CN116804175A - 一株植物乳杆菌xz8-2及其在天麻发酵加工中的应用 - Google Patents
一株植物乳杆菌xz8-2及其在天麻发酵加工中的应用 Download PDFInfo
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- CN116804175A CN116804175A CN202310605147.2A CN202310605147A CN116804175A CN 116804175 A CN116804175 A CN 116804175A CN 202310605147 A CN202310605147 A CN 202310605147A CN 116804175 A CN116804175 A CN 116804175A
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Abstract
本发明公开了一株植物乳杆菌(Lactobacillusplantarum)XZ8‑2及其在天麻发酵加工中的应用,菌株XZ8‑2在中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏编号为CGMCCNo.26382。本发明筛选到的植物乳杆菌XZ8‑2在天麻发酵加工中能够显著提高天麻素和改善天麻风味,将本发明的植物乳杆菌XZ8‑2接种至天麻浆液中进行发酵,能够显著提高发酵液中天麻素的含量,并且显著降低马尿臭气味;为天麻深加工提供了一种新的加工方式,可以加工制备天麻发酵口服液等产品中。
Description
技术领域
本发明属于微生物技术领域,涉及一株植物乳杆菌,具体涉及植物乳杆菌XZ8-2及其在天麻发酵加工中的应用。
背景技术
天麻(Gastrodia elataBl.),又名赤箭、神草、合离草、离母等,是一种富含天麻素(Gastrodin)、对羟基苯甲醇(Hydroxybenzene methanol)、巴利森苷(Parishin)等活性成分的兰科植物天麻的块茎。研究表明,天麻具有多种生理功能,不仅能保护中枢神经系统和心血管系统,还具有抗炎、改善记忆、增强免疫力、延缓衰老等作用。2019年,中国国家卫生健康委将天麻纳入既是食品又是中药材物质试点名单。2022年,重庆市为推动重庆天麻产业发展特制订了《关于对铁皮石斛、灵芝和天麻开展按照传统既是食品又是中药材的物质管理试点工作的实施方案》。天麻现已走进平凡百姓家,炖鸡、煮茶时都可以加入适量天麻食用,而大多数研究以证实,新鲜天麻直接食用营养价值和药用价值并不能达到最高,对天麻进行“杀酶保苷”才能更好的释放出天麻活性成分,从而获得更好的食用和药用价值。
中药炮制是我国传统医药的一大特色,对中药减毒增效、转变药性等方面有独特的作用。古有对天麻曝干、蒺藜制、酒制和姜制等古法炮制,现有润制、蒸制、冷冻干燥、微波干燥等加工新技术。根据大量研究表明,以上加工技术均能提高天麻中天麻素含量,但是,天麻有一种独特的气味,略似马尿,被称之为“马尿臭”,以上加工技术并不能去除或明显减弱天麻“马尿臭”。众所周知,食品的风味决定了消费者是否能够接受该食品的重要因素之一,它是食品生产者与消费者共同关注的焦点。为了能让更多的人拥有良好的食用天麻体验,因此研发一种不仅能够提高天麻素而且还能去除或有效减弱天麻“马尿臭”的加工方式十分必要,以期为未来天麻深加工适宜性提供更多的选择方式。
发明内容
本发明的目的是针对上述问题,提供一种植物乳杆菌(Lactobacillusplantarum)XZ8-2,在中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏编号为CGMCCNo.26382。
本发明还提供了一种菌剂,其活性成分包含上述的植物乳杆菌(Lactobacillusplantarum)XZ8-2。
优选地,所述菌剂为冷冻干燥粉剂。
本发明还提供了上述的植物乳杆菌(Lactobacillusplantarum)XZ8-2或上述的菌剂在天麻发酵加工中的应用。
