CN106244487B - 一株乳杆菌及提高稀有皂苷的人参发酵方法 - Google Patents
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Abstract
本发明公开了一株乳杆菌及提高稀有皂苷的人参发酵方法，它包括：将保藏编号为CCTCC M 2016372乳杆菌的种子发酵液，按1‑6%接种量接种于人参液，发酵条件为：33‑40℃摇床培养，转速为70‑160r/min，发酵18‑72h；所述的人参液为人参加水打浆。与普通乳杆菌相比，人参发酵液Rh1+Rg2、F1、Rh2、20（S）‑Rg3、R0的含量都有很大的提高。
Description
技术领域
本发明属于发酵食品技术领域，尤其涉及一株乳杆菌及提高稀有皂苷的人参发酵方法。
背景技术
人参被誉为“百草之王”，广泛生长于我国的黑龙江、吉林、辽宁，特别是长白山地区。人参在我国一直作为名贵中草药，其营养价值丰富，具有补脾益气的特点。人参的主要化学成分是人参皂苷、人参多糖、人参多肽和挥发油等，除此之外，它还含有多种氨基酸、微量元素、脂肪酸和维生素等营养成分。人参皂苷是由苷元骨架与糖基通过糖苷键相连构成的糖苷类化合物。根据皂苷的结构不同，可分为齐墩果烷型五环三萜皂苷和达玛烷型四环三萜皂苷。齐墩果烷型五环三萜皂苷主要存在于三萜类皂苷的植物中，在自然界中不常见。达玛烷型皂苷包括原人参二醇皂苷如Rb1、Rb2、Rc、Rd、F2等，原人参三醇皂苷如Re、Rf、Rg1、Rg2等。其中，Rg1、Rg3、Rh1、Rh2、F2等含量极少，主要存在于红参中，称为稀有人参皂苷。近年来研究发现，人参皂苷Rg3具有明显的抗疲劳、抗氧化损伤和增强机体免疫、抗炎、抗氧化和免疫佐剂作用。另外，人参皂苷Rg3具有抑制肿瘤细胞的粘附、浸润、增殖以及抗肿瘤新生血管的形成，从而具有显著的抗肿瘤作用。稀有人参皂苷F2是一种稀有的次级人参皂苷，具有抑制肿瘤，抗衰老，抗疲劳等方面的作用，另外对治疗心神不安，惊悸失眠、健忘等方面也有显著提高。
发明内容
本发明的目的是提高人参的稀有皂苷的含量，而提供一种发酵乳杆菌发酵人参液方法。
一株乳杆菌，它的保藏编号为：CCTCC M 2016372。
一株乳杆菌在提高发酵人参液稀有皂苷的含量中的应用。
提高稀有皂苷的人参发酵方法，它包括：将保藏编号为CCTCC M 2016372发酵乳杆菌的种子发酵液，按1-6%接种量接种于人参液，发酵条件为：33-40℃摇床培养，转速为70-160r/min，发酵18-72h；所述的人参液为人参加水打浆。
所述的人参液为选取新鲜人参清洗后晾干，置于高压灭菌锅内，121℃维持40min，打开灭菌锅取出人参；按人参：水=1：1打浆，121℃，20min灭菌；
所述的发酵条件为：36-38℃摇床培养，转速为80-120r/min，发酵18-48h。
本发明提供了一株乳杆菌及提高稀有皂苷的人参发酵方法，它包括：将保藏编号为CCTCC M 2016372乳杆菌的种子发酵液，按1-6%接种量接种于人参液，发酵条件为：33-40℃摇床培养，转速为70-160r/min，发酵18-72h；所述的人参液为人参加水打浆。与普通乳杆菌相比，人参发酵液Rh1+Rg2、F1、Rh2、20（S）-Rg3、R0的含量都有很大的提高。
附图说明
图1菌株的革兰氏染色形态；
图2菌株的PCR 扩增片段图谱；
图3 菌株的同源性比对数据分析；
图4 HPLC-PDA法测定的人参皂苷含量。
具体实施方式
实施例1 乳杆菌的分离与鉴定
（1）乳杆菌的分离
将朝鲜族泡菜（采购于吉林省长春市中东大市场）浸泡于液体灭菌MRS培养基中，37℃恒温培养20-24h，将菌液用划线法涂于MRS固体培养基，37℃恒温培养2d，挑取单独菌落进行生理生化鉴定及16srDNA鉴定，确定为发酵乳杆菌（Lactobacillus fermentum），于2016年7月4日，保存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，邮政编码：430072，编号为CCTCC M 2016372；将发酵乳杆菌CCTCC M 2016372接种于液体的无菌MRS培养基，在37℃温度下培养20-24h，活化后继续传代两次，每次接种量为3%，待菌种充分活化后，4℃冷藏待用。
