CN115595332A - 一种复合酶联合微生物发酵制备银耳发酵液的方法 - Google Patents
一种复合酶联合微生物发酵制备银耳发酵液的方法 Download PDFInfo
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Abstract
本发明提供了一种复合酶联合微生物发酵制备银耳发酵液的方法,属于生物发酵技术领域。所述方法包括如下步骤:(1)将干银耳打成粉末,加入纯净水中,用柠檬酸调节pH为4‑5;(2)向其中加入果胶酶,在50‑60℃下酶解1‑3h;(3)继续向其中加入酸性纤维素酶,在50‑60℃下酶解1‑3h;(4)继续向其中加入中性蛋白酶,在50‑60℃下酶解1‑3h;(5)继续向其中加入复合菌,在25‑40℃下发酵130‑150h。本方法制备得到的银耳发酵液黏度低,多糖浓度高,产率优异,且多糖的分子量低,便于生物体吸收利用。
Description
技术领域
本发明涉及生物发酵技术领域,尤其是涉及一种复合酶联合微生物发酵制备银耳发酵液的方法。
背景技术
银耳作为我国传统的食用菌,历来都是深受广大人民所喜爱的食物。其营养成分相当丰富,在银耳中含有蛋白质、脂肪和多种氨基酸、矿物质、其中所含有的活性成分-银耳多糖更是具有特殊的保健功能。
蛋白质占干银耳重量的6%-10%,银耳含有17种氨基酸,即缬氨酸、脯氨酸、丝氨酸、精氨酸、甘氨酸、赖氨酸、丙氨酸、苏氨酸、亮氨酸、异亮氨酸、酪氨酸、苯丙氨酸、谷氨酸、胱氨酸、天门冬氨酸、甲硫氨酸、组氨酸等,其中谷氨酸含量最高,天冬氨酸次之,人体所必需8种氨基酸中的7种,银耳都可以提供,是良好的蛋白质来源。
银耳含有大约0.6%-1.28%的脂肪,其中不饱和脂肪酸约占总脂肪酸量的75%左右,其中主要成分是亚油酸。银耳中含有4.0%-5.44%的无机盐,如S、P、Ca、Fe、Mg和Na等。银耳中还含有多种维生素,即VB1、VB2、VC和VD等,此外银耳还含有一定量的纤维素。
银耳发酵液中含有蛋白质、氨基酸等有机化合物,其中银耳多糖含量突出,因此,对于银耳功能成分的提取常以银耳多糖的提取率、浓度、分子量等指标作为标准。
目前常用的提取银耳多糖的方法中,热水法提取时间长、收率低,而酸、碱浸提法又极易破坏银耳多糖立体旋光活性结构,酶法分离是研究的趋势。但是,酶分离法有一些难以克服的缺陷,如《酶法提取银耳多糖的研究》(林宇野等,食品与发酵工业,1995年第1期)中,银耳多糖的得率只有16.3%,且多糖的分子量很大,为71000-78000,不利于吸收利用;《银耳多糖的制备工艺及研究进展》(刘蔷等,生物产业技术,2019年9月)中,提到几种研究的银耳多糖得率为19.08-40.57%,生产效率较低;《银耳多糖最适提取工艺条件探析》(孙晨雨等,农产品加工,2018年4月第4期)中,银耳多糖质量浓度最高仅为0.191mg/ml;《银耳深层发酵条件的研究》(吴大康等,食品科学,2002,Vol.23,No.1)中的银耳浓度虽然达到了5.5mg/ml,但是需要加入大量的增稠剂和培养基,制备方法复杂,生产成本高。
有鉴于此,亟需开发一种工艺简单、得率好、浓度高、成本低的银耳发酵液提取方法。
发明内容
针对现有技术存在的上述问题,本发明提供了一种复合酶联合微生物发酵制备银耳发酵液的方法。本方法制备得到的银耳发酵液黏度低,多糖浓度高,产率优异,且多糖的分子量低,便于生物体吸收利用。
本发明的技术方案如下:
