CN109112088A - 一种分解五倍子鞣质的混合菌株及其发酵百药煎的方法 - Google Patents
一种分解五倍子鞣质的混合菌株及其发酵百药煎的方法 Download PDFInfo
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Abstract
本发明涉及一种分解五倍子鞣质的混合菌株及其发酵百药煎的方法,可有效解决百药煎的发酵问题;技术方案是,一种分解五倍子鞣质的混合菌株,包括菌株HMB‑2、菌株HMB‑5、菌株HMY‑1和菌株HMY‑2,发酵百药煎的方法,包括菌株活化、种子液制备、发酵,即得百药煎;本发明利用此混菌组合发酵百药煎,质量稳定,重复性高,大大提高百药煎中的没食子酸的含量,没食子酸含量可达到40%以上,制备工艺简单,节能环保,经济和社会效益显著。
Description
技术领域
本发明涉及微生物发酵领域,特别是一种分解五倍子鞣质的混合菌株及其发酵百药煎的方法。
背景技术
百药煎为五倍子同茶叶等经发酵制成的块状物,具有润肺化痰,生津止渴的功效,临床多用于治疗久咳痰多,咽痛,便血,久痢脱肛,口疮,牙疳,痈肿疮疡。其主要药效成分没食子酸,基本是由五倍子中可水解鞣质经单宁酶水解后转化而来。单宁酶可水解没食子酸单宁中的酯键和缩酚羧键,生成没食子酸和葡萄糖。目前对于百药煎的发酵菌种研究多集中在霉菌,有报道曲霉属黑曲霉是百药煎发酵过程中的关键菌株,五倍子生料固体发酵采用黑曲霉菌株更适合单宁酶的生产,尚未有利用细菌和酵母菌发酵百药煎的研究。
发明内容
针对上述情况,为解决现有技术之缺陷,本发明的目的就是提供一种分解五倍子鞣质的混合菌株及其发酵百药煎的方法,可有效解决百药煎的发酵问题。
本发明解决的技术方案是,一种分解五倍子鞣质的混合菌株,包括菌株HMB-2、菌株 HMB-5、菌株HMY-1和菌株HMY-2,
菌株HMB-2,分类命名为芽胞杆菌科芽孢杆菌属蜡样芽孢杆菌(Bacilluscereus),保藏编号为CCTCC M 2018107;
菌株HMB-5,分类命名为芽胞杆菌科芽孢杆菌属巨大芽孢杆菌(Bacillusmegaterium strain),保藏编号为CCTCC M2018106;
菌株HMY-1,分类命名为伯顿丝孢毕赤酵母(Hyphopichia burtonii),保藏编号为CCTCC M 2018104;
菌株HMY-2,分类命名为马克思克鲁维酵母(Kluyveromyces marxianus),保藏编号为 CCTCC M 2018105;
四株菌株于2018年3月7日保藏于中国典型培养物保藏中心,地址湖北省武汉市武昌区八一路299号武汉大学校内武汉大学保藏中心。
所述分解五倍子鞣质的混合菌株发酵百药煎的方法,包括以下步骤:
(1)菌株活化:
将菌株HMY-1、HMY-2接种于PDA固体培养基上,28-32℃中培养12-18h,得活化的菌株HMY-1、HMY-2;将菌株HMB-2、HMB-5接种于LB固体培养基上,35-40℃中培养12-18h,得活化的菌株HMB-2、HMB-5;
菌株HMY-1、HMY-2活化用培养基为PDA固体培养基,是由马铃薯去皮200g切块,煮至一捣即烂,过滤得滤液,滤液再加葡萄糖20g,琼脂粉15g,煮至沸腾,加蒸馏水至1000ml,121℃高压蒸汽灭菌20min,加入过滤除菌的链霉素,至链霉素终浓度为50μg/mL制成;
菌株HMB-2、HMB-5活化用培养基为LB固体培养基,是由酵母膏5.0g/L,蛋白胨10g/L,氯化钠5.0g/L,琼脂20.0g/L,121℃高压蒸汽灭菌20min,加入过滤除菌的制霉菌素,至链霉素终浓度为50μg/mL制成;
(2)种子液制备:
将活化的菌株HMY-1、HMY-2接种于PDA液体培养基中,将活化的菌株HMB-2、HMB-5接种于LB液体培养基中,于28-32℃、130-170rpm/min振荡培养6-8h,调整各菌株浓度为0.8-1.2×108个/mL,分别得菌株HMY-1、HMY-2、HMB-2和HMB-5种子液;
LB液体培养基:酵母膏5.0g/L,蛋白胨10g/L,氯化钠5.0g/L,121℃20min高压蒸汽灭菌后加入过滤除菌的制霉菌素,至终浓度为50μg/mL;
