CN116098982B - 一种岐黄避瘟方发酵液及其制备方法和应用 - Google Patents
一种岐黄避瘟方发酵液及其制备方法和应用 Download PDFInfo
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- CN116098982B CN116098982B CN202310253900.6A CN202310253900A CN116098982B CN 116098982 B CN116098982 B CN 116098982B CN 202310253900 A CN202310253900 A CN 202310253900A CN 116098982 B CN116098982 B CN 116098982B
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Abstract
本发明提供了一种岐黄避瘟方发酵液及其制备方法和应用,属于医药技术领域,所述岐黄避瘟方发酵液的制备方法包括将复合菌种接种于岐黄避瘟方水提液中进行发酵获得岐黄避瘟方发酵液;所述复合菌种包括植物乳杆菌LZU‑J‑TSL6和植物乳杆菌LZU‑S‑ZCJ;本发明制备获得的岐黄避瘟方发酵液具有抗氧化、清除自由基的作用,同时该发酵液相比于水提液总酸、总多糖含量提高,pH降低、总黄酮含量降低;能够应用于制备抗氧化产品。
Description
技术领域
本发明属于医药技术领域,尤其涉及一种岐黄避瘟方发酵液及其制备方法和应用。
背景技术
岐黄避瘟方是应用于中医药治疗早期COVID-19的基础预防方,主要组方为生黄芪15g、防风9g、麸炒白术15g、连翘10g、贯众6g、芦根9g、沙参15g、生姜6g。
岐黄避瘟方由多种中药组成,据报道,岐黄避瘟方主要活性成分是槲皮素、刺芒柄花素、山奈酚、β-谷甾醇、汉黄芩素、异鼠李素。中药复方对于多种疾病都有较好的疗效,但却因治疗进程较慢、口感苦涩、煎煮时间长等被诟病,且中药的有效成分大多数被包裹在致密的细胞壁中,难以溶出。中药发酵技术是以含有活性成分的中药为培养基,向“培养基”中加入发酵的菌种,中药与菌株相互作用,相互影响。一方面,菌株可以利用“中药培养基”提供的养分自我生长,另一方面,在菌株生长过程中产生的酶,能够加速中药有效成分的溶出,提高中药药效。
但是,如何进一步提高岐黄避瘟方的药物活性,开发岐黄避瘟方的新功效尚未见报道。
发明内容
有鉴于此,本发明的目的在于提供一种岐黄避瘟方发酵液及其制备方法和应用,所述岐黄避瘟方的发酵液具有抗氧化、清除自由基的作用,同时该发酵液相比于水提液总酸、总多糖含量提高,pH降低、总黄酮含量降低。
本发明提供了一种岐黄避瘟方发酵液的制备方法,包括以下步骤:
将复合菌种接种于岐黄避瘟方水提液中进行发酵获得岐黄避瘟方发酵液;
所述复合菌种包括植物乳杆菌Lactobacillusplantarum LZU-J-TSL6和植物乳杆菌Lactobacillusplantarum LZU-S-ZCJ。
优选的,所述复合菌种中植物乳杆菌LZU-J-TSL6和植物乳杆菌LZU-S-ZCJ的体积比为(2~4):1;所述植物乳杆菌LZU-J-TSL6和植物乳杆菌LZU-S-ZCJ的活菌浓度独立为(1~9)×1011CFU/mL。
优选的,所述复合菌种的接种体积为岐黄避瘟方水提液体积的3~5%。
优选的,所述发酵的温度为36~38℃,所述发酵的时间为24~48h。
优选的,所述岐黄避瘟方水提液的制备方法包括以下步骤:
将黄芪、防风、白术、连翘、贯众、芦根、沙参、生姜和水混合,浸泡、煎煮后,固液分离,收集液相组分即得。
优选的,所述浸泡的时间为0.4~0.8h,所述煎煮的时间为0.4~0.8h。
优选的,所述岐黄避瘟方水提液的生药浓度为0.15~0.20g/mL。
本发明提供了所述的制备方法制备获得的岐黄避瘟方发酵液。
本发明还提供了一种岐黄避瘟方发酵制剂,包括所述的岐黄避瘟方发酵液。