（2）乳杆菌的鉴定
从朝鲜族泡菜中筛选的菌株革兰氏染色结果为革兰氏阳性菌，如图1所示，形态为短杆状；通过上海申工公司对菌液进行PCR，扩增后的产物经过琼脂糖凝胶电泳，如图2所示，在1200bp至1500处存在特异性条带。然后对扩增、纯化后的16S rDNA进行测序鉴定，测定该序列长为1354bp；该菌株的16S rDNA序列在核糖体数据库http://rdp.cme.msu.edu/index.jsp上进行同源性比对，结果如图3所示，结果该菌株的16S rDNA 与Lactobacillus fermentum strain的16S rDNA具有较高的同源性，证实该菌株为发酵乳杆菌（Lactobacillus fermentum）。
实施例2 利用菌株发酵提取人参皂苷
1） 人参液的制备
选取新鲜人参清洗后晾干，置于高压灭菌锅内，121℃维持40min，打开灭菌锅取出人参；按人参：水=1：1打浆，121℃，20min灭菌冷藏。
2） 利用发酵乳杆菌发酵人参液
将实施例1中获取的发酵乳杆菌CCTCC M 2016372种子发酵液按3%接种量接种于人参液，进行发酵。发酵条件为：37℃摇床培养，转速为80-120r/min，时间为18-48h。人参发酵液冻干成粉末，待用。
3）人参皂苷的提取
取发酵人参粉末约1g，精密称定，置索氏提取装置中，加三氯甲烷加热回流3小时，弃去三氯甲烷溶液，药渣挥干溶剂，连同滤纸筒移入100ml锥形瓶中，精密加水饱和正丁醇50ml，密塞，放置过夜，超声处理（功率250W，频率50Hz）30分钟，过滤，精密称取滤液25ml，置于蒸发皿中蒸干，残渣加95%甲醇溶液4ml，溶解并转移至5ml容量瓶中，加入95%甲醇稀释至刻度，摇匀，既得人参皂苷样品。
4）人参皂苷含量测定
采用上述方法，对未发酵的人参液、利用发酵乳杆菌标准株CICC 22556和利用发酵乳杆菌CCTCC M 2016372人参发酵液进行皂苷提取，采用二极管阵列检测器高效液相法（HPLC-PDA）测定人参中人参皂苷单体的含量，用Unitary C18色谱柱（4.6mmX250mm，5μm），以乙腈(A)-0.05%磷酸水溶液(B)梯度洗脱(见表1)，流速为1.3ml/min，柱温为30℃，检测波长203nm，结果如图4所示，发酵乳杆菌CCTCC M 2016372人参发酵液中Rh1+Rg2的含量比未发酵的人参液提高了1倍；发酵乳杆菌CCTCC M 2016372人参发酵液中F1的含量比未发酵的人参液提高了1倍；发酵乳杆菌CCTCC M 2016372人参发酵液中Rh2的含量比未发酵的人参液提高了2.1倍；发酵乳杆菌CCTCC M 2016372人参发酵液中20（S）-Rg3的含量比未发酵的人参液提高了2倍且含量高达0.18%；发酵乳杆菌CCTCC M 2016372人参发酵液中R0的含量比未发酵的人参液提高了10倍且含量高达0.31%。
<110> 吉林农业大学
<120> 一株乳杆菌及提高稀有皂苷的人参发酵方法
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gtcgccaacg agtggcggac gggtgagtaa cacgtaggta acctgcccag aagcggggga 60
caacatttgg aaacagatgc taataccgca taacagcgtt gttcgcatga acaacgctta 120
aaagatggct tctcgctatc acttctggat ggacctgcgg tgcattagct tgttggtggg 180
gtaacggcct accaaggcga tgatgcatag ccgagttgag agactgatcg gccacaatgg 240
gactgagaca cggcccatac tcctacggga ggcagcagta gggaatcttc cacaatgggc 300
gcaagcctga tggagcaaca ccgcgtgagt gaagaagggt ttcggctcgt aaagctctgt 360