PDA液体培养基:马铃薯去皮200g切块,煮至一捣即烂,过滤,滤液再加葡萄糖20g,煮至沸腾,补足蒸馏水至1000ml,分装于锥形瓶中,121℃20min高压蒸汽灭菌后加入过滤除菌的链霉素,终浓度为50μg/mL;
(3)发酵:
将绿茶叶5-7g,加13-17倍绿茶叶重量体积的蒸馏水,文火煎煮18-22min,过滤,得绿茶汁;将五倍子细粉80-120g、酒糟20-30g混匀,加入绿茶汁搅拌均匀得软材,置温度32-37℃、湿度80-90%下放置24-48h;再加入软材2-4倍重量体积的无菌水,加入体积0.2-0.5%的菌株 HMB-2、HMB-5、HMY-1、HMY-2种子液;搅拌均匀,在28-32℃的温度、50-60%的湿度下,发酵66-120h,取出,55-75℃烘干,即得百药煎;得所述重量体积是指固体按g计,液体按 mL计。
本发明利用此混菌组合发酵百药煎,质量稳定,重复性高,大大提高百药煎中的没食子酸的含量,没食子酸含量可达到40%以上,制备工艺简单,节能环保,经济和社会效益显著。
具体实施方式
以下结合实施例对本发明的具体实施方式作详细说明。
本发明在具体实施中,由以下实施例实现。
实施例1
分解五倍子鞣质的混合菌株发酵百药煎的方法,包括以下步骤:
(1)菌株活化:
将菌株HMY-1、HMY-2接种于PDA固体培养基上,28℃中培养18h,得活化的菌株HMY-1、HMY-2;将菌株HMB-2、HMB-5接种于LB固体培养基上,35℃中培养18h,得活化的菌株HMB-2、HMB-5;
(2)种子液制备:
将活化的菌株HMY-1、HMY-2接种于PDA液体培养基中,将活化的菌株HMB-2、HMB-5接种于LB液体培养基中,于28℃、170rpm/min振荡培养8h,调整各菌株浓度为0.8×108个 /mL,分别得菌株HMY-1、HMY-2、HMB-2和HMB-5种子液;
(3)发酵:
将绿茶叶5g,加13倍绿茶叶重量体积的蒸馏水,文火煎煮18min,过滤,得绿茶汁;将五倍子细粉80g、酒糟20g混匀,加入绿茶汁搅拌均匀得软材,置温度32℃、湿度80%下放置48h;再加入软材2倍重量体积的无菌水,加入体积0.2%的菌株HMB-2、HMB-5、HMY-1、HMY-2种子液;搅拌均匀,在28℃的温度、50%的湿度下,发酵120h,取出,55℃烘干,即得百药煎。
实施例2
分解五倍子鞣质的混合菌株发酵百药煎的方法,包括以下步骤:
(1)菌株活化:
将菌株HMY-1、HMY-2接种于PDA固体培养基上,30℃中培养16h,得活化的菌株HMY-1、HMY-2;将菌株HMB-2、HMB-5接种于LB固体培养基上,37℃中培养16h,得活化的菌株HMB-2、HMB-5;
(2)种子液制备:
将活化的菌株HMY-1、HMY-2接种于PDA液体培养基中,将活化的菌株HMB-2、HMB-5接种于LB液体培养基中,于30℃、150rpm/min振荡培养7h,调整各菌株浓度为1.0×108个 /mL,分别得菌株HMY-1、HMY-2、HMB-2和HMB-5种子液;
(3)发酵:
将绿茶叶6.2g,加15倍绿茶叶重量体积的蒸馏水,文火煎煮20min,过滤,得绿茶汁;将五倍子细粉100g、酒糟25g混匀,加入绿茶汁搅拌均匀得软材,置温度35℃、湿度85%下放置36h;再加入软材3倍重量体积的无菌水,加入体积0.35%的菌株HMB-2、HMB-5、 HMY-1、HMY-2种子液;搅拌均匀,在30℃的温度、55%的湿度下,发酵90h,取出,60℃烘干,即得百药煎。
实施例3
分解五倍子鞣质的混合菌株发酵百药煎的方法,包括以下步骤:
(1)菌株活化:
将菌株HMY-1、HMY-2接种于PDA固体培养基上,30℃中培养12h,得活化的菌株HMY-1、HMY-2;将菌株HMB-2、HMB-5接种于LB固体培养基上,37℃中培养12h,得活化的菌株HMB-2、HMB-5;
(2)种子液制备:
将活化的菌株HMY-1、HMY-2接种于PDA液体培养基中,将活化的菌株HMB-2、HMB-5接种于LB液体培养基中,于32℃、130rpm/min振荡培养6h,调整各菌株浓度为1.2×108个 /mL,分别得菌株HMY-1、HMY-2、HMB-2和HMB-5种子液;