一种复合酶联合微生物发酵制备银耳发酵液的方法,包括如下步骤:
(1)将干银耳打成粉末,加入纯净水中,用柠檬酸调节pH为4-5;
(2)向其中加入果胶酶,在50-60℃下酶解1-3h;
(3)继续向其中加入酸性纤维素酶,在50-60℃下酶解1-3h;
(4)继续向其中加入中性蛋白酶,在50-60℃下酶解1-3h;
(5)继续向其中加入复合菌,在25-40℃下发酵130-150h。
优选的,步骤(1)所述干银耳的含水率小于15%;将干银耳打成粉末的过至少150目的筛网。
优选的,步骤(1)干银耳和纯净水的质量体积比为1g:40-60ml。
优选的,步骤(2)所述果胶酶的质量为干银耳质量的0.08-0.12%。
优选的,步骤(3)所述酸性纤维素的质量为干银耳质量的0.08-0.12%。
优选的,步骤(4)所述中性蛋白酶的质量为干银耳质量的0.08-0.12%。
优选的,步骤(5)所述复合菌是植物乳杆菌、德氏乳杆菌保加利亚亚种及嗜热链球菌的混合菌种,三者的质量比为1:1.8-2.2:1.8-2.2。
优选的,步骤(5)所述复合菌的质量为干银耳质量的1-1.5%。
本发明有益的技术效果在于:
1、本发明不同于现有技术一次性将几种酶添加进去,而是分步骤加入三种酶的。经过发明人大量实验发现,果胶酶作用于银耳细胞壁上的果胶,使细胞更容易破碎释放胞内多糖;进一步的纤维素酶作用与细胞壁作用,使细胞壁上的大分子多糖变成小分子多糖;最后蛋白酶使细胞内蛋白质变成小分子多糖甚至是氨基酸。
2、本发明得到的银耳发酵液黏度最低为25.5mPa·s,黏度降低后的发酵液便于工业方面后续料液分离;得到的多糖浓度最高可达4.57mg/ml,且多糖的分子量低于10000的占70%以上,便于生物体吸收利用;本发明得率最高为76.17%,是现有技术的1.5-2倍。
3、本发明在复合剂发酵后,只需进行过滤和去渣即可直接利用。银耳发酵液除了含有银耳多糖外,还含有多种氨基酸、矿物质及维生素,用途广泛,可食用,也可作为化妆品原料等。
附图说明
图1为实施例1制备得到的发酵液中的多糖分子量分布;
图2为实施例2在不同发酵温度下所得发酵液的黏度和多糖含量;
图3为实施例3在不同料液比下所得发酵液的黏度和多糖含量;
图4为实施例4在不同发酵时间下所得发酵液的黏度和多糖含量。
具体实施方式
下面结合附图和实施例,对本发明进行具体描述。显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
下述实施例中,果胶酶采用山东隆科特酶制剂有限公司食品级果胶酶,活力6万U/mL;酸性纤维素酶采用山东隆科特酶制剂有限公司食品级酸性纤维素酶,活力20万U/mL;中性蛋白酶采用山东隆科特酶制剂有限公司食品级中性蛋白酶,活力5万U/mL。
所用复合菌种中,植物乳杆菌采用科汉森公司Harvest LB-1,产品编号3580179;德氏乳杆菌保加利亚亚种和嗜热链球菌采用杜邦营养食品配料(北京)有限公司的混合菌种,名称为乳酸菌种TS-H 2084LYO 200DCU,产品编号61033903,该混合菌种中德氏乳杆菌保加利亚亚种和嗜热链球菌的质量比为1:1。
实施例1:
取40g干银耳打粉加入2000mL纯净水混合,加入适量柠檬酸调节pH至4.5;加入果胶酶0.1%,55℃酶解2h;酸性纤维素酶0.1%,55℃酶解2h;中性蛋白酶0.1%,55℃酶解2h;酶解结束后取酶解液检测其多糖含量、多糖分子量及黏度,检测结果见下表1。其中所用干银耳的含水率小于15%,打成粉末后过150目筛。
