CN113576991A - 一种铁皮石斛发酵液的制备方法 - Google Patents
一种铁皮石斛发酵液的制备方法 Download PDFInfo
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- CN113576991A CN113576991A CN202110901480.9A CN202110901480A CN113576991A CN 113576991 A CN113576991 A CN 113576991A CN 202110901480 A CN202110901480 A CN 202110901480A CN 113576991 A CN113576991 A CN 113576991A
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- dendrobium officinale
- fermentation
- liquid
- escherichia coli
- yeast
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Abstract
本发明公开了一种铁皮石斛发酵液的制备方法,属于微生物领域。该方法包括以下步骤:S1:从酵母菌保存斜面上取孢子接种于活化培养基上进行活化,得到活化后的酵母菌;S2:将S1中得到的活化后的酵母菌块接种到种子培养基中进行扩大培养,得到种子液;S3:取出S2中得到的种子液接种到含有铁皮石斛的发酵培养基中进行发酵,得到铁皮石斛发酵物。本发明提供的铁皮石斛发酵液制备方法简单、高效,可对铁皮石斛的发酵产物研究提供新方法,并且发酵液中富含多糖、黄酮与酚等活性物质。
Description
技术领域
本发明涉及一种发酵技术领域,尤其涉及铁皮石斛发酵技术。
背景技术
铁皮石斛(Dendrobium officinale),兰科石斛属,又称为“黑节草”或“铁皮枫斗”,其根为肉质气生根,干燥茎为名贵中药铁皮石斛,具有"植物黄金"的美誉,是中医传统养阴重要用药。铁皮石斛含有多糖、生物碱、氨基酸和微量元素等物质,此外还有联苄类化合物和菲类化合物、香豆素类等物质。它在医学上有免疫调节作用、抗肿瘤作用、降血糖作用、抗肝功能损伤作用,此外还有抗衰老、抗氧化的作用。此外,铁皮石斛具有“益胃”“生津”的功效,“益胃”主要与提高胃蛋白酶活性的作用有关,在生活中铁皮石斛常用来改善肠胃疾病症状;“生津”则是与铁皮石斛具有促进唾液分泌作用有关。
随着生物酶技术的发展,普遍采用酶分离法来获得原生质体。常用酶制剂有果胶酶、纤维素酶、木聚糖酶等酶制剂,而纤维素酶具有使细胞壁被破坏的能力。酵母菌被广泛应用于传统发酵食品行业,是酱油、料酒、馒头、面包等食品的关键发酵菌种。酵母菌在不同培养基以及培养条件下生长速度、产孢丝有所不同。
若能优化高产纤维素酶和酵母菌的混合菌株发酵条件,使铁皮石斛发酵液中的化学物质产出增加,是能为工业应用带来重要经济价值的。
发明内容
本发明的目的在于提供一种铁皮石斛发酵液的制备方法,能够解决铁皮石斛发酵液中的化学物质产出不足的问题。
为了达到上述目的本发明采用如下技术方案:
一种铁皮石斛发酵液的制备方法,包括以下步骤:
S1:从酵母菌与重组大肠杆菌保存斜面上取孢子分别接种于活化培养基上进行活化培养至OD600=0.6,得到活化后的酵母菌与重组大肠杆菌,重组大肠杆菌的基因序列如SEQ ID NO.1所示;
S2:将S1中得到的活化后的酵母菌与重组大肠杆菌菌块分别接种到各自种子培养基中进行扩大培养,得到种子液;
S3:取出S2中得到的种子液接种到含有铁皮石斛的发酵培养基中进行发酵,得到铁皮石斛发酵物。