(3)发酵:
将绿茶叶6.2g,加17倍绿茶叶重量体积的蒸馏水,文火煎煮22min,过滤,得绿茶汁;将五倍子细粉120g、酒糟30g混匀,加入绿茶汁搅拌均匀得软材,置温度37℃、湿度90%下放置24h;再加入软材4倍重量体积的无菌水,加入体积0.5%的菌株HMB-2、HMB-5、 HMY-1、HMY-2种子液;搅拌均匀,在32℃的温度、60%的湿度下,发酵66h,取出,75℃烘干,即得百药煎。
本发明经多次反复实验,均取得了一致的结果,相关实验资料如下:
实验1菌株的分离
采集发酵时间为0h,6h,18h,24h,30h,42h,48h,66h的百药煎样品,编号分别为 C-1~C-8,稀释涂布平板法分别涂布在LB培养基、PDA培养基。将LB培养基倒置于37℃恒温培养箱中培养,将PDA培养基倒置于30℃恒温培养箱中培养。挑取分离培养基表面长出的不同菌落进行划线纯化。共分离出细菌7株,酵母菌7株,丝状真菌3株。各样品中微生物菌群情况见表1。
表1百药煎样品中微生物菌群
实验2菌株的鉴定
为进一步确定各菌种分类学地位,本发明采用传统分类方法和分子生物学分类方法对该菌株进行了分类鉴定。
1传统形态学观察
1.1菌落形态
用接种针挑取单菌落,将细菌接种于LB固体培养基,置37℃恒温培养箱中培养;将真菌接种于PDA固体培养基,置28℃恒温培养箱中培养,观察菌体在培养基中的菌落形态与生长情况。
1.2显微观察
将细菌进行结晶紫染色,置于显微镜下观察细菌结构,同时进行革兰氏染色;将酵母菌用美蓝染色,置于显微镜下观察;将丝状真菌用插片法进行培养,待菌丝长过插片的位置后,取出玻片进行显微镜观察。
2分子生物学鉴定
2.1细菌16sDNA扩增
以菌落为模板,上游引物27F:5’GAGAGTTTGATCCTGGCTCAG3’,下游引物1495R: 5’CTACGGCTACCTTGTTACGA3’完成PCR扩增。
PCR体系为Fx KOD Buffer12.5μL,dNTP0.4μL,引物各0.3μL,Fx KOD酶0.4μL,DMSO1.25μL,ddH2O10μL。
PCR程序为94℃预热5min,94℃变性1min,57℃退火90s,72℃延伸40s,循环30次,72℃保持10min,16℃保温30min。用1%琼脂糖凝胶电泳对PCR结果进行验证,将扩增产物进行测序并将结果进行序列分析。
2.2真菌18sDNA扩增
以提取的真菌基因组DNA为模板,上游引物NS1:5’GTAGTCATATGCTTGTCTC3’,下游引物NS2:5’GGCTGCTGGCACCAGACTTGC3’完成PCR扩增。
基因组提取:取培养的真菌一管,装入陶瓷珠,用FastPrep仪器进行操作(5M/s,10s, 2次)。然后加入500μLDES溶液和5μLβ-巯基乙醇,混匀,65℃水浴30min。加入500μL水饱和苯酚:三氯甲烷:异戊醇(25:24:1),12000rpm离心5min。将上清液转移至1.5mL离心管中,再加500μL水饱和苯酚:三氯甲烷:异戊醇(25:24:1)沉淀一次。12000rpm离心 5min后,将上清液转移至1.5mL离心管中,加入冷的70%乙醇混匀,-20℃沉淀2h。12000rpm 离心5min,挥干残余的乙醇,加入TER溶液溶解。
PCR体系为Easy Taq Buffer2.5μL,dNTP1.0μL,引物各1.0μL,Easy Taq酶0.1μL,DMSO1.25μL,模板0.5μL,ddH2O21μL。
PCR程序为94℃预热5min,94℃变性1min,50℃退火90s,72℃延伸40s,循环30次,72℃保持10min,16℃保温30min。用1%琼脂糖凝胶电泳对PCR结果进行验证。在15000bp 处有目的条带出现的扩增结果为阳性。
2.3测序比对
选取扩增结果阳性的产物进行测序,将测序结果提交NCBI进行blast比对,选取序列同源性在99%以上的菌属初步作为比对菌株的种属。
3实验结果
3.1传统形态学观察结果
样品C1~C8中所分离微生物的传统形态学观察结果分别见表2、表3、表4。
表2样品C1~C8中所分离细菌的形态特征
表3样品C1~C8中所分离酵母菌的形态特征
表4样品C1~C8中所分离丝状真菌的形态特征