本发明提供了所述的岐黄避瘟方发酵液或所述的岐黄避瘟方发酵制剂在制备抗氧化产品中的应用。
与现有技术相比,本发明具有如下有益效果:本发明利用复合菌种对岐黄避瘟方的水提液进行发酵,经过植物乳杆菌LZU-J-TSL6和植物乳杆菌LZU-S-ZCJ的复合发酵后获得的发酵液具有抗氧化作用,较好的自由基清除作用,同时能够提高总酸、总多糖含量,降低pH、总黄酮含量。本发明获得的发酵液对DPPH自由基、超氧阴离子自由基有很好的清除作用,能够应用于制备抗氧化产品。
附图说明
图1为植物乳杆菌LZU-J-TSL6和植物乳杆菌LZU-S-ZCJ单独发酵岐黄避瘟方的水提液的结果;
图2为植物乳杆菌LZU-J-TSL6和植物乳杆菌LZU-S-ZCJ不同比例复合发酵岐黄避瘟方的水提液的结果;
图3为实施例2中总黄酮测定的标准曲线图;
图4为实施例2中总多糖测定的标准曲线图;
图5为岐黄避瘟方的水提液发酵前后超氧阴离子自由基清除活性变化;
图6为岐黄避瘟方的水提液发酵前后DPPH自由基清除活性变化;
图7为岐黄避瘟方的水提液发酵前后pH变化;
图8为岐黄避瘟方的水提液发酵前后总黄酮含量变化;
图9为岐黄避瘟方的水提液发酵前后总多糖含量变化;
图10为岐黄避瘟方的水提液发酵前后总酸含量变化。
生物保藏说明
植物乳杆菌Lactobacillusplantarum LZU-J-TSL6保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:61242,保藏日期为2020年10月23日,保藏地址为广州市先烈中路100号大院59号楼5楼。
植物乳杆菌Lactobacillusplantarum LZU-S-ZCJ保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:61402,保藏日期为2020年12月31日,保藏地址为广州市先烈中路100号大院59号楼5楼。
具体实施方式
本发明提供了一种岐黄避瘟方发酵液的制备方法,包括以下步骤:将复合菌种接种于岐黄避瘟方水提液中进行发酵获得岐黄避瘟方发酵液;所述复合菌种包括植物乳杆菌LZU-J-TSL6和植物乳杆菌LZU-S-ZCJ。
在本发明中,所述复合菌种中植物乳杆菌LZU-J-TSL6和植物乳杆菌LZU-S-ZCJ的体积比优选为(2~4):1,进一步优选为2.5~3.5:1,最优选为3:1;所述植物乳杆菌LZU-J-TSL6和植物乳杆菌LZU-S-ZCJ的活菌浓度独立优选为(1~9)×1011CFU/mL,进一步优选为(3~7)×1011CFU/mL。在本发明中,所述复合菌种的接种体积优选为岐黄避瘟方水提液体积的3~5%,进一步优选为3.5%~4.5%。在本发明中,所述发酵的温度优选为36~38℃,进一步优选为37℃;所述发酵的时间优选为24~48h,进一步优选为30~42h。
在本发明中,植物乳杆菌LZU-J-TSL6保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:61242,保藏日期为2020年10月23日,保藏地址为广州市先烈中路100号大院59号楼5楼。
植物乳杆菌LZU-S-ZCJ保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:61402,保藏日期为2020年12月31日,保藏地址为广州市先烈中路100号大院59号楼5楼。
在本发明中,所述植物乳杆菌LZU-J-TSL6或植物乳杆菌LZU-S-ZCJ优选的通过以下方法分别培养获得:将菌种接种于固体培养基中活化,获得活化菌种;将所述活化菌种接种于液体培养基中进行培养获得一代菌液,将所述一代菌液传代至新的液体培养基中进行培养获得二代菌液,所述二代菌液即为发酵用的菌种。