进一步地,所述活化培养基按重量百分比计,组成为1%胰蛋白胨、1%酵母粉、1%葡萄糖、0.5%氯化钠、0.1%磷酸氢二钾,余量用双蒸水溶解定容;活化条件是37℃、220rpm。
进一步地,所述发酵培养基包括:碳源、铁皮石斛、蒸馏水、种子液即是混菌,混菌包含酵母菌与重组大肠杆菌。
进一步地,所述碳源选自葡萄糖、果糖、麦芽糖、乳糖、甘油,其用量是体积分数2%-10%;
所述铁皮石斛、蒸馏水的料液比g/mL是1:3~5;
所述酵母菌与重组大肠杆菌的体积比是1~3:1~3。
进一步地,所述碳源是乳糖;
所述发酵培养基包括:乳糖,铁皮石斛,蒸馏水,混合菌液;
各成分用量比例为:乳糖体积分数是6%,铁皮石斛5g,铁皮石斛:蒸馏水g/mL=1:4.5,混菌5mL,酵母菌菌液:大肠杆菌菌液mL=2:3。
进一步地,所述发酵的条件为温度4~50℃,发酵天数是1~5d。
进一步地,所述发酵的条件为温度37℃,发酵天数4d。
本发明的优点包括:本发明的铁皮石斛发酵酶液的制备方法简单,进一步优化发酵培养基,将筛选合适的碳源浓度、料液比、菌株配比、发酵温度以及发酵天数,使整体方案达到最优,促进铁皮石斛的化学物质的渗出,以及其发酵液抗氧化活性的提高,可对铁皮石斛的发酵产物研究提供新方法。
附图说明
此处所说明的附图用来提供对本发明的进一步理解,构成本申请的一部分,并不构成对本发明的不当限定,在附图中:
图1是不同碳源的发酵物对羟基自由基清除效果实验统计图;
图2是不同乳糖加入量的发酵物对羟基自由基清除效果实验统计图;
图3是不同菌种配比的发酵物对羟基自由基清除效果实验统计图;
图4是不同发酵温度的发酵物对羟基自由基清除效果实验统计图;
图5是不同料液比的发酵物对羟基自由基清除效果实验统计图;
图6是不同发酵时间的发酵物对羟基自由基清除效果实验统计图。
具体实施方式
下面将结合附图以及具体实施例来详细说明本发明,在此以本发明的示意性实施例及说明用来解释本发明,但并不作为对本发明的限定。
本说明书中采用的实验材料、试剂和方法如下:
1实验材料,试剂和仪器
材料:铁皮石斛,产于浙江衢州;酵母菌,重组大肠杆菌,均保存于广东药科大学基础学院分子生物学实验室。
试剂:水杨酸、乙醇、FeSO4、30%过氧化氢、葡萄糖、苯酚、没食子酸、 Folin试剂、无水Na2CO3、浓硫酸、亚硝酸钠、硝酸铝、氢氧化钠、透明质酸酶、透明质酸钠、冰醋酸、乙酰丙酮、对二甲氨基苯甲醛、氯化钙、一水合柠檬酸、十二水合磷酸二氢钠、酪氨酸酶、左旋多巴。去离子水、蒸馏水本实验室自制。
仪器:分析天平,紫外可见光分光光度计,台式高速冷冻离心机,恒温恒湿培养箱,双人单面净化工作台,高压蒸汽灭菌锅,pH计,电热鼓风干燥箱。
2实验方法
铁皮石斛发酵液的制备方法,包括以下步骤:
S1:从酵母菌与重组大肠杆菌保存斜面上取孢子分别接种于活化培养基上进行活化,得到活化后的酵母菌与重组大肠杆菌;
重组大肠杆菌的基因序列已提交至美国国立生物技术信息中心数据库 (NCBI),登录号为MT653325,核苷酸序列如SEQ ID NO.1所示。
SEQ ID NO.1
ATGGGATCCG AACATCATCA TCATCATCAT GAAGTGTCCG CACCTCTCAC
CCTGCCCACC GGCCCCGACT TCCTCTGGGG GGCGTCGACC GCCGCACACC
AGATCGAGGG CGGCAACGTC AACAGCGACT GGTGGGCCAA GGAGCACGTC
CCCGGCACGC TGTGCGCCGA GAGCTCGGGC GACGCCGTCG ACAGCTACCA
CCGTTACCCC GAGGACGTCC GCCTGCTCGC CGACGCCGGC CTCGACACCT
ACCGGTTCTC GGTCGAGTGG GCGCGCATCG AGCCGGCGGA GGGCGAGTTC
