CN113308501A - 一种乳酸菌胞外多糖的制备方法 - Google Patents
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Abstract
本发明公开了一种乳酸菌胞外多糖的制备方法,包括如下步骤:将活化后的乳酸菌置于添加有大豆小肽的MRS培养液,37℃,180rpm摇床培养12h后,将所得的乳酸菌发酵液以3~6%的接种量接种于添加有精胺、月光花素的MRS液体培养基中,调节初始pH值为5.5~7,培养8~15h后,将所得的粗乳酸菌胞外多糖经DEAE‑52纤维素阴离子交换柱、Sepharose CL‑6B凝胶柱、截留分子量为290~350道尔顿的纳滤膜、截流分子量为3500Da的超滤管纯化后,浓缩,乙醇溶液沉淀,取沉淀物冷冻干燥,即得。本发明所得的乳酸菌胞外多糖具有优良的生物活性,且可以显著提高乳酸菌胞外多糖的产量。
Description
技术领域
本发明涉及微生物领域,具体涉及一种乳酸菌胞外多糖的制备方法。
背景技术
胞外多糖(exopolysaccharide,EPS)是微生物(如细菌、酵母、真菌)在生长过程中分泌到细胞外的粘液或荚膜多糖。在众多产胞外多糖的微生物中,乳酸菌(lactic acidbacteria,LAB)具有发酵周期短、营养要求简单等有点,从而成为了微生物胞外多糖的理想来源。
乳酸菌胞外多糖作为稳定剂、增稠剂广泛运用于酸奶、奶酪等发酵奶制品,具有降低胆固醇、抗氧化、抗肿瘤、对消化道的调节等作用。
乳酸菌发酵产生胞外多糖受到多种因素相互作用,主要是培养基成分和培养条件对胞外多糖合成量和组成存在较大相互作用。虽然乳酸菌产生的胞外多糖的功能及应用广泛,但是现有的方法产量低,如何提高乳酸菌胞外多糖的产量是现在面临的主要问题。
发明内容
为解决上述技术问题,本发明提供了一种乳酸菌胞外多糖的制备方法,可以显著提高乳酸菌胞外多糖的产量。
为实现上述目的,本发明采取的技术方案为:
一种乳酸菌胞外多糖的制备方法,包括如下步骤:
S1、将活化后的乳酸菌置于添加有大豆小肽的MRS培养液,37℃,180rpm摇床培养12h,得到乳酸菌发酵液;
S2、将乳酸菌发酵液以3~6%的接种量接种于添加有精胺、月光花素的MRS液体培养基中,调节初始pH值为5.5~7,于30~40℃下,培养8~15h,得粗乳酸菌胞外多糖;
S3、将粗乳酸菌胞外多糖经DEAE-52纤维素阴离子交换柱分离纯化,收集含有乳酸菌胞外粗多糖的第一洗脱峰的洗脱液,冷冻浓缩至5~6mL后,再经Sepharose CL-6B凝胶柱层析分离纯化,收集含有乳酸菌胞外粗多糖的第二洗脱峰的洗脱液,经截留分子量为290~350道尔顿的纳滤膜纳滤脱盐,再经截流分子量为3500Da的超滤管,以5000rpm的转速离心超滤15~60min,浓缩,90~95%的乙醇溶液沉淀,取沉淀物冷冻干燥,得到乳酸菌胞外纯多糖。
进一步地,所述步骤S1中,MRS液体培养基的组成为:按重量百分比,碳源2%,氮源1%,磷酸氢二钾0.1~0.7%,牛肉膏1%,酵母粉0.45%,柠檬氢二胺0.3%,乙酸钠0.6%,硫酸镁0.068%,硫酸锰0.035%,大豆小肽 1~2%,蒸馏水余量;组成的MRS液体培养基的碳氮比为3:1~1:3。
进一步地,所述步骤S2中,MRS液体培养基的组成为:按重量百分比,碳源2%,氮源1%,磷酸氢二钾0.1~0.7%,牛肉膏1%,酵母粉0.45%,柠檬氢二胺0.3%,乙酸钠0.6%,硫酸镁0.068%,硫酸锰0.035%,精胺 0.2~0.3%,月光花素 0.4~0.6%,蒸馏水余量;组成的MRS液体培养基的碳氮比为3:1~1:3。
进一步地,所述的碳源为葡萄糖;所述的氮源为浓缩乳清蛋白。
本发明具有以下有益效果:
1)可以显著提高乳酸菌胞外多糖的产量;
2)所得的乳酸菌胞外多糖具有优良的生物活性。
