CN107801905A - 一种发酵薏米富集川芎嗪和溶纤酶功效成分的方法 - Google Patents
一种发酵薏米富集川芎嗪和溶纤酶功效成分的方法 Download PDFInfo
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Abstract
本发明涉及保健食品技术领域,尤其是一种发酵薏米富集川芎嗪和溶纤酶功效成分的方法,采用枯草芽孢杆菌发酵薏米,不仅能够获得具有高纤溶活性的纤溶酶,而且还能够获得可以作为钙离子拮抗药的川芎嗪以及用途广泛的乙偶姻。与同菌株发酵黄豆中的川芎嗪相比,发酵薏米中川芎嗪含量是黄豆的150倍以上,乙偶姻是其的4倍以上,溶纤酶相当。
Description
技术领域
本发明涉及保健食品技术领域,尤其是一种发酵薏米富集川芎嗪和溶纤酶功效成分的方法。
背景技术
薏米又名薏苡仁或薏仁米,俗称“药王米”、“回回米”、“六谷米”,自古以来就是一种声誉卓著的滋补食品。据化验分析,薏米含蛋白质高达18-21%、脂肪4-6%、碳水化合物79.2%,还富含多种维生素(尤其是B族维生素)、人体所需的氨基酸及矿物质、薏苡素及薏苡酯等多种活性成分,营养非常丰富。薏米具有消食、利尿、化脓、镇痛、消肿、润肤、美容、消除疲劳、预防高血压及促消化等功效,特别是其中含有的薏苡酯能很好地抑制某些癌细胞的生长。所以薏米是很好的药食两用功能性食品原料,正日益成为人们理想的健康营养食品。
川芎嗪(Tetramethylpyrazine,TMP),又名四甲基吡嗪,最早是从川芎中提取的具有生物活性成分的药效成分。川芎嗪具有扩张血管、改善微循环及抑制血小板集聚等药理作用,同时也能抑制肿瘤细胞生长及转移、调控细胞生长、抑制细胞凋亡,缺血/再灌注损伤保护作用、保护视力及预防白内障、预防糖尿病及并发症等作用。
溶纤酶(Fibrinolytic Enzyme)是在豆豉发酵过程由枯草杆菌产生的一种丝氨酸蛋白酶,具有明显溶栓作用和高纤溶活性的激酶,并能刺激静脉内皮细胞产生纤溶酶原激活剂,从而更有效地发挥溶栓效果。可用于治疗和预防血栓病,它可经口服直接溶栓,同时激活体内的纤溶酶原而增加内源性纤溶酶的活性,不仅溶栓能力强,而且无毒副作用。
乙偶姻(Acetoin)是众多微生物的代谢产物,也是川芎嗪合成的前体物质,可广泛应用于制药行业、化工行业等。乙偶姻和川芎嗪可由再生原料发酵产生。1962年,Kos-uge和Kamiya第一次报道利用微生物发酵产生川芎嗪。法国Besson等人利用B.subtilis IFO3013以大豆为培养基固态发酵生产川芎嗪,通过添加适量的乙偶姻,发酵14天后,川芎嗪的产量可达2.5g/kg。
为此,现有技术中,有研究者采用枯草芽孢杆菌发酵大豆合成川芎嗪,得到川芎嗪含量为0.92mg/100g;如专利公开号为CN101503718A。可是,现有技术中,并未有研究者采用枯草芽孢杆菌发酵薏米,富集川芎嗪、溶纤酶等活性成分的研究。
发明内容
为了解决现有技术中存在的上述技术问题,本发明提供一种发酵薏米富集川芎嗪和溶纤酶功效成分的方法,采用枯草芽孢杆菌或纳豆芽孢杆菌接种到熟化薏米中发酵,富集川芎嗪和溶纤酶功效成分。
具体是通过以下技术方案得以实现的:
发酵薏米富集川芎嗪和溶纤酶功效成分的方法,包括以下步骤:
(1)将枯草芽孢杆菌菌种活化,并培养,得到种子液;
(2)将种子液接种到熟化薏米中,于37℃恒温培养3天,得到含有川芎嗪、溶纤酶和乙偶姻的薏米发酵产物。
在上述枯草芽孢杆菌菌种活化是将枯草芽孢杆菌接种到LB斜面培养基上,在37℃恒温活化培养18-22h;再将其转接到LB液体培养基中,在180rpm、37℃下,于恒温恒压床上培养18-22h;得到种子液,该种子液中,活菌数为106-107cfu/ml。
上述的LB斜面培养基是100mL培养基中,含有0.5g氯化钠,0.3g牛肉膏,1g蛋白胨,1.5~2g琼脂,余量为水,pH自然。
上述的LB液体培养基是100mL培养基中,含有0.5g氯化钠,0.3g牛肉膏,1g蛋白胨,余量为水,pH自然。
上述的熟化薏米,是将20g薏米浸泡过夜,清洗,将薏米与蒸馏水按照1:1~3(g/ml),优选为1:1.5(g/ml)混合,置于250ml三角瓶中,优选在121℃下蒸煮灭菌处理30min。
