CN112335885A - 一种多菌联合发酵制备益生菌膳食补充剂的方法 - Google Patents
一种多菌联合发酵制备益生菌膳食补充剂的方法 Download PDFInfo
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Abstract
本发明涉及发酵益生菌的技术领域,特别是涉及一种多菌联合发酵制备益生菌膳食补充剂的方法,配料常见易于获得,无需过多的复杂工序,产品营养均衡,包括以下步骤:S1、制取酶解液,备用;S2、制取培养基,备用;S3、制取优势枯草芽孢杆菌和优势乳酸菌菌种,备用;S4、将步骤S3中制得的优势枯草芽孢杆菌接种至步骤S2中培养基中进行好氧发酵,接种比例为培养基重量的1%‑2%,发酵温度37摄氏度,发酵时间48小时,制得发酵液A;S5、将步骤S3中制得的优势乳酸菌接种至步骤S4发酵液A中进行厌氧发酵,制得发酵液B;S6、发酵液B进行离心,收集菌泥,将菌泥重悬于生理盐水,得到重悬液;S7、步骤S6中重悬液加入冻干保护剂。
Description
技术领域
本发明涉及发酵益生菌的技术领域,特别是涉及一种多菌联合发酵制备益生菌膳食补充剂的方法。
背景技术
膳食补充剂是一种旨在补充膳食的产品(而非烟草),它可能含有一种或多种如下膳食成分,一种维生素、一种矿物质、一种草本(草药)或其他植物、一种氨基酸、一种用以增加每日总摄入量来补充膳食的食物成分,或以上成分的一种浓缩物、代谢物、成分、提取物或组合产品等,也包括在得到批准、发证、许可前已作为膳食补充剂或食品上市的、已批准的新药、维生素或生物制剂,DSHEA对膳食补充剂作为定义规定了其组成内容和标记要求:产品形式可以使丸剂、胶囊、片剂或液体。
益生菌是通过定殖在人体内,改变宿主某一部位菌群组成的一类对宿主有益的活性微生物。通过调节宿主黏膜与系统免疫功能或通过调节肠道内菌群平衡,促进营养吸收保持肠道健康的作用,从而产生有利于健康作用的单微生物或组成明确的混合微生物。
人类对微生物资源的利用曾经经历过天然混菌培养至纯种培养2个阶段,纯培养技术使得研究者摆脱了面对多种微生物共存的复杂局面能够不受干扰的对单一目的菌株进行研究,但是,在长期的试验和生产实践中,发现很多重要的生化过程紧靠单种微生物不能完成或者只能微弱的进行,必须依靠2种或多种微生物共同培养来完成。
现有技术中,益生菌膳食补充剂多为多种物质的混合品,对身体健康影响不大,并且制作成本较高。
发明内容
为解决上述技术问题,本发明提供一种多菌联合发酵制备益生菌膳食补充剂的方法,配料常见易于获得,无需过多的复杂工序,产品营养均衡,降低制作成本。
本发明的一种多菌联合发酵制备益生菌膳食补充剂的方法,包括以下步骤:
S1、取干净的豆粕、花生粕加入到高混机内,均匀混合后,粉碎,过筛,加水拌匀,添加豆粕和花生粕总重2%的丝氨酸蛋白酶,加入添加氢氧化钠溶液将pH调节至9.0,并升温至55摄氏度,水解3-6小时,然后降温至50摄氏度,添加豆粕和花生粕总重3%的中性蛋白酶,添加稀盐酸将pH调节至6.5,水解2-3小时,然后用120目滤网过滤除去杂质,并通过添加稀盐酸或者氢氧化钠溶液将pH调节至6.5-7.0,然后在温度60摄氏度、均质压力20-25Mpa的条件下,用高压均质机均质3min,降温至37摄氏度,制得酶解液备用;