3.2分子生物学鉴定结果
将细菌16sDNA的PCR测序结果提交至NCBI,与Gene Bank数据库中已知基因序列进行Blast比对,分析待测菌株与模式菌株的同源性。并结合《伯杰细菌分类手册》和《微生物分类学》等有关资料,对所分细菌进行初步鉴定。将酵母菌、丝状真菌的18sRNA序列比对分析结果、形态学特点,结合《真菌鉴定手册》和《常用和常见真菌》等有关资料进行初步鉴定,结果分别见表5、6、7。
表5百药煎样品所分离细菌种属鉴定结果
表6百药煎中所分离酵母菌菌属鉴定
表7百药煎中所分离丝状真菌鉴定结果
3混菌组合的筛选
采用平板透明圈初筛的方法初筛具有降解鞣质特性的菌株,通过比较所分离单菌不同混菌组合降解鞣质生成没食子酸能力的特性,获得能用于百药煎发酵的最佳混菌组合,即 HMB-2、HMB-5、HMY-1、HMY-2,其发酵百药煎中没食子酸含量可达到40%以上。
实验3混菌组合发酵百药煎
1、菌种活化HMB-2、HMB-5活化于LB培养基上,37℃培养箱中培养12h;HMY-1、HMY-2活化于PDA培养基上30℃培养箱中培养12h.
2、种子液制备分别用接种环刮取适量HMB-2、HMB-5菌种于LB液体培养基中,HMY-1、 HMY-2于PDA液体培养基中,30℃、150rpm/min振荡培养6h。
3、混菌组合发酵五倍子细粉100g、酒糟25g、绿茶叶6.2g,绿茶叶加15倍量蒸馏水煎煮 20min,过滤。五倍子细粉与酒糟混匀后加茶汁适量制软材,置温度35℃、湿度85%48h,加入200mL无菌水,分别接种HMB-2、HMB-5、HMY-1、HMY-2菌种种子液各0.2%,搅拌均匀,置于温度30℃、湿度60%的条件下,静置培养66h,取出,60℃烘干。经检测,没食子酸含量41.3%,远高于传统百药煎发酵。
实验4五倍子及本发明制百药煎样品鞣质及没食子酸含量比较
将五倍子及本发明制百药煎样品按照2015版药典通则2202测定鞣质含量以及对其没食子酸含量进行测定,结果表明五倍子经发酵鞣质含量明显降低,没食子酸含量明显升高。结果见下表8。
表8五倍子及本发明制百药煎样品鞣质及没食子酸含量测定表
实验5本发明制百药煎样品与传统制法百药煎样品体外抑菌活性比较
1、样品溶液制备将混菌组合发酵百药煎样品与传统炮制品粉碎过80目筛,分别称取25g,用水100mL煎煮2次,每次30min。合并滤液浓缩成质量浓度为0.25g/mL的药液备用。
2、菌液的制备将金黄色葡萄球菌(菌落)制成菌液,PBS缓冲液调整浓度为0.5麦氏比浊度。
3、抑菌活性比较先在无菌平皿内加入已灭菌的M-H琼脂培养基20mL,作为底层,待凝固后涂布金黄色葡萄球菌菌液100μL,每个平板中放置5个牛津杯,其中4个加入同种药液200μL,其中1个加入相应的溶剂作阴性对照。培养皿直接放置于37℃恒温培养箱中培养24h,观察菌落生产情况,用游标卡尺测量抑菌圈,计算平均值,进行对比研究,百药煎混菌组合发酵样品对金黄色葡萄球菌抑制活性优于百药煎传统炮制品。
实验6本发明制百药煎样品与传统炮制法百药煎组镇咳作用比较
取昆明小鼠40只,体重18~22g,雌雄各半,随机分为4组:空白组,本发明制百药煎样品组(8g/kg),传统炮制品(8g/kg),阳性组;以复方甘草片做阳性对照(0.9g/kg),空白组给予等体积的生理盐水,各组均灌胃给予相应药物,第七天给药后30分钟,将小鼠放入预先用0.3ml浓氨水饱和2min的玻璃罩内,观察小鼠从喷雾开始到出现腹肌收缩张嘴咳嗽的潜伏期以及3分钟内的咳嗽次数。对浓氨水引咳小鼠咳嗽潜伏期和咳嗽次数的影响效果分析见表9。
表9本发明制百药煎样品与传统炮制品镇咳作用比较(X±S n=10)
注:与空白组比较:*P<0.05**P<0.01
由表可知,本发明方法制百药煎样品能延长浓氨水引咳小鼠咳嗽的潜伏期,同时也能明显减少小鼠3min内的咳嗽次数,与空白组比较,差异具有显著性意义(P<0.01),与传统炮制品相比,本发明方法对浓氨水引咳小鼠咳嗽潜伏期更长和咳嗽次数更少,对浓氨水刺激具有更显著的耐受性。
本发明利用此混菌组合发酵百药煎,质量稳定,重复性高,大大提高百药煎中的没食子酸的含量,没食子酸含量可达到40%以上,制备工艺简单,节能环保,经济和社会效益显著。