tgttaaagaa gaacacgtat gagagtaact gttcatacgt tgacggtatt taaccagaaa 420
gtcacggcta actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttatccgga 480
tttattgggc gtaaagagag tgcaggcggt tttctaagtc tgatgtgaaa gccttcggct 540
taaccggaga agtgcatcgg aaactggata acttgagtgc agaagagggt agtggaactc 600
catgtgtagc ggtggaatgc gtagatatat ggaagaacac cagtggcgaa ggcggctacc 660
tggtctgcaa ctgacgctga gactcgaaag catgggtagc gaacaggatt agataccctg 720
gtagtccatg ccgtaaacga tgagtgctag gtgttggagg gtttccgccc ttcagtgccg 780
gagctaacgc attaagcact ccgcctgggg agtacgaccg caaggttgaa actcaaagga 840
attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagct acgcgaagaa 900
ccttaccagg tcttgacatc ttgcgccaac cctagagata gggcgtttcc ttcgggaacg 960
caatgacagg tggtgcatgg tcgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc 1020
gcaacgagcg caacccttgt tactagttgc cagcattaag ttgggcactc tagtgagact 1080
gccggtgaca aaccggagga aggtggggac gacgtcagat catcatgccc cttatgacct 1140
gggctacaca cgtgctacaa tggacggtac aacgagtcgc gaactcgcga gggcaagcaa 1200
atctcttaaa accgttctca gttcggactg caggctgcaa ctcgcctgca cgaagtcgga 1260
atcgctagta atcgcggatc aagcaatgcc gcggtgaata cgttcccggg ccttgtacaa 1320
caccgcccgt caacaccaat gaaggagttt gtaa 1354
Claims (5)
1.一株发酵乳杆菌Lactobacillus fermentum，它的保藏编号为：CCTCC M 2016372。
2.权利要求1所述的一株发酵株乳杆菌在提高发酵人参液稀有皂苷的含量中的应用。
3.提高稀有皂苷的人参发酵方法，其特征在于，它包括：将保藏编号为CCTCC M2016372的发酵乳杆菌的种子发酵液，按1-6%接种量接种于人参液，发酵条件为：33-40℃摇床培养，转速为70-160r/min，发酵18-72h；所述的人参液为人参加水打浆。
4.根据权利要求3所述的提高稀有皂苷的人参发酵方法，其特征在于：所述的人参液为选取新鲜人参清洗后晾干，置于高压灭菌锅内，121℃维持40min，打开灭菌锅取出人参；按人参：水=1：1打浆，121℃，20min灭菌。
5.根据权利要求3或4所述的提高稀有皂苷的人参发酵方法，其特征在于：发酵条件为：36-38℃摇床培养，转速为80-120r/min，发酵18-48h。
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| CN109182441A (zh) * | 2018-08-31 | 2019-01-11 | 吉林农业大学 | 人参皂苷Rb1发酵液的制备方法及其应用 |
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