S2、在步骤S1中酶解液再加入蛋白胨、琼脂、磷酸二氢钾、脱脂乳粉、白砂糖、柠檬酸以及硫酸亚铁,并通过添加稀盐酸或者氢氧化钠溶液将pH调节至6.5-7.0,然后在高温蒸汽下灭菌,降温后制得培养基,备用;
S3、将枯草芽孢杆菌接种在步骤S2中培养基中进行划线培养,选取优势菌种,然后对优势菌种进行扩大培养,备用;将乳酸菌接种在步骤S2中培养基中进行划线培养,选取优势菌种,然后对优势菌种进行扩大培养,备用;
S4、将步骤S3中制得的优势枯草芽孢杆菌接种至步骤S2中培养基中进行好氧发酵,接种比例为培养基重量的1%-2%,发酵温度37摄氏度,发酵时间48小时,制得发酵液A;
S5、将步骤S3中制得的优势乳酸菌接种至步骤S4发酵液A中进行厌氧发酵,接种比例为培养基重量的6%-9%,发酵温度37摄氏度,发酵时间48小时,制得发酵液B;
S6、发酵液B进行离心,收集菌泥,将菌泥重悬于生理盐水,得到重悬液;
S7、步骤S6中重悬液加入冻干保护剂,充分混合后,添加稀盐酸者氢氧化钠溶液调节pH至6.5-7.0,真空冷冻干燥,然后粉碎装袋制得多菌联合发酵制备益生菌膳食补充剂。
本发明的一种多菌联合发酵制备益生菌膳食补充剂的方法,按重量份数计,步骤S1中豆粕50-100份,花生粕50-100份。
本发明的一种多菌联合发酵制备益生菌膳食补充剂的方法,按重量份数计,步骤S2中蛋白胨10-20份、琼脂10-20份、磷酸二氢钾2-5份、脱脂乳粉10-20份、白砂糖10-20份、柠檬酸2-5份以及硫酸亚铁2-5份。
本发明的一种多菌联合发酵制备益生菌膳食补充剂的方法,所述步骤S6中,菌泥与生理盐水重量比例为1:1-1.6。
本发明的一种多菌联合发酵制备益生菌膳食补充剂的方法,步骤S7重悬液中还加入调味剂。
本发明的一种多菌联合发酵制备益生菌膳食补充剂的方法,所述步骤S3中乳酸菌包括任意比例的植物乳杆菌、瑞士乳杆菌、嗜酸乳杆菌、长双歧杆菌、两歧双歧杆菌、短双歧杆菌。
与现有技术相比本发明的有益效果为:本发明的主要原料为豆粕和花生粕,都具有价格低廉的优势,豆粕蛋白质含量高,氨基酸种类繁多,但是存在多种抗营养分子,影响使用者吸收,花生粕含有的抗营养分子较少,并且富含粗蛋白质和粗纤维,但是通过添加枯草芽孢杆菌以及乳酸菌,好氧发酵过程中,枯草芽孢杆菌快速生长,产生丰富的蛋白酶、纤维素酶等水解酶类,将豆粕和花生粕中的纤维素和蛋白质降解为易吸收的小分子营养物,易于使用者进行快速吸收,并且同时消除豆粕中的抗营养分子,不会对人体产生伤害,厌氧发酵过程中,植物乳杆菌、瑞士乳杆菌、嗜酸乳杆菌、长双歧杆菌、两歧双歧杆菌、短双歧杆菌快速生长,生成的乳酸以及厌氧条件能够较好的抑制有害细菌的生长,其中两歧双歧杆菌、婴儿双歧杆菌和长双歧杆菌是人体的健康标志,是对人体有诸多好处的有益菌群,植物乳杆菌和瑞士乳杆菌具有调节肠道菌群、增强免疫力、保护胃黏膜、改善肠道功能、通便、防治腹泻、促进消化、抗肿瘤、抗氧化等作用,制得富含多种益生菌的膳食补充剂,大大降低生产成本,且工序简单,制得的膳食补充剂营养均衡,适合大范围推广。
具体实施方式
下面结合实施例,对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明,但不用来限制本发明的范围。
实施例1:
本发明的一种多菌联合发酵制备益生菌膳食补充剂的方法,包括以下步骤:
S1、取干净的豆粕50份、花生粕100份加入到高混机内,均匀混合后,粉碎,过筛,加水拌匀,添加豆粕和花生粕总重2%的丝氨酸蛋白酶,加入添加氢氧化钠溶液将pH调节至9.0,并升温至55摄氏度,水解3-6小时,然后降温至50摄氏度,添加豆粕和花生粕总重3%的中性蛋白酶,添加稀盐酸将pH调节至6.5,水解2-3小时,然后用120目滤网过滤除去杂质,并通过添加稀盐酸或者氢氧化钠溶液将pH调节至6.5-7.0,然后在温度60摄氏度、均质压力20-25Mpa的条件下,用高压均质机均质3min,降温至37摄氏度,制得酶解液备用;
S2、在步骤S1中酶解液再加入蛋白胨20份、琼脂20份、磷酸二氢钾5份、脱脂乳粉10份、白砂糖20份、柠檬酸5份以及硫酸亚铁5份,并通过添加稀盐酸或者氢氧化钠溶液将pH调节至6.5-7.0,然后在高温蒸汽下灭菌,降温后制得培养基,备用;
S3、将枯草芽孢杆菌接种在步骤S2中培养基中进行划线培养,选取优势菌种,然后对优势菌种进行扩大培养,备用;将乳酸菌(植物乳杆菌20%、瑞士乳杆菌10%、嗜酸乳杆菌20%、长双歧杆菌20%、两歧双歧杆菌10%、短双歧杆菌20%)接种在步骤S2中培养基中进行划线培养,选取优势菌种,然后对优势菌种进行扩大培养,备用;
S4、将步骤S3中制得的优势枯草芽孢杆菌接种至步骤S2中培养基中进行好氧发酵,接种比例为培养基重量的2%,发酵温度37摄氏度,发酵时间48小时,制得发酵液A;
S5、将步骤S3中制得的优势乳酸菌接种至步骤S4发酵液A中进行厌氧发酵,接种比例为培养基重量的6%,发酵温度37摄氏度,发酵时间48小时,制得发酵液B;
S6、发酵液B进行离心,收集菌泥,将菌泥重悬于生理盐水,菌泥与生理盐水重量比例为1:1,得到重悬液;
S7、步骤S6中重悬液加入冻干保护剂和调味剂,充分混合后,添加稀盐酸者氢氧化钠溶液调节pH至6.5-7.0,真空冷冻干燥,然后粉碎装袋制得多菌联合发酵制备益生菌膳食补充剂。
实施例2:
与实施例1主要区别在于:S1中豆粕和花生粕份数,步骤S2中蛋白胨、琼脂、磷酸二氢钾、脱脂乳粉、白砂糖、柠檬酸以及硫酸亚铁的份数,步骤S4中优势枯草芽孢杆菌接种比例,步骤S5中优势乳酸菌接种比例,步骤S6中菌泥与生理盐水重量比例;
实施例3:
与实施例1主要区别在于:S1中豆粕和花生粕份数,步骤S2中蛋白胨、琼脂、磷酸二氢钾、脱脂乳粉、白砂糖、柠檬酸以及硫酸亚铁的份数,步骤S4中优势枯草芽孢杆菌接种比例,步骤S5中优势乳酸菌接种比例,步骤S6中菌泥与生理盐水重量比例;
实施例1、实施例2以及实施例3区别点详见表1:
表1
对比例1:
与实施例1区别在于:未添加优势枯草芽孢杆菌;
S1、取干净的豆粕50份、花生粕100份加入到高混机内,均匀混合后,粉碎,过筛,加水拌匀,添加豆粕和花生粕总重2%的丝氨酸蛋白酶,加入添加氢氧化钠溶液将pH调节至9.0,并升温至55摄氏度,水解3-6小时,然后降温至50摄氏度,添加豆粕和花生粕总重3%的中性蛋白酶,添加稀盐酸将pH调节至6.5,水解2-3小时,然后用120目滤网过滤除去杂质,并通过添加稀盐酸或者氢氧化钠溶液将pH调节至6.5-7.0,然后在温度60摄氏度、均质压力20-25Mpa的条件下,用高压均质机均质3min,降温至37摄氏度,制得酶解液备用;
S2、在步骤S1中酶解液再加入蛋白胨20份、琼脂20份、磷酸二氢钾5份、脱脂乳粉10份、白砂糖20份、柠檬酸5份以及硫酸亚铁5份,并通过添加稀盐酸或者氢氧化钠溶液将pH调节至6.5-7.0,然后在高温蒸汽下灭菌,降温后制得培养基,备用;