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<110> 河南中医药大学
<120> 一种分解五倍子鞣质的混合菌株及其发酵百药煎的方法
<130> 11
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 514
<212> DNA
<213> CCTCC M 2018107 HMY-1
<400> 1
gctagtataa gcatttatac agtgaaactg cgaatggctc attaaatcag ttatcgttta 60
tttgatagta cctactagat ggataacctc ggtaaatctg acgctaatac atgcataaaa 120
tcccaacctc tggaagggat gtatttatta gataaaaaat caatgccttc gggctctttg 180
atgattcata ataacttgtc gaaccgcatg gctttagctg gcggtggttc attcaaattt 240
ctgccctatc aactttcgat ggtaggatag tggcctacca tggtttcaac gggtaacggg 300
gaataagggt tcgattccgg agaggcagcc tgagaaacgg ctaccacatc caaggaaggc 360
agcaggcgcg caaattaccc aatcccgaca cggggaggta gtgacaataa ataacgatac 420
agggcccttt cgggtcttgt aattggaatg agtacaatgt aaatacctta acgaggaaca 480
attggagggc aagtctggtg ccagcagcca acag 514
<210> 2
<211> 555
<212> DNA
<213> CCTCC M 2018105 HMY-2
<400> 2
aggccggtaa gtgaaactgc gaatggctca ttaaatcagt tatggttcct ttggtcgctc 60
gctcctctcc tacttggata actgtggtaa ttctagagct aatacatgcc gacgggcgct 120
gacccccttc gcggggggga tgcgtgcatt tatcagatca aaaccaaccc ggtcagcccc 180
tctccggccc cggccggggg gcgggcgccg gcggctttgg tgactctaga taacctcggg 240
ccgatcgcac gccccccgtg gcggcgacga cccattcgaa cgtctgccct atcaactttc 300
gatggtagtc gccgtgccta ccatggtgac cacgggtgac ggggaatcag ggttcgattc 360
cggagaggga gcctgagaaa cggctaccac atccaaggaa ggcagcaggc gcgcaaatta 420
cccactcccg acccggggag gtagtgacga aaaataacaa tacaggactc tttcgaggcc 480
ctgtaattgg aatgagtcca ctttaaatcc tttaacgagg atccattgga gggcaagtct 540
ggtgccagca gccag 555
<210> 3
<211> 1081
<212> DNA
<213> CCTCC M 2018107 HMB-2
<400> 3
ccatgcaagt cgagcgaatg gattaagagc ttgctcttat gaagttagcg gcggacgggt 60
gagtaacacg tgggtaacct gcccataaga ctgggataac tccgggaaac cggggctaat 120
accggataac attttgaacc gcatggttcg aaattgaaag gcggcttcgg ctgtcactta 180