在本发明中,所述固体培养基优选为MRS琼脂培养基,所述活化的温度优选为36~38℃,进一步优选为37℃;所述活化的时间优选为24~48h。在本发明中,所述液体培养基为MRS肉汤培养基,所述MRS肉汤培养基的浓度优选为50~55g/L,进一步优选为52~53g/L,更进一步优选为52.24g/L。在本发明中,培养获得一代菌液的时间优选为20~28h,进一步优选为22~26h,培养获得二代菌液的时间优选为20~28h,进一步优选为22~26h。在本发明中,一代菌液和二代菌液的培养体系体积优选为9~11ml,进一步优选为10ml。
在本发明中,所述岐黄避瘟方水提液的制备方法包括以下步骤:将黄芪、防风、白术、连翘、贯众、芦根、沙参、生姜和水混合,浸泡、煎煮后,固液分离,收集液相组分即得。
本发明对所述黄芪、防风、白术、连翘、贯众、芦根、沙参、生姜的用量没有特殊限定,按照岐黄避瘟方中药材的比例设定即可。在本发明中,所述黄芪、防风、白术、连翘、贯众、芦根、沙参、生姜的总质量与水的比例为1:5~10,进一步优选为1:6~7;在本发明中,所述浸泡的时间优选为0.4~0.8h,进一步优选为0.45~0.6h,更进一步优选为0.5h;所述煎煮的时间优选为0.4~0.8h,进一步优选为0.45~0.6h,更进一步优选为0.5h。在本发明中,所述固液分离优选的采用过滤实现,所述过滤优选的采用过滤网过滤;本发明在所述煎煮后,过滤前优选的还包括灭菌的步骤,所述灭菌优选的采用煮沸的方式实现。在本发明中,通过上述方法制备获得的所述岐黄避瘟方水提液的生药浓度优选为0.15~0.20g/mL,进一步优选为0.17~0.19g/mL,更进一步优选为0.18g/mL。
本发明还提供了所述的制备方法制备获得的岐黄避瘟方发酵液,所述岐黄避瘟方发酵液能够清除DPPH、超氧阴离子,提高总酸、总多糖含量,降低pH、总黄酮含量。
本发明还提供了一种岐黄避瘟方发酵制剂,包括所述的岐黄避瘟方发酵液。在本发明中,所述岐黄避瘟方发酵制剂优选的包括岐黄避瘟方发酵液药学上能够接受的辅料,本发明对所述辅料的具体种类没有限定。本发明对所述岐黄避瘟方发酵制剂的剂型没有特殊限定,采用本领域常规剂型即可,包括但不限于液剂、颗粒剂、胶囊剂、丸剂。
本发明还提供了所述的岐黄避瘟方发酵液或所述的岐黄避瘟方发酵制剂在制备抗氧化产品中的应用。在本发明中,所述抗氧化产品优选为体外抗氧化产品。
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
植物乳杆菌为Lactobacillus plantarum LUZ-J-TSL6于2020年10月23日保藏于广东省微生物菌种保藏中心,保藏编号为:GDMCC No:61242,植物乳杆菌Lactobacillusplantarum LZU-S-ZCJ于2020年12月31日保藏于广东省微生物菌种保藏中心,保藏编号为:GDMCC No:61402。
植物乳杆菌LUZ-J-TSL6菌液的制备方法为:
(1)将植物乳杆菌在固体培养基上,37℃培养活化48h;
(2)将活化后的植物乳杆菌单菌落接种于10ml含52.24gMRS肉汤/L的液体培养基,37℃培养24h,得一代菌液;
(3)取400μL一代菌液接种于9.6mL步骤(2)所述液体培养基中,37℃培养24h,得二代菌液,二代菌液即为接种用的植物乳杆菌菌液。
植物乳杆菌LZU-S-ZCJ菌液的制备方法与植物乳杆菌LUZ-J-TSL6一致,在此不再赘述。
制备获得的植物乳杆菌LUZ-J-TSL6和植物乳杆菌LZU-S-ZCJ菌液的活菌浓度为1×108CFU/ml。
岐黄避瘟方水提液的制备方法:
黄芪、防风、白术、连翘、贯众、芦根、沙参、生姜按照中药复方比例(15:9:15:9:6:9:15:3)混合,加6.5倍水浸泡0.5h后,煎煮0.5h,煮沸灭菌,滤网过滤收集滤液,灭菌灌装,得岐黄避瘟方水提液。
将制备获得的植物乳杆菌LUZ-J-TSL6和植物乳杆菌LZU-S-ZCJ菌液的制备方法分别单独接种岐黄避瘟方水提液、以1:1、1:3和3:1的体积比例复合后接种于岐黄避瘟方水提液培养72h,接种菌种和岐黄避瘟方水提液的体积比为3:100。结果如图1和2所示,以植物乳杆菌LUZ-J-TSL6和植物乳杆菌LZU-S-ZCJ菌液3:1的体积比例复合后接种获得的发酵液中活菌数最高。
