CN113826888A - 一种提高发酵乳杆菌dali02发酵液抑菌能力的制备方法 - Google Patents
一种提高发酵乳杆菌dali02发酵液抑菌能力的制备方法 Download PDFInfo
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- CN113826888A CN113826888A CN202111148264.8A CN202111148264A CN113826888A CN 113826888 A CN113826888 A CN 113826888A CN 202111148264 A CN202111148264 A CN 202111148264A CN 113826888 A CN113826888 A CN 113826888A
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- dali02
- lactobacillus fermentum
- oligosaccharide
- fermentation
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Classifications
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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Abstract
本发明提供一种提高发酵乳杆菌DALI02发酵液抑菌能力的制备方法，通过在乳清蛋白酶解液中添加复合低聚糖，经发酵乳杆菌DALI02发酵剂发酵，得到提高抑菌能力的发酵乳杆菌DALI02发酵液；所述乳清蛋白酶解液的制备方法为：乳清蛋白粉添加量为8%~12%w/w，调节pH为6~8，添加木瓜蛋白酶，加酶量为1000~2000 U/g，水解温度为60~70℃，酶解时间为4~8 h，经离心去除沉淀物，高温灭酶后，冷却得到乳清蛋白酶解液；所述复合低聚糖为低聚果糖、低聚半乳糖、菊粉、水苏糖、低聚木糖按0.2：1：1：1：1比例混合而成；通过本发明，发酵乳杆菌DALI02发酵液抑菌能力显著提升了。
Description
技术领域
本发明提供一种提高发酵乳杆菌DALI02发酵液抑菌能力的制备方法，属于食品生物发酵与功能食品领域。
技术背景
发酵乳杆菌DALI02（Lactobacillus fermentum DALI02）属乳杆菌属，为革兰氏阳性菌杆菌，兼性厌氧。发酵乳杆菌是肠道固有乳酸杆菌之一，对宿主健康有着重要影响的益生菌。
益生菌被定义为“当以足够的量摄入时，能给宿主带来健康益处的活微生物”，而益生元是“被宿主微生物选择性利用并带来健康益处的底物”。另一方面，益生元是不可存活的底物，作为宿主所含有益微生物的营养物质，包括施用的益生菌菌株和常驻微生物。在益生元的最新定义中，预计它可以通过活宿主微生物的选择性利用，引发任何宿主微生物生态系统的变化，而不仅仅是肠道。
大量研究表明，益生菌能够抑制致病菌（革兰氏阴性菌和过氧化氢酶阳性细菌）的生长和繁殖，进而调节肠道微生态菌群结构平衡。临床研究表明某些益生菌可以通过调节肠道菌群结构，辅助治疗轮状病毒、梭菌及抗生素等引起的多种腹泻和代谢功能紊乱。
发明内容
本发明的目的是针对上述现有技术存在的缺陷，提供一种提高发酵乳杆菌DALI02发酵液抑菌能力的制备方法。
本发明是这样实现的：一种提高发酵乳杆菌DALI02发酵液抑菌能力的制备方法，其特征在于：通过在乳清蛋白酶解液中添加复合低聚糖，经发酵乳杆菌DALI02发酵剂发酵，得到提高抑菌能力的发酵乳杆菌DALI02发酵液；
所述乳清蛋白酶解液的制备方法为：乳清蛋白粉添加量为8%~12%w/w，调节pH为6~8，添加木瓜蛋白酶，加酶量为1000~2000 U/g，水解温度为60~70℃，酶解时间为4~8 h，经离心去除沉淀物，高温灭酶后，冷却得到乳清蛋白酶解液；
所述复合低聚糖为低聚果糖、低聚半乳糖、菊粉、水苏糖、低聚木糖按0.2：1：1：1：1比例混合而成；
发酵乳杆菌DALI02发酵剂制备时，将1代菌株按3%接种量接种于MRS液体培养基，置于37℃培养箱培养18 h，得到发酵乳杆菌DALI02发酵剂；