tggatggacc cgcgtcgcat tagctagttg gtgaggtaac ggctcaccaa ggcaacgatg 240
cgtagccgac ctgagagggt gatcggccac actgggactg agacacggcc cagactccta 300
cgggaggcag cagtagggaa tcttccgcaa tggacgaaag tctgacggag caacgccgcg 360
tgagtgatga aggctttcgg gtcgtaaaac tctgttgtta gggaagaaca agtgctagtt 420
gaataagctg gcaccttgac ggtacctaac cagaaagcca cggctaacta cgtgccagca 480
gccgcggtaa tacgtaggtg gcaagcgtta tccggaatta ttgggcgtaa agcgcgcgca 540
ggtggtttct taagtctgat gtgaaagccc acggctcaac cgtggagggt cattggaaac 600
tgggagactt gagtgcagaa gaggaaagtg gaattccatg tgtagcggtg aaatgcgtag 660
agatatggag gaacaccagt ggcgaaggcg actttctggt ctgtaactga cactgaggcg 720
cgaaagcgtg gggagcaaac aggattagat accctggtag tccacgccgt aaacgatgag 780
tgctaagtgt tagagggttt ccgcccttta gtgctgaagt taacgcatta agcactccgc 840
ctggggagta cggccgcaag gctgaaactc aaaggaattg acgggggccc gcacaagcgg 900
tggagcatgt gggtttaatt cgaagcaacg cggagaacct taccaggtct gacatcctct 960
gacaccccta gagatagggc ttctccttcg gagcagagtg acaacgtgtg catgatgtcg 1020
tcagctcgtg tcgtgagatg tgggttagtc ccgcaacgag ccgcaaccct tgaatcctta 1080
a 1081
<210> 4
<211> 1032
<212> DNA
<213> CCTCC M 2018106 HMB-5
<400> 4
ggctataatg cagtcgagcg aactgattag aagcttgctt ctatgacgtt agcggcggac 60
gggtgagtaa cacgtgggca acctgcctgt aagactggga taacttcggg aaaccgaagc 120
taataccgga taggatcttc tccttcatgg gagatgattg aaagatggtt tcggctatca 180
cttacagatg ggcccgcggt gcattagcta gttggtgagg taacggctca ccaaggcaac 240
gatgcatagc cgacctgaga gggtgatcgg ccacactggg actgagacac ggcccagact 300
cctacgggag gcagcagtag ggaatcttcc gcaatggacg aaagtctgac ggagcaacgc 360
cgcgtgagtg atgaaggctt tcgggtcgta aaactctgtt gttagggaag aacaagtacg 420
agagtaactg ctcgtacctt gacggtacct aaccagaaag ccacggctaa ctacgtgcca 480
gcagccgcgg taatacgtag gtggcaagcg ttatccggaa ttattgggcg taaagcgcgc 540