TCGCGGGCGC AGCTCGCGCA CTACCGGCGC ATGATCGACA CCTGCCTGGC
CGCGGGCGTC ACGCCGATGG TCACGCTGCT GCACTTCACC CTGCCGCGCT
GGTTCGCCGA GCGGGGCGGC TGGCTCGCGC CCGACGCCGT CGACACCTAC
CTGCGGTACG TCGAGACCGT CGCGACGATC CTCGACGGCG TGCGGCACGT
CGTGACGATC AACGAGCCCA ACATCACCTC GATGATGCTC GGCGCCAGCC
GGCGCACCGA CCTCGACGCG GCCGGGCTGG CCGCGCCGGA CCAGGAGGCC
TCCGCCGTCC AGGCCCGCGC CCACCGCGGC GCCGTCGACA TCCTCCGGGC
CCGCGGCCAC CAGGCCGGGT GGACGGTCGC CAACCAGGTG TTCCAGGCCG
CGCCGGGCGC CGAGGCGGCC CGCGACGCCT ACGCCTACCC GCGCGAGGAC
TTCTTCCTCG AGGTGTCCCG CGACGACGAC TTCGTCGGGG TCCAGAGCTA
CCTGCGCACC ATCATCGGGC CCGACGGCGA GCCGGTGCCG TTCGGCGAA
按重量百分比计,活化培养基的组成为:1%胰蛋白胨、1%酵母粉、1%葡萄糖、0.5%氯化钠、0.1%磷酸氢二钾,余量用双蒸水溶解定容,121℃,20min 高压灭菌后,分别加入酵母菌与重组大肠杆菌37℃、220rpm活化培养至 OD600=0.6。
S2:将S1中得到的活化后的酵母菌与重组大肠杆菌菌块分别接种到各自种子培养基中进行扩大培养,得到种子液;
S3:取出S2中得到的种子液(混菌)接种到含有铁皮石斛的发酵培养基中进行发酵,得到铁皮石斛发酵物;
得到的发酵培养基包括:碳源,混菌的用量5mL,铁皮石斛5g,蒸馏水。
3水杨酸法测定发酵液的羟自由基清除能力,具体方法步骤如下:
以Fenton反应法测定,H2O2+Fe2+=OH-+H2O+Fe3+,羟基自由基与水杨酸反应生成焦儿茶酸,其在510nm处有最大吸收波长。各溶液的加入量如表1 所示,在试管中依次加入9mmol/LFeSO4,9mmol/L乙醇-水杨酸,发酵物样品,接着加入适量蒸馏水,最后加入8.8mmol/L H2O2后摇匀,37℃水浴加热15min 后取出,测其吸光度,记为A0、Ax、Ax0。
清除率计算公式:羟自由基清除率(%)=A0-(Ax-Ax0)/A0×100%
表1清除羟自由基实验办法(单位mL)
4考察发酵培养基中各因素影响,具体方法步骤如下:
首先选取不同类型碳源(葡萄糖、果糖、麦芽糖、乳糖、甘油),以体积分数10%的浓度添加入石斛发酵体系中,发酵结束后,测定羟基自由基清除率,以确定较优碳源,结果如图1所示,其中最优碳源是乳糖,并在0%~10%范围内对其进行浓度寻优,结果如图2所示,其中最优浓度为6%。确定最优碳源后,依次改变菌种配比(酵母菌:重组大肠杆菌mL=3:1、3:2、1:1、2:3、1:3)、发酵温度(4℃、25℃、37℃、50℃)、料液比(铁皮石斛:蒸馏水g/mL=1:3、1:3.5、1:4、1:4.5、1:5)、发酵天数(1d~5d)等因素的取值水平,考察各因素对铁皮石斛发酵液抗氧化活性的影响,结果如图3-6所示,最优菌种体积配比为酵母菌:重组大肠杆菌=2:3;最优温度为37℃;最优料液比(g:mL)为1: 4.5;最优发酵天数是4d。
实施例1
综上实验可知,最佳制备方法如下:
S1、S2的步骤同上文“2实验方法”中铁皮石斛发酵液的制备方法,
S3步骤中,发酵培养基包括:乳糖6%(体积比v/v),铁皮石斛5g,蒸馏水,其中铁皮石斛:蒸馏水(g/mL)=1:4.5,混菌的用量5mL,其中酵母菌菌液:大肠杆菌菌液(mL)=2:3;