所述应用为将植物乳杆菌XZ8-2接种到天麻发酵液中进行发酵。
所述应用包括如下步骤:
(1)制备天麻浆液;
(2)取植物乳杆菌菌株XZ8-2菌种活化后将菌液接种至天麻浆液中进行发酵。
步骤(1)中所述天麻浆液是采用新鲜天麻洗净后加水打浆制备得到。
步骤(2)中植物乳杆菌菌株XZ8-2的活化方法为取XZ8-2菌种,接种在MRS培养基中36-38℃扩大培养。
优选地,步骤(2)中取植物乳杆菌菌株XZ8-2菌种活化后将菌液接种至天麻浆液中进行发酵,菌株XZ8-2的接种量为1.5-2.5%,在28-32℃下静置发酵24-72小时。
进一步优选地,菌株XZ8-2的接种量为1.8-2.3%,在29-31℃下静置发酵44-52小时,优选接种量为2%,在30℃下静置发酵48小时。
本发明的有益效果是:本发明筛选到的植物乳杆菌XZ8-2在天麻发酵加工中能够显著提高天麻素和改善天麻风味,将本发明的植物乳杆菌XZ8-2接种至天麻浆液中进行发酵,能够显著提高发酵液中天麻素的含量,并且显著降低马尿臭气味;为天麻深加工提供了一种新的加工方式,可以加工制备天麻发酵口服液等产品中。
附图说明
图1是菌株XZ8-2在培养基中的形态图。
图2是XZ8-2菌株进行形态染色观察图。
图3是实验用天麻样品。
图4是天麻加水打浆得到的浆液。
图5是天麻发酵液中挥发性物质含量结果。
图6是天麻发酵液中挥发性物质数量结果。
图7是天麻风味感官评价结果。
具体实施方式
下面结合实施例对本发明作进一步说明,但并不因此而限制本发明。
下述实施例中的实验方法,如无特别说明,均为常规方法;所用化学、生物试剂如无特殊说明,均为本领域常规试剂,均可通过商购获得。
本发明实施例中用到的主要试剂和仪器设备如下表1、2所示:
表1试验试剂
表2试验仪器与设备
实施例1菌株XZ8-2的分离、纯化和鉴定
1.传统发酵泡菜的采集
采样地位于重庆市毛哥食品开发有限公司,采样时间为2022年4月10日,采样目标为传统发酵泡菜。用无菌勺吸取30mL泡菜发酵液于50mL的无菌离心管中,迅速旋紧管盖并放入低温采样箱中。
2.菌株的分离和纯化
步骤1:准备培养基
使用常规的MRS培养基,具体配方如下:
10g蛋白胨、5g牛肉粉、4g酵母粉、葡萄糖2g、1mL吐温80、2g磷酸氢二钾、5g乙酸钠、2g柠檬酸三铵、0.2g硫酸镁、0.05g硫酸锰、15g琼脂粉、1000mL蒸馏水。将配好的培养基灭菌处理,冷却至55-60℃时倒入培养皿中(每皿15mL左右,厚度3-5mm)。
步骤2:制备菌悬液
泡菜发酵液样品带回实验室后首先对其进行乳酸菌富集培养。在超净工作台中将样品泡菜汁以5%的接种量接种于无菌MRS肉汤中,在恒温培养箱中37℃静置培养18-24h,当MRS肉汤变浑浊,并且在其底部有白色菌体沉淀时,说明得到了较好的富集,可进行下一步的分离纯化。
步骤3:涂布平板,恒温培养
分别取富集完成的MRS肉汤菌悬液1mL,在无菌状态下用无菌生理盐水稀释至10-3、10-4、10-5。分别取10-3、10-4、10-5稀释度的菌悬液100μL接种到MRS培养基中央位置,用无菌涂布棒在培养基表面轻轻地涂布均匀,室温下静置5-10min,待菌液吸附进培养基后,培养皿倒置放入恒温培养箱37℃培养48h-72h。
步骤4:平板划线纯化
用接种环挑取平板上菌落形态不同的菌平板划线接种到新的MRS培养基进行划线分离(尽量挑去单菌落中心处的细菌),如此重复直至分离纯化得到形态一致的纯的单菌落,然后进行革兰氏染色,淘汰非目标菌株。
步骤5:菌种保存
经过筛选后,保留目标菌,用移液枪吸取0.5mL菌悬液和0.5mL 20%灭菌后的甘油,将其均匀混合在灭菌过的菌种保藏管中,并保存在-80℃冰箱中。
3.菌株的形态鉴定
将菌株在培养基上培养至对数生长期,按试剂盒说明书(索莱宝公司)染色后于光学显微镜下观察菌株XZ8-2的革兰氏阴阳性、有无荚膜、有无鞭毛等形态特征。
4.菌株的分子鉴定