实施例2
以实施例1中植物乳杆菌LUZ-J-TSL6和植物乳杆菌LZU-S-ZCJ菌液以3:1的体积比例复合后接种获得的发酵液为研究对象,检测发酵前后性质变化。
1.超氧阴离子自由基清除活性测定
参考试剂盒说明书操作测定。制备对照管:加入1mL试剂一、0.05mL双蒸水、0.1mL试剂二、三、四;制备标准管:加入1mL试剂一、0.05mL Vc标准品、0.1mL试剂二、三、四;制备测定管:加入1mL试剂一、0.05mL样品、0.1mL试剂二、三、四。将以上混匀,置37℃水浴40min,加入显色剂2.0mL,混匀,静置10min,使用酶标仪测定550nm处吸光度。
2.DPPH自由基清除活性测定
按试剂盒说明书操作测定。制备对照管:加入400μL样本和600μL80%甲醇;制备空白管:加入400μL 80%甲醇和600μL工作液;制备样品管:加入400μL样品和600μL工作液。将以上混匀,静置30min,4000/rpm离心5min,使用酶标仪测定517nm处吸光度。
3.pH测定
使用pH测定仪直接测定发酵前后pH变化,吸取10mL试液,使用测定仪,读出pH值。
4.总黄酮测定
制备样品:取水提液、发酵液各10mL,离心后吸取上清液,加入30mL乙酸乙酯多次萃取,得到水提液和发酵液中的总黄酮,将水提液、发酵液的萃取液挥干后,用甲醇定容至50mL。采用AlCl3比色法测定样品总黄酮含量;制备对照品:取10mg芦丁对照品,置于50mL容量瓶,加入15mL甲醇,超声溶解,冷却后定容,摇匀,即0.2mg/mL的芦丁对照品溶液;
制备试剂:醋酸钠-醋酸缓冲液,取1.76g无水醋酸钠,纯化水溶解至100mL,得到0.2mol/L醋酸钠溶液。
量取1.15mL冰醋酸,纯化水稀释至100mL,得到0.2mol/L冰醋酸溶液。将两者配成pH5.2的缓冲溶液。
AlCl3溶液:取1.34gAlCl3,置于100mL容量瓶,定容,即0.1mol/LAlCl3溶液。
筛选吸光度:取芦丁对照品溶液1mL,置于10mL容量瓶,加入2mL0.1mol/LAlCl3溶液和1mLpH5.2的醋酸钠-醋酸缓冲液,甲醇定容,40℃水浴10min显色,在200-600nm范围扫描,芦丁在421nm处有最大吸收;
取1mL样品溶液,置于10mL容量瓶,加入2mL 0.1mol/LAlCl3溶液和1mLpH5.2的醋酸钠-醋酸缓冲液,甲醇定容,40℃水浴10min显色,在200-600nm范围扫描,样品在421nm处有最大吸收;
测定吸光度:取芦丁标准品溶液0.2、0.4、0.6、0.8、1.0、1.2mL,置于10mL容量瓶中,加入2mL 0.1mol/LAlCl3溶液和1mLpH 5.2的醋酸钠-醋酸缓冲液,甲醇定容,40℃水浴10min显色,421nm处测定吸光度,绘制标准曲线见表。
取样品溶液0.2mL,置于10mL容量瓶中,加入2mL 0.1mol/LAlCl3溶液和1mLpH5.2的醋酸钠-醋酸缓冲液,甲醇定容,40℃水浴10min显色,421nm处测定吸光度,根据标准曲线计算总黄酮含量。
表1总黄酮标准曲线
样品 | 0D421 | 总黄酮含量(mg/mL) |
0.2mL芦丁 | 0.088 | 0.040 |
0.4mL芦丁 | 0.144 | 0.080 |
0.6mL芦丁 | 0.192 | 0.120 |
0.8mL芦丁 | 0.225 | 0.160 |
1.0mL芦丁 | 0.277 | 0.200 |
1.2mL芦丁 | 0.333 | 0.240 |
5.总多糖测定
1.取水提液、发酵液各10mL,离心后吸取上清液,加入30mL无水乙醇反复振摇多次,4℃下静置过夜后再次离心,沉淀加入80%乙醇混匀,静置10min,5500r/min离心5min,弃去上清液,沉淀加水加热溶解后定容,采用蒽酮-硫酸比色法测定样品总多糖含量。
2.5g蒽酮溶解,置于100mL容量瓶定容,即5%蒽酮溶液,4℃冷藏避光备用。