S3、将乳酸菌(植物乳杆菌20%、瑞士乳杆菌10%、嗜酸乳杆菌20%、长双歧杆菌20%、两歧双歧杆菌10%、短双歧杆菌20%)接种在步骤S2中培养基中进行划线培养,选取优势菌种,然后对优势菌种进行扩大培养,备用;
S5、将步骤S3中制得的优势乳酸菌接种至步骤S2中培养基中进行厌氧发酵,接种比例为培养基重量的6%,发酵温度37摄氏度,发酵时间48小时,制得发酵液B;
S6、发酵液B进行离心,收集菌泥,将菌泥重悬于生理盐水,菌泥与生理盐水重量比例为1:1,得到重悬液;
S7、步骤S6中重悬液加入冻干保护剂和调味剂,充分混合后,添加稀盐酸者氢氧化钠溶液调节pH至6.5-7.0,真空冷冻干燥,然后粉碎装袋制得多菌联合发酵制备益生菌膳食补充剂。
对比例2:
与实施例1区别在于:未进行步骤S1中水解;
S1、取干净的豆粕50份、花生粕100份加入到高混机内,均匀混合后,粉碎,过筛,加水拌匀,然后用120目滤网过滤除去杂质,并通过添加稀盐酸或者氢氧化钠溶液将pH调节至6.5-7.0,然后在温度60摄氏度、均质压力20-25Mpa的条件下,用高压均质机均质3min,降温至37摄氏度,制得酶解液备用;
S2、向步骤S1产物中计入蛋白胨20份、琼脂20份、磷酸二氢钾5份、脱脂乳粉10份、白砂糖20份、柠檬酸5份以及硫酸亚铁5份,并通过添加稀盐酸或者氢氧化钠溶液将pH调节至6.5-7.0,然后在高温蒸汽下灭菌,降温后制得培养基,备用;
S3、将枯草芽孢杆菌接种在步骤S2中培养基中进行划线培养,选取优势菌种,然后对优势菌种进行扩大培养,备用;将乳酸菌(植物乳杆菌20%、瑞士乳杆菌10%、嗜酸乳杆菌20%、长双歧杆菌20%、两歧双歧杆菌10%、短双歧杆菌20%)接种在步骤S2中培养基中进行划线培养,选取优势菌种,然后对优势菌种进行扩大培养,备用;
S4、将步骤S3中制得的优势枯草芽孢杆菌接种至步骤S2中培养基中进行好氧发酵,接种比例为培养基重量的2%,发酵温度37摄氏度,发酵时间48小时,制得发酵液A;
S5、将步骤S3中制得的优势乳酸菌接种至步骤S4发酵液A中进行厌氧发酵,接种比例为培养基重量的6%,发酵温度37摄氏度,发酵时间48小时,制得发酵液B;
S6、发酵液B进行离心,收集菌泥,将菌泥重悬于生理盐水,菌泥与生理盐水重量比例为1:1,得到重悬液;
S7、步骤S6中重悬液加入冻干保护剂和调味剂,充分混合后,添加稀盐酸者氢氧化钠溶液调节pH至6.5-7.0,真空冷冻干燥,然后粉碎装袋制得多菌联合发酵制备益生菌膳食补充剂。
由实施例1-3以及对比例1-2制得的多菌联合发酵制备益生菌膳食补充剂,对枯草芽孢杆菌和植物乳杆菌数量进行检测:
枯草芽孢杆菌计数:采用GB/T 4789.15-2010;
植物乳杆菌计数:采用GB/T 4789.35-2010;
得到表2中数据:
表2
由实施例1-3以及对比例1-2中数据可知,实施例1-3中均具有含量较高的枯草芽孢杆菌和植物乳杆菌,并由此可推断具有含量较高的瑞士乳杆菌、嗜酸乳杆菌、长双歧杆菌、两歧双歧杆菌以及短双歧杆菌,这些细菌都是对人体有益的细菌,通过摄入这些细菌,可使身体健康发展;
对比例1中由于未添加枯草芽孢杆菌,导致植物乳杆菌的含量大幅降低,主要因为枯草芽孢杆菌好氧发酵过程中快速生长,产生丰富的蛋白酶、纤维素酶等水解酶类,将豆粕和花生粕中的纤维素和蛋白质降解为易吸收的小分子营养物,同时消除豆粕中的抗营养分子,使得乳酸菌快速繁殖;