在上述所得酶解液中加入0.5g复合菌,在30℃发酵144h。发酵结束后取发酵液检测其多糖含量、多糖分子量及黏度,检测结果如下表1所示。
发酵液中的多糖分子量分布如图1所示,从图中可以看到产物的分子量大幅变小,其分子量分布都在1万以下,主要分布在9000分子量。
对比例1:
在实施例1的酶解液中加入0.5g植物乳杆菌,在30℃发酵144h。发酵结束后取发酵液检测其多糖含量、多糖分子量及黏度,检测结果如下表1所示。所述植物乳杆菌的菌种从国家菌种保藏管理中心获得,保藏号:CGMCC1.12732。
表1
实施例2:
在实施例1中的酶解液中加入复合菌0.5g后,分别在10℃、20℃、30℃、40℃、50℃发酵144h,实验结果如图2所示。从图2中可以看到,在发酵温度在30℃时,多糖含量达到最高,且此时溶液粘度最小。
实施例3:
将实施例1中的发酵料液比(即银耳与水的质量体积比)分别设定为1:30、1:40、1:50、1:60、1:70,pH为4.5,复合酶制剂酶解后,加入0.5g复合菌30℃发酵144h,实验结果如图3所示。从图3可以看到,在发酵料液比为1:50时,多糖含量较高,且溶液粘度较低。
实施例4:
在实施例1中的酶解液中加入0.5g复合菌,在30℃分别发酵96h、120h、144h、168h、192h,实验结果如图4所示。从图4可以看到,在发酵时间在144h时,多糖含量最高,且黏度整体无太大变化。
尽管本发明的实施方案已公开如上,但其并不仅仅限于说明书和实施方式中所列运用,它完全可以被适用于各种适合本发明的领域,对于熟悉本领域的人员而言,对于本领域的普通技术人员而言,在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,因此在不背离权利要求及等同范围所限定的一般概念下,本发明并不限于特定的细节。
Claims (8)
1.一种复合酶联合微生物发酵制备银耳发酵液的方法,其特征在于,包括如下步骤:
(1)将干银耳打成粉末,加入纯净水中,用柠檬酸调节pH为4-5;
(2)向其中加入果胶酶,在50-60℃下酶解1-3h;
(3)继续向其中加入酸性纤维素酶,在50-60℃下酶解1-3h;
(4)继续向其中加入中性蛋白酶,在50-60℃下酶解1-3h;
(5)继续向其中加入复合菌,在25-40℃下发酵130-150h。
2.根据权利要求1所述的方法,其特征在于,步骤(1)所述干银耳的含水率小于15%;将干银耳打成粉末的过至少150目的筛网。
3.根据权利要求1所述的方法,其特征在于,步骤(1)干银耳和纯净水的质量体积比为1g:40-60ml。
4.根据权利要求1所述的方法,其特征在于,步骤(2)所述果胶酶的质量为干银耳质量的0.08-0.12%。
5.根据权利要求1所述的方法,其特征在于,步骤(3)所述酸性纤维素的质量为干银耳质量的0.08-0.12%。
6.根据权利要求1所述的方法,其特征在于,步骤(4)所述中性蛋白酶的质量为干银耳质量的0.08-0.12%。
7.根据权利要求1所述的方法,其特征在于,步骤(5)所述复合菌是植物乳杆菌、德氏乳杆菌保加利亚亚种及嗜热链球菌的混合菌种,三者的质量比为1:1.8-2.2:1.8-2.2。
8.根据权利要求1所述的方法,其特征在于,步骤(5)所述复合菌的质量为干银耳质量的1-1.5%。
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