发酵的条件为温度37℃,发酵天数4d(96小时)。
参比实施例1
S1、S2的步骤同实施例1,不同的是S3中使用蒸馏水代替混菌种子液进行发酵,其他条件不变。
参比实施例2
S1、S2的步骤同实施例1,不同的是S2中只将酵母菌菌块接种到种子培养基中进行扩大培养,S3只加入酵母菌,其他条件不变。
参比实施例3
S1、S2的步骤同实施例1,不同的是S2中只将重组大肠杆菌菌块接种到种子培养基中进行扩大培养,S3只加入重组大肠杆菌,其他条件不变。
试验例1最适发酵条件下的总多糖含量测定
多糖在硫酸的作用下,先水解成单糖,并脱水生成糖醛衍生物,与苯酚生成橙黄色溶液,其在490nm处有最大吸收,吸收值与糖含量呈线性关系。
实验步骤:(1)标准曲线绘制:准确称取葡萄糖50mg于500mL容量瓶中配成0.1mg/mL的标准溶液母液,用蒸馏水分别配成0、0.02、0.04、0.06、0.08、0.1mg/mL的标准液。不同浓度的1mL标准液分别加入5%苯酚1mL,并迅速加入浓硫酸5mL,静置10min。摇匀,30℃放置30min后于490nm测定OD 值,以葡萄糖含量为横坐标,OD值为纵坐标,绘制标准曲线。
(2)样品含量测定:吸取1.0mL样品液,按上述步骤测定OD值,并代入标准曲线计算样品中的总糖含量,测定结果如表2所示。铁皮石斛总多糖得率测定:铁皮石斛投料量为A,所得铁皮石斛总多糖量为A1,总多糖得率计算为 B,计算公式为B=A1/A×100%。
表2
由表2可知,实施例1的多糖得率最高。
试验例2
最适发酵条件下的总酚含量测定,具体方法步骤如下:
以没食子酸为对照品,Folin-Cioealteau显色法测定多酚含量。没食子酸的标准曲线绘制:采用准确称取0.011g没食子酸,先加入少量甲醇溶解,再加蒸馏水定容到100mL,得到适当浓度的没食子酸溶液,分别取0.4、0.6、0.8、1.0、 1.2、1.4、1.6mL体积置于试管中,加入2%Na2CO3溶液,混匀,2min后加入 0.5mL 50%Folin-Cioealteau试剂,补水至5mL,混匀,静置30min,于750nm 测定OD值。样品总酚含量测定的步骤参照标准测定没食子酸溶液的方法操作,每个样品做3次重复,总酚含量根据标准物没食子酸标准曲线确定的方程进行计算,测定结果如表3所示。铁皮石斛总酚得率测定:铁皮石斛投料量为A,所得铁皮石斛总酚量为A1,总酚得率计算为B,计算公式为B=A1/A×100%。
表3
由表3可知,实施例1的总酚得率最高。
试验例3
最适发酵条件下的总黄酮含量测定,具体方法步骤如下:
标准曲线绘制:称取芦丁对照品10mg,80%乙醇溶解并定容到50mL,制备成浓度为0.2mg/mL,准确吸取0.00、0.20、0.40、0.60、0.80、1.00mL标准液,补水至2mL,加0.2mL亚硝酸钠溶液,混匀,放置6min,加0.2mL硝酸铝溶液,混匀,放置6min,加0.8mL氢氧化钠溶液,加水至5mL,摇匀,放置15min。在波长490nm处测定吸光度值。以吸光度为纵坐标,芦丁质量浓度为横坐标绘制标准曲线。
样品总黄酮含量测定:吸取2mL样品液,加0.2mL亚硝酸钠溶液,混匀,放置6min,加0.2mL硝酸铝溶液,混匀,放置6min,加0.8mL氢氧化钠溶液,加水至5mL,摇匀,放置15min。在波长490nm处测定吸光度值,结果如表4 所示。铁皮石斛总黄酮得率测定:铁皮石斛投料量为A,所得铁皮石斛总黄酮量为A1,总黄酮得率计算为B,计算公式为B=A1/A×100%。
表4
由表4可知,实施例1的总黄酮得率最高。
试验例4
铁皮石斛发酵液对透明质酸酶抑制试验,具体方法步骤如下:
(1)试剂配制
透明质酸酶:浓度500U/ml,现配现用,不能过夜,用醋酸缓冲液做溶剂;