其中，MRS液体培养基制备时，大豆蛋白胨10 g、牛肉膏10 g、酵母膏5 g、葡萄糖20g、Tweeen 80 1 mL、磷酸二氢钠2 g、无水乙酸钠5 g、柠檬酸三胺2 g、硫酸锰0.02 g、硫酸镁 0.1 g加入蒸馏水1 L，调节pH至6.2左右，121℃、15 min灭菌，得到MRS液体培养基。
在酶解乳清蛋白酶解液中，添加3.0%~4.6% w/w的复合低聚糖，经常规灭菌后，接种2%~5% w/w的发酵乳杆菌DALI02发酵剂，经37℃培养18~28 h制备而成，得到提高抑菌能力的发酵乳杆菌DALI02发酵液。
利用该方法制备的提高抑菌能力的发酵乳杆菌DALI02发酵液用于制备强抑菌能力的发酵乳饮料、浓缩发酵液、冻干发酵粉产品。
乳清蛋白酶解液的制备时，将8%~12% w/w的乳清蛋白粉溶于50℃的水中，用0.1mol/L的NaOH调节pH为6~8，添加木瓜蛋白酶，加酶量为1000~2000 U/g，酶解温度为60~70℃，酶解时间为4~6 h，经离心去除沉淀物，高温灭酶后，冷却得到乳清蛋白酶解液。
发酵乳杆菌DALI02（Lactobacillus fermentum DALI02）在2018年7月5日保藏于中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物探究所，保藏号为CGMCC 16064。
本发明方法先进科学，通过本发明，一种提高发酵乳杆菌DALI02发酵液抑菌能力的制备方法，本发明所用的发酵乳杆菌DALI02（Lactobacillus fermentum DALI02）在2018年7月5日保藏于中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物探究所，保藏号为CGMCC 16064。
本发明所用指示菌株大肠杆菌（Escherichia coli CICC10012）、沙门氏菌（Salmonella WX29）、枯草芽孢杆菌（Bacillus subtilis）、金黄色葡萄球菌（Staphylococcus aureus CICC10201）购自中国工业微生物菌种保存管理中心。
本发明涉及以下培养基：
1、LB固体培养基：胰蛋白胨10 g、酵母粉5 g、氯化钠10 g、琼脂20 g、蒸馏水1 L，调节pH至7.0左右，121℃、15 min灭菌。
2、LB液体培养基：胰蛋白胨10 g、酵母粉5 g、氯化钠10 g、蒸馏水1 L，调节pH至7.0左右，121℃、15 min灭菌。
3、MRS液体培养基：大豆蛋白胨10 g、牛肉膏10 g、酵母膏5 g、葡萄糖20 g、Tweeen80 1 mL、磷酸二氢钠2 g、无水乙酸钠5 g、柠檬酸三胺2 g、硫酸锰0.02 g、硫酸镁 0.1 g，蒸馏水1 L，调节pH至6.2左右，121℃、15 min灭菌。
4、MRS固体培养基：大豆蛋白胨10 g、牛肉膏10 g、酵母膏5 g、葡萄糖20 g、Tweeen80 1 mL、磷酸二氢钠2 g、无水乙酸钠5 g、柠檬酸三胺2 g、硫酸锰0.02 g、硫酸镁 0.1 g，琼脂15 g，蒸馏水1 L，调节pH至6.2左右，121℃、15 min灭菌。
一、菌株的活化传代；
1、发酵乳杆菌DALI02：将1代菌株按3%接种量接种于MRS液体培养基，置于37℃培养箱培养18 h，备用。测定乳酸菌活菌数量采用MRS固体培养基。
2、大肠杆菌（Escherichia coli CICC10012）、沙门氏菌（Salmonella WX29）、枯草芽孢杆菌（Bacillus subtilis）、金黄色葡萄球菌（Staphylococcus aureus CICC10201）：将1代菌株按3%接种量接种于LB液体培养基，置于37℃摇床培养12 h，备用。测定抑制作用采用LB固体培养基。
二、乳清发酵液的制备；
将8%~12%的乳清蛋白粉溶于50℃的水中，用0.1 mol/L的NaOH调节pH为6~8，添加木瓜蛋白酶，加酶量为1000~2000 U/g，酶解温度为60~70℃，酶解时间为4~6 h，经离心去除沉淀物，高温灭酶后，冷却备用。
三、发酵原料的制备；