对疑似乳酸菌的菌株进行DNA提取及16S rDNAPCR扩增。将已纯化的疑似目标菌株接种于MRS肉汤中,37℃培养18-24h后,采用细菌基因组DNA提取试剂盒进行DNA提取。
将提取得到的DNA进行PCR扩增,其中浓度为10μM的上游引物27F(5'-AGAGTTTGATCC TGGCTCAG-3',SEQ ID NO.1)1μL、浓度为10μM的下游引物1495R(5'-CTA CGGCTACCTTGT TAC GA-3',,SEQ ID NO.2)1μL,2×Taq plus Buffer 12.5μL、模板DNA 1μL,用无菌dd H2O将体系补足至25μL。并以无菌超纯水替代模板DNA作为阴性对照。扩增条件为:94℃5min;94℃30s,55℃30s,72℃1min,共29个循环,最后72℃延伸5min。回收目标DNA片段,然后送往北京擎科生物技术有限公司进行测序。测序结果如SEQ ID NO.3所示:
ATTCTGTGCCCTTAGACGGCTAGCTCCTAAAAGGTTACCCCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTTGCAGCCTACAGTCCGAACTGAGAATGGTTTTAAGAGATTAGCTTAACCTCGCGGTCTCGCGACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCGTCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACTAGAGTGCCCAACTTAATGCTGGCAACTAGTAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTCTGTCCCCGAAGGGAACCTCTAATCTCTTAGACTGTCAGAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCTTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGATTACTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGTAATCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCTCCCAGTTTCCAATGCACTTCTTCGGTTGAGCCGAAGGCTTTCACATTAGACTTAAAAGACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGGTAAATACCGTCACTGGGGAAACCGTTA。
5.菌株的形态及分子鉴定结果
对通过平板划线纯化得来的疑似乳酸菌的菌落进行形态观察,发现该菌落在培养基中的形态特征为乳白色,表面光滑,易挑落(见图1)。对其菌株进行形态染色观察发现该菌株为革兰氏阳性菌,有荚膜,无鞭毛(见图2)。采用菌体直接扩增16S rDNA的方法,测序结果经BLAST比对,该菌株为乳酸菌属(Lactobacillus),16S rDNA同源性分析结果显示,与Gene Bank数据库中已知植物乳杆菌(Lactobacillusplantarum)的同源性达100%,该分离得到的该植物乳杆菌菌株命名为XZ8-2,将其进行保藏,保藏信息如下:
将菌株XZ8-2于2023年1月送中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC)进行保藏,保藏单位地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所;保藏日期:2023年1月03日;保藏编号:CGMCC No.26382;分类命名:植物乳杆菌Lactobacillusplantarum。
实施例2菌株XZ8-2对天麻活性成分天麻素影响的研究
1.天麻采集
天麻样品为乌天麻,样品采集于重庆云阳县乌天麻培育示范基地,海拔为1700m,采样时间为2021年10月。