透明质酸钠:0.5mg/ml,一次配制,多次使用,用醋酸缓冲液做溶剂;
Buffer:溶液A 0.2mol/L醋酸(11.55ml冰醋酸溶于1L蒸馏水中)4.8ml
溶液B 0.2mol/L醋酸钠(16.4g无水醋酸钠或27.2g三水合醋酸钠溶于1L 蒸馏水中)45.2ml,混合稀释至100ml,配制成PH=5.6的醋酸缓冲液;
乙酰丙酮溶液:50ml 1.0mol/L碳酸钠溶液和3.5ml乙酰丙酮溶液混合均匀(临用现配)
P-DAB显色剂:0.8g对二甲氨基苯甲醛溶于15ml浓盐酸和15ml无水乙醇混合均匀。
氯化钙溶液CaCl2:2.5mol/L
氢氧化钠溶液NaOH:5mol/L
(2)试验步骤
透明质酸酶抑制率(%)=[(C-D)-(A-B)]/(C-D)×100%
式中:
A—(透明质酸酶+样品+透明质酸钠)试样溶液的OD值;
B—(醋酸缓冲液+样品+醋酸缓冲液)试样空白的OD值;
C—(透明质酸酶+去离子水+透明质酸钠)对照溶液的OD值;
D—(醋酸缓冲液+去离子水+醋酸缓冲液)对照空白的OD值;
结果如下表5所示:
表5
由表5可知,实施例1的透明质酸酶抑制率最高
试验例5
铁皮石斛发酵液对酪氨酸酶抑制试验,具体方法步骤如下:
(1)试剂配制
pH6.8的磷酸氢二钠-柠檬酸缓冲液:
0.2mol/L Na2HPO4·12H2O:14.33g溶于200mL水,用玻棒搅拌至溶解;
②0.1mol/L一水合柠檬酸:2.1g溶于100mL水,用玻棒搅拌至溶解;
③pH6.8磷酸氢二钠-柠檬酸缓冲液:0.2mol/L Na2HPO4·12H2O 154.5mL,加入0.1mol/L一水合柠檬酸45.5mL,配制成200mL的磷酸氢二钠-柠檬酸缓冲液;
酪氨酸酶,活力≥1000unit/mg固体;
左旋多巴,纯度≥98%;
酪氨酸酶溶液:用pH6.8磷酸氢二钠-柠檬酸缓冲液配制,100u/mL,临用配制;
左旋多巴溶液:用pH6.8磷酸氢二钠-柠檬酸缓冲液配制,1mg/mL,避光保存;
(2)试验步骤
计算酪氨酸酶抑制率:
抑制率(%)=[1-(T-T0)/(C-C0)]×100%…………………………(1)
(1)式中:
T—样品管吸光值,即样品与酪氨酸酶反应后溶液吸光值;
T0—样品本底吸光值;
C—酶反应管吸光值3次平均值,即未加样品时酪氨酸酶和多巴反应的吸光值;
C0—溶剂本底吸光值T-T0
实验结果见表6
表6
由表6可知,实施例1的酪氨酸酶抑制率最高。
以上对本发明实施例所提供的技术方案进行了详细介绍,本文中应用了具体个例对本发明实施例的原理以及实施方式进行了阐述,以上实施例的说明只适用于帮助理解本发明实施例的原理;同时,对于本领域的一般技术人员,依据本发明实施例,在具体实施方式以及应用范围上均会有改变之处,综上所述,本说明书内容不应理解为对本发明的限制。
序列表
<110> 广东药科大学 、广州雅纯化妆品制造有限公司
<120> 一种铁皮石斛发酵液的制备方法
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 849
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgggatccg aacatcatca tcatcatcat gaagtgtccg cacctctcac cctgcccacc 60
ggccccgact tcctctgggg ggcgtcgacc gccgcacacc agatcgaggg cggcaacgtc 120
aacagcgact ggtgggccaa ggagcacgtc cccggcacgc tgtgcgccga gagctcgggc 180