3.20mg无水葡萄糖,置于100mL容量瓶定容,即0.2g/L的葡萄糖标准溶液。
4.取上述葡萄糖标准溶液1.0mL、3.0mL、5.0mL、7.0mL、9.0mL,定容至25mL;各取2mL加入5%蒽酮溶液1mL,摇匀;迅速加入浓硫酸5mL,摇匀,室温静置10min;沸水浴15min,冷却至室温;测定OD490,制作标准曲线见图3。
5.5mg待测多糖溶解,定容至100mL,取2mL同步骤4测定OD490值,得到待测多糖中葡萄糖含量。
表2总多糖标准曲线
样品名称 | OD490 | 含糖量(mg/mL) |
s-1 | 0.190 | 0.008 |
s-2 | 0.271 | 0.024 |
s-3 | 0.336 | 0.040 |
s-4 | 0.414 | 0.056 |
s-5 | 0.454 | 0.072 |
6.总酸测定
按照GB 12456-2021《食品安全国家标准食品中总酸的测定》方法,采用电位滴定法测定。量取一定量样品溶液,按下pH开关,使用含有氢氧化钠滴定仪滴定至终点,读出滴定液剂量,通过公式X=c*(v1-v2)*K*F/m*1000,得到总酸。
结果如图5~图10所示,发酵液中总酸、总多糖含量显著高于水提液,发酵液的pH、总黄酮含量低于水提液,发酵液对DPPH自由基、超氧阴离子自由基有很好的清除作用。
表3发酵前后岐黄避瘟方水提液的性质对比
实施例3
以500mL体系为小试实验;20L体系为中试实验,进行岐黄避瘟方发酵液的制备,并测定发酵前后有效成分的含量以及pH。岐黄避瘟方发酵液的具体制备方法参见实施例1记载。
1.pH测定
使用pH仪直接测定,得到pH。
2.可溶性固形物测定
吸取一定量液体,滴于手持式折光仪上,测定可溶性固形物含量。
3.总酸测定
按照GB 12456-2021《食品安全国家标准食品中总酸的测定》方法,采用电位滴定法测定。量取一定量样品溶液,按下pH开关,使用含有氢氧化钠滴定仪滴定至终点,读出滴定液剂量,通过公式X=c*(v1-v2)*K*F/m*1000,得到总酸。
4.总多糖测定
测定样品液OD490,代入标准曲线中,得到结果,详细见实验例2中总多糖测定描述。
5.总黄酮测定
测定样品液OD421,代入标准曲线中,得到结果,详细见实验例2中总黄酮测定描述。
结果如表4所示。
表4不同试验体系发酵前后有效成分的含量以及pH
由以上实施例可知,本发明提供的岐黄避瘟方发酵液具有抗氧化、清除自由基的作用,同时该发酵液相比于水提液总酸、总多糖含量提高,pH降低、总黄酮含量降低;能够应用于制备抗氧化产品。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (5)
1.一种岐黄避瘟方发酵液在制备抗氧化药物中的应用,其特征在于,所述岐黄避瘟方发酵液的制备方法如下:
将复合菌种接种于岐黄避瘟方水提液中进行发酵获得岐黄避瘟方发酵液;所述复合菌种包括植物乳杆菌LZU-J-TSL6和植物乳杆菌LZU-S-ZCJ;
所述植物乳杆菌LZU-J-TSL6和植物乳杆菌LZU-S-ZCJ的活菌浓度独立为(1~9)×1011CFU/mL;
所述发酵的温度为36~38℃,所述发酵的时间为24~48h;
所述岐黄避瘟方水提液的制备方法如下:
将黄芪、防风、白术、连翘、贯众、芦根、沙参、生姜和水混合,浸泡、煎煮后,固液分离,收集液相组分即得。
2.根据权利要求1所述的应用,其特征在于,所述复合菌种中植物乳杆菌LZU-J-TSL6和植物乳杆菌LZU-S-ZCJ的体积比为(2~4):1。
3.根据权利要求1或2所述的应用,其特征在于,所述复合菌种的接种体积为岐黄避瘟方水提液体积的3~5%。
4.根据权利要求3所述的应用,其特征在于,所述浸泡的时间为0 .4~0 .8h,所述煎煮的时间为0.4~0.8h。
5.根据权利要求3所述的应用,其特征在于,所述岐黄避瘟方水提液的生药浓度为0.15~0.20g/mL。
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