2.天麻发酵
(1)天麻样品处理
将鲜天麻洗净打浆,取300g左右洗净后的鲜天麻按质量比1∶1加入饮用水打浆得到天麻浆,放入发酵罐中,灭菌,-20℃保存备用。天麻处理前后如图3(处理前)和图4(处理后)所示。
(2)菌种活化
取出实验室保藏的植物乳杆菌菌株XZ8-2,接种在装有10mL MRS肉汤培养基的摇菌管中,在37℃培养箱中以150r/min的条件下振荡培养48h后,转接至第2代,再以相同条件培养48h,以确保菌株充分恢复活力。然后分别吸取10μL菌液在MRS固体培养基上涂布培养,37℃恒温培养2d,挑取长势较好的单菌落于MRS肉汤培养基中扩大培养,活化1~2次,充分活化后的菌液在4℃、7500r/min条件下离心10min,在超净台中用无菌生理盐水重悬,使用细菌浊度仪将菌液浓度调至3×108CFU/mL,备用。
(3)发酵终点确认
将调节好浓度的菌液按照2%的接种量,接种于前面制得的天麻浆中,在30℃下静置发酵5天,每天定点取样,测定天麻素含量,以此来确认发酵终点。
3.天麻素测定方法
称取不同发酵天数的天麻发酵液样品约2.000g于锥形瓶中,加入体积分数为50%乙醇溶液50mL,称重,用锡纸密封,进行30min超声处理(功率为240W,频率为40kHz),冷却至室温,再称重,用体积分数为50%的乙醇溶液补足超声损失的重量,量取续滤液10mL,然后浓缩至近干,用体积分数为3%的乙腈溶液定容至25mL容量瓶中,得待测溶液。
色谱条件:采用ThermoFisher-C18(4.6mm×250mm,5μm)色谱柱,流动相为乙腈与0.1%磷酸溶液体积比3∶97,流速为0.8mL/min,检测波长为220nm,柱温为30℃,进样量为20μL。
洗脱程序如下:
所得标准曲线如表3中所示:
表3天麻素标准曲线
4.试验结果
由表4所示,在发酵第0-2d,天麻素含量呈明显上升趋势,在第2d天麻素含量达到最大值,为1.14mg/g,在第2-4d内呈急速下降趋势。因为本发明的目的在于提高提天麻素含量,因此,确认第2d为发酵终点。
表4发酵天麻过程中天麻素的变化/(mg/g)
注:1.不同字母表示有显著性差异(P<0.05),下同;2.除水分外,所有指标按干基计算,下同。
由表5可知,相较于不接种植物乳杆菌进行天麻发酵的对照,添加植物乳杆菌菌株XZ8-2进行天麻发酵处理2天后天麻素含量得到提高,其中,天麻素含量显著增多(P<0.05),为未处理天麻的3.27倍,XZ8-2菌株发酵天麻能有效提高天麻素含量。
表5发酵天麻前后的天麻素含量/(mg/g)
实施例3菌株XZ8-2对天麻风味影响的研究
1.挥发性成分测定方法
萃取方法:取实施例2中发酵2天的天麻样品,准确称取5.000g样品置于20mL顶空进样瓶中,加入2-辛醇(10μL,浓度为300mg/L),加盖密封。将装有100μm聚二甲基硅氧烷萃取头的手动进样器插入瓶中,80℃顶空吸附40min。吸附完成后,迅速拔出手动进样器放置于气相色谱仪进样口(250℃),推出萃取头解析5min后进行数据采集。
GC条件:DB-5MS毛细管柱(30m×0.25mm,0.25μm);40℃保持3min,以10℃/min速度升温到100℃,保持2min,再以3℃/min速度升温到160℃,保持5min,最后以10℃/min速度升温到250℃;不分流进样;高纯氮气流速为1mL/min。
MS条件:离子源温度250℃,电子能量70eV,接口温度250℃,四极杆质量分析器,质量扫描范围为40~400m/z。
通过NIST17 Library谱库检索所得挥发性成分(匹配度>80%),再利用内标物质对各挥发性成分进行定量分析。
2.感官评价
感官评价小组由经过培训的8名志愿者组成,结合感官小组意见,最终选择4种气味属性描述词,分别为马尿臭(天麻独特的气味)、青香味(新鲜青草、叶子香气)、酸腥味(海水、水产等的气味)和泥土味(湿的泥土味、新鲜蘑菇气味),在0-5分间进行定量打分评价,0表示无嗅闻到该气味,3表示气味强度中等,5表示气味强度很大。