gacgccgtcg acagctacca ccgttacccc gaggacgtcc gcctgctcgc cgacgccggc 240
ctcgacacct accggttctc ggtcgagtgg gcgcgcatcg agccggcgga gggcgagttc 300
tcgcgggcgc agctcgcgca ctaccggcgc atgatcgaca cctgcctggc cgcgggcgtc 360
acgccgatgg tcacgctgct gcacttcacc ctgccgcgct ggttcgccga gcggggcggc 420
tggctcgcgc ccgacgccgt cgacacctac ctgcggtacg tcgagaccgt cgcgacgatc 480
ctcgacggcg tgcggcacgt cgtgacgatc aacgagccca acatcacctc gatgatgctc 540
ggcgccagcc ggcgcaccga cctcgacgcg gccgggctgg ccgcgccgga ccaggaggcc 600
tccgccgtcc aggcccgcgc ccaccgcggc gccgtcgaca tcctccgggc ccgcggccac 660
caggccgggt ggacggtcgc caaccaggtg ttccaggccg cgccgggcgc cgaggcggcc 720
cgcgacgcct acgcctaccc gcgcgaggac ttcttcctcg aggtgtcccg cgacgacgac 780
ttcgtcgggg tccagagcta cctgcgcacc atcatcgggc ccgacggcga gccggtgccg 840
ttcggcgaa 849
Claims (7)
1.一种铁皮石斛发酵液的制备方法,其特征在于:
包括以下步骤:
S1:从酵母菌与重组大肠杆菌保存斜面上取孢子分别接种于活化培养基上进行活化培养至OD600=0.6,得到活化后的酵母菌与重组大肠杆菌,重组大肠杆菌的基因序列如SEQ IDNO.1所示;
S2:将S1中得到的活化后的酵母菌与重组大肠杆菌菌块分别接种到各自种子培养基中进行扩大培养,得到种子液;
S3:取出S2中得到的种子液接种到含有铁皮石斛的发酵培养基中进行发酵,得到铁皮石斛发酵物。
2.根据权利要求1所述的一种铁皮石斛发酵液的制备方法,其特征在于:
所述活化培养基按重量百分比计,组成为1%胰蛋白胨、1%酵母粉、1%葡萄糖、0.5%氯化钠、0.1%磷酸氢二钾,余量用双蒸水溶解定容;活化条件是37℃、220rpm。
3.根据权利要求1所述的一种铁皮石斛发酵液的制备方法,其特征在于:
所述发酵培养基包括:碳源、铁皮石斛、蒸馏水、种子液即是混菌,混菌包含酵母菌与重组大肠杆菌。
4.根据权利要求3所述的一种铁皮石斛发酵液的制备方法,其特征在于:
所述碳源选自葡萄糖、果糖、麦芽糖、乳糖、甘油,其用量是体积分数2%-10%;
所述铁皮石斛、蒸馏水的料液比g/mL是1:3~5;
所述酵母菌与重组大肠杆菌的体积比是1~3:1~3。
5.根据权利要求4所述的一种铁皮石斛发酵液的制备方法,其特征在于:
所述碳源是乳糖;
所述发酵培养基包括:乳糖,铁皮石斛,蒸馏水,混菌;
各成分用量比例为:乳糖体积分数是6%,铁皮石斛5g,铁皮石斛:蒸馏水g/mL=1:4.5,混菌5mL,酵母菌菌液:大肠杆菌菌液mL=2:3。
6.根据权利要求1所述的一种铁皮石斛发酵液的制备方法,其特征在于:
所述发酵的条件为温度4~50℃,发酵天数是1~5d。
7.根据权利要求1所述的一种铁皮石斛发酵液的制备方法,其特征在于:
所述发酵的条件为温度37℃,发酵天数4d。
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