gcaggcggtt tcttaagtct gatgtgaaag cccacggctc aaccgtggag ggtcattgga 600
aactggggaa cttgagtgca gaagagaaaa gcggaattcc acgtgtagcg gtgaaatgcg 660
tagagatgtg gaggaacacc agtggcgaag gcggcttttt ggtctgtaac tgacgctgag 720
gcgcgaaagc gtggggagca aacaggatta gataccctgg tagtccacgc cgtaaacgat 780
gagtgctaag tgttagaggg tttccgccct ttagtgctgc agctaacgca ttaagcactc 840
cgcctgggga gtacggtcgc aagactgaaa ctcaaaggaa ttgacggggg cccgcacaag 900
cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac cttaccagtc ttgacatcct 960
ctgacactct agagatagag cgttcccctt cggggacaga gtgacaaggt ggtgcatggt 1020
tgtcgtcagc tc 1032
Claims (5)
1.一种分解五倍子鞣质的混合菌株,包括菌株HMB-2、菌株HMB-5、菌株HMY-1和菌株HMY-2,其特征在于,
菌株HMB-2,分类命名为芽胞杆菌科芽孢杆菌属蜡样芽孢杆菌(Bacillus cereus),保藏编号为CCTCC M 2018107;
菌株HMB-5,分类命名为芽胞杆菌科芽孢杆菌属巨大芽孢杆菌(Bacillus megateriumstrain),保藏编号为CCTCC M 2018106;
菌株HMY-1,分类命名为伯顿丝孢毕赤酵母(Hyphopichia burtonii),保藏编号为CCTCC M 2018104;
菌株HMY-2,分类命名为马克思克鲁维酵母(Kluyveromyces marxianus),保藏编号为CCTCC M 2018105;
四株菌株于2018年3月7日保藏于中国典型培养物保藏中心,地址湖北省武汉市武昌区八一路299号武汉大学校内武汉大学保藏中心。
2.权利要求1所述分解五倍子鞣质的混合菌株发酵百药煎的方法,其特征在于,包括以下步骤:
(1)菌株活化:
将菌株HMY-1、HMY-2接种于PDA固体培养基上,28-32℃中培养12-18h,得活化的菌株HMY-1、HMY-2;将菌株HMB-2、HMB-5接种于LB固体培养基上,35-40℃中培养12-18h,得活化的菌株HMB-2、HMB-5;
菌株HMY-1、HMY-2活化用培养基为PDA固体培养基,是由马铃薯去皮200g切块,煮至一捣即烂,过滤得滤液,滤液再加葡萄糖20g,琼脂粉15g,煮至沸腾,加蒸馏水至1000ml,121℃高压蒸汽灭菌20min,加入过滤除菌的链霉素,至链霉素终浓度为50μg/mL制成;
菌株HMB-2、HMB-5活化用培养基为LB固体培养基,是由酵母膏5.0g/L,蛋白胨10g/L,氯化钠5.0g/L,琼脂20.0g/L,121℃高压蒸汽灭菌20min,加入过滤除菌的制霉菌素,至链霉素终浓度为50μg/mL制成;
(2)种子液制备:
将活化的菌株HMY-1、HMY-2接种于PDA液体培养基中,将活化的菌株HMB-2、HMB-5接种于LB液体培养基中,于28-32℃、130-170rpm/min振荡培养6-8h,调整各菌株浓度为0.8-1.2×108个/mL,分别得菌株HMY-1、HMY-2、HMB-2和HMB-5种子液;
所述的LB液体培养基:酵母膏5.0g/L,蛋白胨10g/L,氯化钠5.0g/L,121℃ 20min高压蒸汽灭菌后加入过滤除菌的制霉菌素,至终浓度为50μg/mL;
所述的PDA液体培养基:马铃薯去皮200g切块,煮至一捣即烂,过滤,滤液再加葡萄糖20g,煮至沸腾,补足蒸馏水至1000ml,分装于锥形瓶中,121℃ 20min高压蒸汽灭菌后加入过滤除菌的链霉素,终浓度为50μg/mL;
(3)发酵:
将绿茶叶5-7g,加13-17倍绿茶叶重量体积的蒸馏水,文火煎煮18-22min,过滤,得绿茶汁;将五倍子细粉80-120g、酒糟20-30g混匀,加入绿茶汁搅拌均匀得软材,置温度32-37℃、湿度80-90%下放置24-48h;再加入软材2-4倍重量体积的无菌水,加入体积0.2-0.5%的菌株HMB-2、HMB-5、HMY-1、HMY-2种子液;搅拌均匀,在28-32 ℃的温度、50-60%的湿度下,发酵66-120h,取出,55-75℃烘干,即得百药煎;得所述重量体积是指固体按g计,液体按mL计。
3.根据权利要求2所述分解五倍子鞣质的混合菌株发酵百药煎的方法,其特征在于,包括以下步骤:
(1)菌株活化:
将菌株HMY-1、HMY-2接种于PDA固体培养基上,28℃中培养18h,得活化的菌株HMY-1、HMY-2;将菌株HMB-2、HMB-5接种于LB固体培养基上,35℃中培养18h,得活化的菌株HMB-2、HMB-5;
(2)种子液制备:
将活化的菌株HMY-1、HMY-2接种于PDA液体培养基中,将活化的菌株HMB-2、HMB-5接种于LB液体培养基中,于28℃、170rpm/min振荡培养8h,调整各菌株浓度为0.8×108个/mL,分别得菌株HMY-1、HMY-2、HMB-2和HMB-5种子液;
(3)发酵:
将绿茶叶5g,加13倍绿茶叶重量体积的蒸馏水,文火煎煮18min,过滤,得绿茶汁;将五倍子细粉80g、酒糟20g混匀,加入绿茶汁搅拌均匀得软材,置温度32℃、湿度80%下放置48h;再加入软材2倍重量体积的无菌水,加入体积0.2%的菌株HMB-2、HMB-5、HMY-1、HMY-2种子液;搅拌均匀,在28℃的温度、50%的湿度下,发酵120h,取出,55℃烘干,即得百药煎。
4.根据权利要求2所述分解五倍子鞣质的混合菌株发酵百药煎的方法,其特征在于,包括以下步骤:
(1)菌株活化:
将菌株HMY-1、HMY-2接种于PDA固体培养基上,30℃中培养16h,得活化的菌株HMY-1、HMY-2;将菌株HMB-2、HMB-5接种于LB固体培养基上,37℃中培养16h,得活化的菌株HMB-2、HMB-5;
(2)种子液制备:
将活化的菌株HMY-1、HMY-2接种于PDA液体培养基中,将活化的菌株HMB-2、HMB-5接种于LB液体培养基中,于30℃、150rpm/min振荡培养7h,调整各菌株浓度为1.0×108个/mL,分别得菌株HMY-1、HMY-2、HMB-2和HMB-5种子液;
(3)发酵:
将绿茶叶6.2g,加15倍绿茶叶重量体积的蒸馏水,文火煎煮20min,过滤,得绿茶汁;将五倍子细粉100g、酒糟25g混匀,加入绿茶汁搅拌均匀得软材,置温度35℃、湿度85%下放置36h;再加入软材3倍重量体积的无菌水,加入体积0.35%的菌株HMB-2、HMB-5、HMY-1、HMY-2种子液;搅拌均匀,在30℃的温度、55%的湿度下,发酵90h,取出,60℃烘干,即得百药煎。
5.根据权利要求2所述分解五倍子鞣质的混合菌株发酵百药煎的方法,其特征在于,包括以下步骤:
(1)菌株活化:
将菌株HMY-1、HMY-2接种于PDA固体培养基上,30℃中培养12h,得活化的菌株HMY-1、HMY-2;将菌株HMB-2、HMB-5接种于LB固体培养基上,37℃中培养12h,得活化的菌株HMB-2、HMB-5;
(2)种子液制备:
将活化的菌株HMY-1、HMY-2接种于PDA液体培养基中,将活化的菌株HMB-2、HMB-5接种于LB液体培养基中,于32℃、130rpm/min振荡培养6h,调整各菌株浓度为1.2×108个/mL,分别得菌株HMY-1、HMY-2、HMB-2和HMB-5种子液;
(3)发酵:
将绿茶叶6.2g,加17倍绿茶叶重量体积的蒸馏水,文火煎煮22min,过滤,得绿茶汁;将五倍子细粉120g、酒糟30g混匀,加入绿茶汁搅拌均匀得软材,置温度37℃、湿度90%下放置24h;再加入软材4倍重量体积的无菌水,加入体积0.5%的菌株HMB-2、HMB-5、HMY-1、HMY-2种子液;搅拌均匀,在32 ℃的温度、60%的湿度下,发酵66h,取出,75℃烘干,即得百药煎。
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