生菌混合菌液进行发酵，37℃发酵48小时后，将发酵液进行过滤，滤液备用，将药渣加入8~12倍的水再次煎煮浓缩至原体积1/4~1/5，加入浓缩液1/10~2/10倍体积的混合菌液，进行二次发酵，发酵后再次过滤，合并两次滤液，加入0.5~1.0g/L山梨酸钾作保护剂，最终得到该液态中药微生态制剂；
所述七味中药是丹参、党参、大青叶、甘草、茯苓、猪苓、黄芪，所述丹参、党参、大青叶、甘草、茯苓、猪苓、黄芪的质量比为3~5∶2~4∶2~4∶2~4∶1~3∶1~3∶1~3；
所述益生菌混合菌液由植物乳杆菌、双歧杆菌、丁酸梭菌和解淀粉芽孢杆菌按一定体积比组成，所述；其中，植物乳杆菌MⅢ 2~4份、双歧杆菌2~4份、解淀粉芽孢杆菌1~3份、丁酸梭菌1~3份，皆为体积份。
2.如权利要求1所述的液态发酵中药微生态制剂，其特征在于：所述解淀粉芽孢杆菌为解淀粉芽孢杆菌L2，该菌株分离自健康鸡的盲肠，具有蛋白酶和淀粉酶活性，保藏在中国典型培养物保藏中心，保藏编号为CCTCC NO：M 2020570。
3.如权利要求2所述的液态发酵中药微生态制剂，其特征在于：植物乳杆菌MⅢ的活菌数为1.93×109CFU/mL，双歧杆菌的活菌数为2.54×109CFU/mL，丁酸梭菌的活菌数为1×107CFU/mL，解淀粉芽孢杆菌L2的活菌数为1.1×109CFU/mL。
4.权利要求1-3任一项所述液态发酵中药微生态制剂用于预防或治疗冠状病毒PEDV所引起的猪流行性腹泻。
5.如权利要求1-3任一项所述的液态中药微生态制剂在防治和治疗猪冠状病毒方面的应用。
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