3.试验结果
采用GC-MS对添加菌株XZ8-2发酵的天麻和未添加菌株XZ8-2发酵的天麻进行挥发性成分检测。如图5和图6所示,2组样品共检出67种挥发性化合物,其中主要包括13种醛类、14种酯类、8种酸类、11种醇类、4种萜类、4种酮类、4种酚类、4种烷烃类和3种其他化合物。
醛类物质主要源于脂肪氧化降解,CK组中醛类物质含量在已知物质含量中占比较大,为26.12%。己醛具有生油脂和青草气味,庚醛呈强烈和不愉快脂肪气味,反-2-辛烯醛、壬醛、2,4-癸二烯醛和月桂醛具有强烈的脂肪气息,对天麻总体风味组成有重要贡献。酯类物质主要带来水果香,虽然在天麻气味组成中含量不是特别高,但对天麻气味组成起到了不可忽略的作用。酮类和醇类物质具有花香和木质香气,给天麻带来一种独特的清香气味。
在XZ8-2组中醛类化合物含量显著减少,从543.58μg/kg减少到37.65μg/kg;酯类化合物数量和含量均减少,并且化合物构成变化较大,辛酸甲酯、2-甲基己二酸二甲酯、棕榈酸乙酯等大部分酯类化合物未检出;同样醇类化合物结构组成也发生了变化,在XZ8-2处理后的天麻样品中新增了苯乙醇、二乙二醇、三乙二醇和1-十一醇等化合物,丰富了天麻风味;酸类化合物在增多,因为乳酸菌发酵产酸较多,从而使天麻气味更加丰富;萜类化合物新增了橙花醇和法尼醇,萜类物质具有柑橘香、药草香、柠檬香、胡椒香、辛香、木香等香气;酮类化合物明显减少;酚类化合物显著增加,含量从39.04μg/kg增加到939.68μg/kg,其中含量最高的是对甲酚,对甲酚有焦香、烟熏香气,并且阈值较低,对天麻风味组成贡献较大;醚类化合物未检出。
感官评价实验结果如图7所示,XZ8-2组泥土味、马尿臭、青香味得分大幅降低,而酸腥味得分明显变高,结合GC-MS数据结果来看,酸类和醇类物质较为丰富,从而可能导致酸腥味更重,天麻马尿臭味变化最大,气味得分明显降低,说明经过发酵后的天麻,气味组成发生较大变化。因此,发酵可以使天麻马尿臭问题得到显著改善。
由以上实施案例可知,本发明提供的一株从传统发酵泡菜中分离出的植物乳杆菌XZ8-2,具有提高天麻素和改善天麻风味的作用,可以提供一种新的天麻深加工方式。
Claims (10)
1.植物乳杆菌(Lactobacillusplantarum)XZ8-2,在中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏编号为CGMCCNo.26382。
2.一种菌剂,其活性成分包含权利要求1所述的植物乳杆菌(Lactobacillusplantarum)XZ8-2。
3.如权利要求2所述的菌剂,其特征在于:所述菌剂为冷冻干燥粉剂。
4.如权利要求1所述的植物乳杆菌(Lactobacillusplantarum)XZ8-2或权利要求2或3所述的菌剂在天麻发酵加工中的应用。
5.根据权利要求4所述的应用,其特征在于:所述应用为将植物乳杆菌XZ8-2接种到天麻发酵液中进行发酵。
6.根据权利要求5所述的应用,其特征在于:所述应用包括如下步骤:
(1)制备天麻浆液;
(2)取植物乳杆菌菌株XZ8-2菌种活化后将菌液接种至天麻浆液中进行发酵。
7.根据权利要求6所述的应用,其特征在于:步骤(1)中所述天麻浆液是采用新鲜天麻洗净后加水打浆制备得到。
8.根据权利要求6所述的应用,其特征在于:步骤(2)中植物乳杆菌菌株XZ8-2的活化方法为取XZ8-2菌种,接种在MRS培养基中36-38℃扩大培养。
9.根据权利要求6所述的应用,其特征在于:步骤(2)中取植物乳杆菌菌株XZ8-2菌种活化后将菌液接种至天麻浆液中进行发酵,菌株XZ8-2的接种量为1.5-2.5%,在28-32℃下静置发酵24-72小时。
10.根据权利要求9所述的应用,其特征在于:菌株XZ8-2的接种量为1.8-2.3%,在29-31℃下静置发酵44-52小时,优选接种量为2%,在30℃下静置发酵48小时。
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