上述种子液与熟化薏米的重量比为1~10:100(ml/g),优选为7:100(ml/g)。
将得到的薏米发酵产物,经过如下处理,实现活性成分的提取:
将薏米发酵产物称取一部分,加入2倍重量的生理盐水,浸泡24h,离心,取上清液,即得含溶纤酶的溶液;采用纤维蛋白平板法测定溶纤酶酶活。
将薏米发酵产物称取一部分,加入有机溶剂,超声提取,并离心,取上清液,得到含乙偶姻和川芎嗪的有机液。
薏米发酵产物与有机溶剂的比为1:5~20(g/ml),优选1:10(g/ml),分别提取两次,保证充分提取川芎嗪。
上述的有机溶剂是乙醇和水的混合溶液,乙醇质量用量为40-80%,余量为水;乙醇优选80%。
上述的超声提取,超声功率为220-600w,优选400w,超声处理时间为10-60min,优选30min,次数为2。
对含乙偶姻和川芎嗪的有机液检测分析:
经过微孔膜处理后,采用Agilent 1260液相色谱仪进行定量检测,其检测条件如下:流动相,甲醇:超纯水(含1%乙酸,0.05%三氟乙酸)=30:70,色谱柱Phecda C18(4.6×250mm,5ul),柱温:45℃,流速:0.8ml/min,检测波长:297nm,进样量:20ul。
采用枯草芽孢杆菌发酵薏米,不仅能够获得具有高纤溶活性的纤溶酶,而且还能够获得可以作为钙离子拮抗药的川芎嗪以及用途广泛的乙偶姻。相比同菌株发酵黄豆中的川芎嗪相比,发酵薏米中川芎嗪含量是黄豆的150倍以上(发酵黄豆中川芎嗪含量为0.032mg/g,薏米中含量为4.69mg/g),乙偶姻是其的4倍以上(发酵黄豆中乙偶姻为16.35mg/g,薏米中含量为69.80mg/g),溶纤酶相当(发酵黄豆中溶纤酶酶活4135U/g,薏米中酶活为3826.41U/g)。
在发酵薏米过程中,原料来源丰富易取,发酵过程无污染,安全性高,易于控制且成本较低。本发明为薏米高活性、高附加值的开发利用提供了新途径,同时为乙偶姻和川芎嗪的生物合成提供了新思路和新理论。
具体实施方式
下面结合具体的实施方式来对本发明的技术方案做进一步的限定,但要求保护的范围不仅局限于所作的描述。
实施例1
(1)菌种活化:
配制LB斜面培养基:每100ml培养基中含有0.5g氯化钠,1g蛋白胨,0.3g牛肉膏,1.5-2.0g琼脂,其余为水,pH自然。
灭菌处理:选择在121℃下灭菌处理20min;
接种培养:在无菌条件下,将枯草芽孢杆菌接种于LB培养基上,于37℃培养活化18-22h。
(2)种子液培养:
配制LB液体培养基:每100ml培养基中含有0.5g氯化钠,1g蛋白胨,0.3g牛肉膏,其余为水,pH自然。
灭菌处理:选择在121℃下灭菌处理20min,在无菌条件下,将LB斜面培养基中的枯草芽孢杆菌接种与三角瓶中的LB液体培养基中,在37℃,180rmp的摇床上,恒温恒压,培养20-22h,得到种子液。
(3)发酵:
将20g薏精米,采用蒸馏水浸泡过夜,清洗,添加25mL蒸馏水,置于250mL的广口三角瓶中,高温灭菌处理,灭菌条件为21℃下蒸煮灭菌处理30min;
将种子液0.2ml接种到上述广口三角瓶中,在37℃培养箱中静态培养3天,得到含川芎嗪、溶纤酶和乙偶姻的薏精米发酵产物。
(4)溶纤酶测定:
将薏精米发酵产物,称取5g于试管中,加入10ml生理盐水,置于冰箱中,冷冻浸泡24h,离心,取上清液10ul,采用纤维蛋白平板测量溶纤酶酶活,结果为1368.52U/g。
(5)测定川芎嗪、乙偶姻:
①干燥:
将含川芎嗪、溶纤酶和乙偶姻的薏精米发酵产物置于低温冰箱中,迅速冷冻干燥24h,将干燥粉末过60目筛;
②提取:
将过筛粉末称取3g,与有机溶剂,即就是80%的乙醇水溶液按照质量比为1:10比例混合,在400W功率的超声波下处理30min,再在8000rmp/min的转速上,离心15min,取上清液,将滤渣再按以上操作重复一次,收集上清液;
③测定:
将上清液进行HPLC检测,测得薏精米发酵产物中川芎嗪含量为1.742mg/g(干基),乙偶姻含量为68.41mg/g。
实施例2
将实施例1中的种子液接种到广口三角瓶中的量调整为1ml;发酵步骤中,对于灭菌条件为121℃处理30min;提取步骤中,过筛粉末与有机溶剂质量比为1:5;其他均不变,并按照实施例1的操作方式进行检测,结果为:溶纤酶酶活为3280.26U/g。