对比例2中,未将豆粕和花生粕进行水解,营养物质有限,导致枯草芽孢杆菌繁殖速度较慢,进而影响到植物乳杆菌的繁殖速度。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明技术原理的前提下,还可以做出若干改进和变型,这些改进和变型也应视为本发明的保护范围。
Claims (6)
1.一种多菌联合发酵制备益生菌膳食补充剂的方法,其特征在于,包括以下步骤:
S1、取干净的豆粕、花生粕加入到高混机内,均匀混合后,粉碎,过筛,加水拌匀,添加豆粕和花生粕总重2%的丝氨酸蛋白酶,加入添加氢氧化钠溶液将pH调节至9.0,并升温至55摄氏度,水解3-6小时,然后降温至50摄氏度,添加豆粕和花生粕总重3%的中性蛋白酶,添加稀盐酸将pH调节至6.5,水解2-3小时,然后用120目滤网过滤除去杂质,并通过添加稀盐酸或者氢氧化钠溶液将pH调节至6.5-7.0,然后在温度60摄氏度、均质压力20-25Mpa的条件下,用高压均质机均质3min,降温至37摄氏度,制得酶解液备用;
S2、在步骤S1中酶解液再加入蛋白胨、琼脂、磷酸二氢钾、脱脂乳粉、白砂糖、柠檬酸以及硫酸亚铁,并通过添加稀盐酸或者氢氧化钠溶液将pH调节至6.5-7.0,然后在高温蒸汽下灭菌,降温后制得培养基,备用;
S3、将枯草芽孢杆菌接种在步骤S2中培养基中进行划线培养,选取优势菌种,然后对优势菌种进行扩大培养,备用;将乳酸菌接种在步骤S2中培养基中进行划线培养,选取优势菌种,然后对优势菌种进行扩大培养,备用;
S4、将步骤S3中制得的优势枯草芽孢杆菌接种至步骤S2中培养基中进行好氧发酵,接种比例为培养基重量的1%-2%,发酵温度37摄氏度,发酵时间48小时,制得发酵液A;
S5、将步骤S3中制得的优势乳酸菌接种至步骤S4发酵液A中进行厌氧发酵,接种比例为培养基重量的6%-9%,发酵温度37摄氏度,发酵时间48小时,制得发酵液B;
S6、发酵液B进行离心,收集菌泥,将菌泥重悬于生理盐水,得到重悬液;
S7、步骤S6中重悬液加入冻干保护剂,充分混合后,添加稀盐酸者氢氧化钠溶液调节pH至6.5-7.0,真空冷冻干燥,然后粉碎装袋制得多菌联合发酵制备益生菌膳食补充剂。
2.如权利要求1所述的一种多菌联合发酵制备益生菌膳食补充剂的方法,其特征在于,按重量份数计,步骤S1中豆粕50-100份,花生粕50-100份。
3.如权利要求2所述的一种多菌联合发酵制备益生菌膳食补充剂的方法,其特征在于,按重量份数计,步骤S2中蛋白胨10-20份、琼脂10-20份、磷酸二氢钾2-5份、脱脂乳粉10-20份、白砂糖10-20份、柠檬酸2-5份以及硫酸亚铁2-5份。
4.如权利要求3所述的一种多菌联合发酵制备益生菌膳食补充剂的方法,其特征在于,所述步骤S6中,菌泥与生理盐水重量比例为1:1-1.6。
5.如权利要求4所述的一种多菌联合发酵制备益生菌膳食补充剂的方法,其特征在于,步骤S7重悬液中还加入调味剂。
6.如权利要求5所述的一种多菌联合发酵制备益生菌膳食补充剂的方法,其特征在于,所述步骤S3中乳酸菌包括任意比例的植物乳杆菌、瑞士乳杆菌、嗜酸乳杆菌、长双歧杆菌、两歧双歧杆菌、短双歧杆菌。
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