1、 复合低聚糖的制备：按低聚果糖:低聚半乳糖:菊粉:水苏糖:低聚木糖为0.2:1:1:1:1比例，充分混匀后，备用。
2、 向酶解乳清蛋白水解液中添加3.0%~4.6%的复合低聚糖，然后经过灭菌后，冷却到37℃左右，备用。
四、发酵；
接种2%~5%的发酵乳杆菌DALI02发酵剂到灭菌的混合料中，在37℃培养18~28 h，即为发酵乳杆菌DALI02乳清发酵液。
五、抑制病原指示菌能力测定；
1、 将致病菌活化二代，以生理盐水调整菌液浓度至OD600=0.5，冷藏备用，无菌操作下，将灭菌LB固体培养基倾倒至平板，冷却至培养基完全凝固，后将活化好的2代致病菌稀释100倍，分别取稀释后的大肠杆菌、沙门氏菌、枯草芽孢杆菌以及金黄色葡萄球菌100 μL注于完全凝固平板上，用无菌涂布棒涂抹均匀，用打孔器在每个平板上均匀打孔，吸取发酵液样品200 μL加入小孔中，将加样平板正置于18℃培养箱扩散12 h，待完全扩散结束后移至37℃培养箱培养12 h，用游标卡尺测量并记录抑菌圈直径。
2、 抑制大肠杆菌能力；
添加复合低聚糖的发酵乳杆菌DALI02乳清发酵液抑制大肠杆菌的能力得到显著增强（p<0.05），其抑菌圈直径达到19.82±0.06mm，显著高于未添加低聚糖发酵乳杆菌DALI02乳清发酵液的抑菌圈直径（p<0.05）。
3、抑制沙门氏菌能力；
添加复合低聚糖的发酵乳杆菌DALI02乳清发酵液抑制沙门氏菌的能力得到显著增强（p<0.05），其抑菌圈直径达到12.75±0.12mm，显著高于未添加低聚糖发酵乳杆菌DALI02乳清发酵液的抑菌圈直径（p<0.05）。
4、抑制枯草芽孢杆菌能力；
添加复合低聚糖的发酵乳杆菌DALI02乳清发酵液抑制枯草芽孢杆菌的能力得到显著增强（p<0.05），其抑菌圈直径达到16.09±0.11mm，显著高于未添加低聚糖发酵乳杆菌DALI02乳清发酵液的抑菌圈直径（p<0.05）。
5、抑制金黄色葡萄球菌能力；
添加复合低聚糖的发酵乳杆菌DALI02乳清发酵液抑制大肠杆菌的能力得到显著增强（p<0.05），其抑菌圈直径达到22.18±0.22mm，显著高于未添加低聚糖发酵乳杆菌DALI02乳清发酵液的抑菌圈直径（p<0.05）。
本发明涉及一种提高发酵乳杆菌DALI02发酵液抑菌能力的制备方法，该方法是通过添加复合低聚糖与发酵乳杆菌DALI02在乳清蛋白水解液中进行发酵，从而提高了发酵乳杆菌DALI02发酵液的抑菌能力。本发明将益生元、益生菌与乳清水解液进行紧密结合，测定发酵乳杆菌DALI02发酵液对腐败或致病菌的抑菌能力，发现发酵乳杆菌DALI02发酵液抑菌能力显著提升了，且具有广谱抑菌能力。本发明可用于具有良好抑菌能力的发酵乳饮料等食品、功能性食品与药品的生产。
具体实施方式
实施例1：利用发酵乳杆菌DALI02制备高抑菌能力的发酵液；
将低聚果糖:低聚半乳糖:菊粉:水苏糖:低聚木糖按0.2:1:1:1:1的比例混合制备成复合低聚糖。将复合低聚糖按3.5%的添加量添加到50℃的酶解乳清液中，充分混匀后，经95℃热处理10 min，冷却至37℃，按2%接种量接入发酵乳杆菌DALI02发酵剂，在37℃下发酵22 h，冷却，采用无菌灌装，得到具有较强抑菌能力的发酵液。
实施例2：利用发酵乳杆菌DALI02制备冻干粉活菌制剂；
将低聚果糖:低聚半乳糖:菊粉:水苏糖:低聚木糖按0.2:1:1:1:1的比例混合制备成复合低聚糖。将复合低聚糖按3.5%的添加量添加到50℃的酶解乳清液中，充分混匀后，经95℃热处理10 min，冷却至37℃，按2%接种量接入发酵乳杆菌DALI02发酵剂，在37℃下发酵22 h，冷却，采用无菌灌装，得到发酵液，经过冷却离心，收集菌泥，加入保护剂，真空冻干至含水量小于3%，制备冻干粉活菌制剂。也可以压碎成粉末状，然后再与奶粉、风味剂等制成粉状或片状等产品形式。
实施例1和2中的乳清水解液的制备方法；
所述的乳清水解液的制备方法为乳清蛋白粉添加量为8%~12%w/w，调节pH为6~8，添加木瓜蛋白酶，加酶量为1000~2000 U/g，水解温度为60~70℃，酶解时间为4~8 h，经离心去除沉淀物，高温灭酶后，冷却备用。
发酵乳杆菌DALI02发酵剂制备时，将1代菌株按3%接种量接种于MRS液体培养基，置于37℃培养箱培养18 h，得到发酵乳杆菌DALI02发酵剂；