具体实施方式
为了使本发明的目的及优点更加清楚明白,以下结合实施例对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
实施例1
一种乳酸菌胞外多糖的制备方法,包括如下步骤:
S1、将活化后的鼠李糖乳杆菌M9置于添加有大豆小肽的MRS培养液,37℃,180rpm摇床培养 12h,得到乳酸菌发酵液;其中,MRS液体培养基的组成为:按重量百分比,葡萄糖2%,浓缩乳清蛋白1%,磷酸氢二钾0.6%,牛肉膏1%,酵母粉0.45%,柠檬氢二胺0.3%,乙酸钠0.6%,硫酸镁0.068%,硫酸锰0.035%,大豆小肽 1.5%,蒸馏水余量;组成的MRS液体培养基的碳氮比为3:1~1:3;
S2、将乳酸菌发酵液以3~6%的接种量接种于添加有精胺、月光花素的MRS液体培养基中,调节初始pH值为6.5,于30~40℃下,培养8~15h,得粗乳酸菌胞外多糖;其中,MRS液体培养基的组成为:按重量百分比,葡萄糖2%,浓缩乳清蛋白1%,磷酸氢二钾0.4%,牛肉膏1%,酵母粉0.45%,柠檬氢二胺0.3%,乙酸钠0.6%,硫酸镁0.068%,硫酸锰0.035%,精胺 0.25%,月光花素 0.5%,蒸馏水余量;组成的MRS液体培养基的碳氮比为2:1;
S3、将粗乳酸菌胞外多糖经DEAE-52纤维素阴离子交换柱分离纯化,收集含有乳酸菌胞外粗多糖的第一洗脱峰的洗脱液,冷冻浓缩至5~6mL后,再经Sepharose CL-6B凝胶柱层析分离纯化,收集含有乳酸菌胞外粗多糖的第二洗脱峰的洗脱液,经截留分子量为290~350道尔顿的纳滤膜纳滤脱盐,再经截流分子量为3500Da的超滤管,以5000rpm的转速离心超滤35min,浓缩,90~95%的乙醇溶液沉淀,取沉淀物冷冻干燥,得到乳酸菌胞外纯多糖。
实施例2
一种乳酸菌胞外多糖的制备方法,包括如下步骤:
S1、将活化后的干酪乳杆菌KW3置于添加有大豆小肽的MRS培养液,37℃,180rpm摇床培养 12h,得到乳酸菌发酵液;其中,MRS液体培养基的组成为:按重量百分比,葡萄糖2%,浓缩乳清蛋白1%,磷酸氢二钾0.1~0.7%,牛肉膏1%,酵母粉0.45%,柠檬氢二胺0.3%,乙酸钠0.6%,硫酸镁0.068%,硫酸锰0.035%,大豆小肽 2%,蒸馏水余量;组成的MRS液体培养基的碳氮比为3:1~1:3;
S2、将乳酸菌发酵液以6%的接种量接种于添加有精胺、月光花素的MRS液体培养基中,调节初始pH值为7,于40℃下,培养8h,得粗乳酸菌胞外多糖;其中,MRS液体培养基的组成为:按重量百分比,葡萄糖2%,浓缩乳清蛋白1%,磷酸氢二钾0.7%,牛肉膏1%,酵母粉0.45%,柠檬氢二胺0.3%,乙酸钠0.6%,硫酸镁0.068%,硫酸锰0.035%,精胺0.3%,月光花素 0.6%,蒸馏水余量;组成的MRS液体培养基的碳氮比为1:3;
S3、将粗乳酸菌胞外多糖经DEAE-52纤维素阴离子交换柱分离纯化,收集含有乳酸菌胞外粗多糖的第一洗脱峰的洗脱液,冷冻浓缩至5~6mL后,再经Sepharose CL-6B凝胶柱层析分离纯化,收集含有乳酸菌胞外粗多糖的第二洗脱峰的洗脱液,经截留分子量为290~350道尔顿的纳滤膜纳滤脱盐,再经截流分子量为3500Da的超滤管,以5000rpm的转速离心超滤35min,浓缩,90~95%的乙醇溶液沉淀,取沉淀物冷冻干燥,得到乳酸菌胞外纯多糖。
实施例3
一种乳酸菌胞外多糖的制备方法,包括如下步骤:
S1、将活化后的植物乳杆菌LP6置于添加有大豆小肽的MRS培养液,37℃,180rpm摇床培养 12h,得到乳酸菌发酵液;其中,MRS液体培养基的组成为:按重量百分比,葡萄糖2%,浓缩乳清蛋白1%,磷酸氢二钾0.1%,牛肉膏1%,酵母粉0.45%,柠檬氢二胺0.3%,乙酸钠0.6%,硫酸镁0.068%,硫酸锰0.035%,大豆小肽 1%,蒸馏水余量;组成的MRS液体培养基的碳氮比为3:1~1:3;