测得薏精米发酵产物中川芎嗪含量为5.167mg/g(干基),乙偶姻含量为91.43mg/g。
实施例3
将实施例1中的种子液接种到广口三角瓶中的量调整为1.4ml;提取步骤中,过筛粉末与有机溶剂质量比为1:5;其他均不变,并按照实施例1的操作方式进行检测,结果为:溶纤酶酶活为3866.51U/g。测得薏精米发酵产物中川芎嗪含量为6.731mg/g(干基),乙偶姻含量为132.86mg/g。
实施例4
将实施例1中的薏精米调整薏糙米;发酵步骤中,对于灭菌条件为121℃处理30min;向250ml广口三角瓶中加入蒸馏水的量为28ml,其他均不变,并按照实施例1的操作方式进行检测,结果为:溶纤酶酶活为3361.25U/g。测得薏糙米发酵产物中川芎嗪含量为5.167mg/g(干基),129.64mg/g。
实施例5
将实施例1中的薏精米调整薏碎米;发酵步骤中,对于灭菌条件为121℃处理30min;向250ml广口三角瓶中加入蒸馏水的量为32ml,其他均不变,并按照实施例1的操作方式进行检测,结果为:溶纤酶酶活为3156.81U/g。测得薏碎米发酵产物中川芎嗪含量为4.33mg/g(干基),66.84mg/g。
实施例6
其他按照实施例1,将实施例1中的枯草芽孢杆菌采用纳豆芽孢杆菌替换;将实施例1中的种子液接种到广口三角瓶中的量调整为1ml;发酵步骤中,对于灭菌条件为121℃处理30min;接种种子液后的发酵温度为30℃,其他均不变,并按照实施例1的操作方式进行检测,结果为:纳豆激酶酶活为3959.46u/g。测得薏米发酵产物中川芎嗪含量为3.562mg/g(干基),乙偶姻41.37mg/g。
Claims (10)
1.一种发酵薏米富集川芎嗪和溶纤酶功效成分的方法,其特征在于,采用枯草芽孢杆菌或纳豆芽孢杆菌接种到熟化薏米中发酵,促使川芎嗪和溶纤酶功效成分富集。
2.如权利要求1所述的发酵薏米富集川芎嗪和溶纤酶等功效成分的方法,其特征在于,包括以下步骤:
(1)将枯草芽孢杆菌菌种活化并培养,得到种子液;
(2)将种子液接种到熟化薏米中,于37℃恒温培养3天,得到含有川芎嗪、溶纤酶和乙偶姻的薏米发酵产物。
3.如权利要求2所述的发酵薏米富集川芎嗪和溶纤酶功效成分的方法,其特征在于,所述的菌种活化,是将枯草芽孢杆菌接种于LB斜面培养基上,在37℃恒温活化培养18-22h。
4.如权利要求2所述的发酵薏米富集川芎嗪和溶纤酶功效成分的方法,其特征在于,所述的种子液培养,是将活化后的菌种接种到LB液体培养基中,在180rpm、37℃下,于恒温恒压床上培养18-22h。
5.如权利要求3所述的发酵薏米富集川芎嗪和溶纤酶功效成分的方法,其特征在于,所述的LB斜面培养基是100mL培养基中,含有0.5g氯化钠,0.3g牛肉膏,1g蛋白胨,1.5~2g琼脂,余量为水,自然pH。
6.如权利要求4所述的发酵薏米富集川芎嗪和溶纤酶功效成分的方法,其特征在于,所述的LB液体培养基是100mL培养基中,含有0.5g氯化钠,0.3g牛肉膏,1g蛋白胨,余量为水,自然pH。
7.如权利要求2所述的发酵薏米富集川芎嗪和溶纤酶功效成分的方法,其特征在于,所述的种子液,其活菌数为1×106~1×107cfu/mL。
8.如权利要求1或2所述的发酵薏米富集川芎嗪和溶纤酶功效成分的方法,其特征在于,所述的熟化薏米,是将薏米浸泡过夜后,清洗,并加1.5倍蒸馏水后,置于灭菌锅中,在121℃下蒸煮灭菌处理30min。
9.一种发酵薏米富集川芎嗪和溶纤酶功效成分并提取的方法,其特征在于,包括以下步骤:
(1)按照权利要求2中的步骤(1)、步骤(2)对薏米进行发酵,得到含有川芎嗪、溶纤酶和乙偶姻的薏米发酵产物;
(2)将薏米发酵产物置于离心管中,加入2倍质量的生理盐水,静置24h,离心,得到含溶纤酶的溶液;或者将薏米发酵产物迅速冷冻后,干燥,将干燥粉末过60目筛,并加入有机溶剂,超声提取,离心,去除渣,得到含川芎嗪和乙偶姻的溶液。
10.如权利要求9所述的发酵薏米富集川芎嗪和溶纤酶功效成分并提取的方法,其特征在于,所述的有机溶剂,是质量浓度为80%的乙醇;所述的超声提取是料液质量比为1:10,于20-50℃下,超声提取30min,在8000rmp/min下离心处理15min,重复提取两次,即得。
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