其中，MRS液体培养基制备时，大豆蛋白胨10 g、牛肉膏10 g、酵母膏5 g、葡萄糖20g、Tweeen 80 1 mL、磷酸二氢钠2 g、无水乙酸钠5 g、柠檬酸三胺2 g、硫酸锰0.02 g、硫酸镁 0.1 g加入蒸馏水1 L，调节pH至6.2左右，121℃、15 min灭菌，得到MRS液体培养基。
本发明并不限于上述实施方式，在不背离本发明实质内容的情况下，本领域技术人员可以想到的任何变形、改进、替换均落入本发明的保护范围。
Claims (5)
1.一种提高发酵乳杆菌DALI02发酵液抑菌能力的制备方法，其特征在于：通过在乳清蛋白酶解液中添加复合低聚糖，经发酵乳杆菌DALI02发酵剂发酵，得到提高抑菌能力的发酵乳杆菌DALI02发酵液；
所述乳清蛋白酶解液的制备方法为：乳清蛋白粉添加量为8%~12%w/w，调节pH为6~8，添加木瓜蛋白酶，加酶量为1000~2000 U/g，水解温度为60~70℃，酶解时间为4~8 h，经离心去除沉淀物，高温灭酶后，冷却得到乳清蛋白酶解液；
所述复合低聚糖为低聚果糖、低聚半乳糖、菊粉、水苏糖、低聚木糖按0.2：1：1：1：1比例混合而成；
发酵乳杆菌DALI02发酵剂制备时，将1代菌株按3%接种量接种于MRS液体培养基，置于37℃培养箱培养18 h，得到发酵乳杆菌DALI02发酵剂；
其中，MRS液体培养基制备时，大豆蛋白胨10 g、牛肉膏10 g、酵母膏5 g、葡萄糖20 g、Tweeen 80 1 mL、磷酸二氢钠2 g、无水乙酸钠5 g、柠檬酸三胺2 g、硫酸锰0.02 g、硫酸镁0.1 g加入蒸馏水1 L，调节pH至6.2左右，121℃、15 min灭菌，得到MRS液体培养基。
2.根据权利要求1所述的一种提高发酵乳杆菌DALI02发酵液抑菌能力的制备方法，其特征在于：
在酶解乳清蛋白酶解液中，添加3.0%~4.6% w/w的复合低聚糖，经常规灭菌后，接种2%~5% w/w的发酵乳杆菌DALI02发酵剂，经37℃培养18~28 h制备而成，得到提高抑菌能力的发酵乳杆菌DALI02发酵液。
3.根据权利要求1或2所述的一种提高发酵乳杆菌DALI02发酵液抑菌能力的制备方法，其特征在于：利用该方法制备的提高抑菌能力的发酵乳杆菌DALI02发酵液用于制备强抑菌能力的发酵乳饮料、浓缩发酵液、冻干发酵粉产品。
4. 根据权利要求1所述的一种提高发酵乳杆菌DALI02发酵液抑菌能力的制备方法，其特征在于：乳清蛋白酶解液的制备时，将8%~12% w/w的乳清蛋白粉溶于50℃的水中，用0.1mol/L的NaOH调节pH为6~8，添加木瓜蛋白酶，加酶量为1000~2000 U/g，酶解温度为60~70℃，酶解时间为4~6 h，经离心去除沉淀物，高温灭酶后，冷却得到乳清蛋白酶解液。
5. 根据权利要求1所述的一种提高发酵乳杆菌DALI02发酵液抑菌能力的制备方法，其特征在于：发酵乳杆菌DALI02（Lactobacillus fermentum DALI02）在2018年7月5日保藏于中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物探究所，保藏号为CGMCC 16064。
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| CN112708577A (zh) * | 2020-12-31 | 2021-04-27 | 扬州大学 | 一种具有高肠道粘附力和免疫调节功能的发酵乳杆菌dali02及其应用 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112708577A (zh) * | 2020-12-31 | 2021-04-27 | 扬州大学 | 一种具有高肠道粘附力和免疫调节功能的发酵乳杆菌dali02及其应用 |
| CN112708577B (zh) * | 2020-12-31 | 2022-04-05 | 扬州大学 | 一种具有高肠道粘附力和免疫调节功能的发酵乳杆菌dali02及其应用 |
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