S2、将乳酸菌发酵液以3%的接种量接种于添加有精胺、月光花素的MRS液体培养基中,调节初始pH值为5.5,于30℃下,培养15h,得粗乳酸菌胞外多糖;其中,MRS液体培养基的组成为:按重量百分比,葡萄糖2%,浓缩乳清蛋白1%,磷酸氢二钾0.1%,牛肉膏1%,酵母粉0.45%,柠檬氢二胺0.3%,乙酸钠0.6%,硫酸镁0.068%,硫酸锰0.035%,精胺0.2%,月光花素 0.4%,蒸馏水余量;组成的MRS液体培养基的碳氮比为3:1;
S3、将粗乳酸菌胞外多糖经DEAE-52纤维素阴离子交换柱分离纯化,收集含有乳酸菌胞外粗多糖的第一洗脱峰的洗脱液,冷冻浓缩至5~6mL后,再经Sepharose CL-6B凝胶柱层析分离纯化,收集含有乳酸菌胞外粗多糖的第二洗脱峰的洗脱液,经截留分子量为290~350道尔顿的纳滤膜纳滤脱盐,再经截流分子量为3500Da的超滤管,以5000rpm的转速离心超滤35min,浓缩,90~95%的乙醇溶液沉淀,取沉淀物冷冻干燥,得到乳酸菌胞外纯多糖。
采用苯酚-硫酸法实现多糖含量的测定,结果显示,较CN201110191610.0所述的乳酸菌胞外纯多糖的制备方法,实施例1~实施例3的多糖含量显著提高,约提高了21.7%左右。
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,在不脱离本发明原理的前提下,还可以作出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (5)
1.一种乳酸菌胞外多糖的制备方法,其特征在于,包括如下步骤:
S1、将活化后的乳酸菌置于添加有大豆小肽的MRS培养液,37℃,180rpm摇床培养 12h,得到乳酸菌发酵液;
S2、将乳酸菌发酵液以3~6%的接种量接种于添加有精胺、月光花素的MRS液体培养基中,调节初始pH值为5.5~7,于30~40℃下,培养8~15h,得粗乳酸菌胞外多糖;
S3、将粗乳酸菌胞外多糖经DEAE-52纤维素阴离子交换柱分离纯化,收集含有乳酸菌胞外粗多糖的第一洗脱峰的洗脱液,冷冻浓缩至5~6mL后,再经Sepharose CL-6B凝胶柱层析分离纯化,收集含有乳酸菌胞外粗多糖的第二洗脱峰的洗脱液,经截留分子量为290~350道尔顿的纳滤膜纳滤脱盐,再经截流分子量为3500Da的超滤管,以5000rpm的转速离心超滤15~60min,浓缩,90~95%的乙醇溶液沉淀,取沉淀物冷冻干燥,得到乳酸菌胞外纯多糖。
2.如权利要求1所述的一种乳酸菌胞外多糖的制备方法,其特征在于,所述步骤S1中,MRS液体培养基的组成为:按重量百分比,碳源2%,氮源1%,磷酸氢二钾0.1~0.7%,牛肉膏1%,酵母粉0.45%,柠檬氢二胺0.3%,乙酸钠0.6%,硫酸镁0.068%,硫酸锰0.035%,大豆小肽 1~2%,蒸馏水余量;组成的MRS液体培养基的碳氮比为3:1~1:3。
3.如权利要求1所述的一种乳酸菌胞外多糖的制备方法,其特征在于,所述步骤S2中,MRS液体培养基的组成为:按重量百分比,碳源2%,氮源1%,磷酸氢二钾0.1~0.7%,牛肉膏1%,酵母粉0.45%,柠檬氢二胺0.3%,乙酸钠0.6%,硫酸镁0.068%,硫酸锰0.035%,精胺 0.2~0.3%,月光花素 0.4~0.6%,蒸馏水余量;组成的MRS液体培养基的碳氮比为3:1~1:3。
4.如权利要求2或3所述的一种乳酸菌胞外多糖的制备方法,其特征在于,所述的碳源为葡萄糖。
5.如权利要求2或3所述的一种乳酸菌胞外多糖的制备方法,其特征在于,所述的氮源为浓缩乳清蛋白。
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