瑞士乳杆菌发酵剂制备及其发酵产物生物活性的评价
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摘要
本文对瑞士乳杆菌发酵剂制备的关键技术进行了研究,筛选了增殖培养基和高效冻干保护剂,优化了乳酸菌增殖培养、细胞浓缩分离等工艺条件,在此基础上,评价了其发酵产物的降血压功能和免疫功能,旨在初探瑞士乳杆菌的生物活性作用机理及其功能成分和量效关系,为工业化开发含有瑞士乳杆菌的功能性乳制品提供必要理论和技术基础。
实验确定了瑞士乳杆菌的最佳增殖培养基组成为:酶水解11%脱脂乳中加0.5%乳糖,0.5%胰蛋白胨,0.3%酵母膏,体积分数为1%KH_2PO_4-NaOH,40℃培养14 h,此培养基中活菌数为5.87×10~9cfu/mL,分别比在11%脱脂乳培养基和酶解脱脂乳培养基中提高100倍和15.4倍。将瑞士乳杆菌发酵液离心,选择最佳离心条件:转速为4000r/min,离心时间为20 min,添加复合保护剂10%海藻糖、11%脱脂乳、4%谷氨酸钠制成菌悬液,经过真空冷冻干燥,冻干存活率高达83%。
在制备了浓缩型发酵剂的基础上,利用8周龄先天性高血压大鼠(SHR)观察瑞士乳杆菌发酵乳及乳清对尾部动脉血压和心率,血脂可能产生的影响。发现瑞士乳杆菌发酵乳及乳清可以改善血清中总胆固醇和甘油三酯的含量;灌胃6周发酵乳的SHR大鼠血压在第四周与对照组比有明显下降(P＜0.05),收缩压最多下降了11.4±1.7 mmHg,且随灌胃时间的延长,与对照组间的差值缓缓增加,这些结果表明,长期服用瑞士乳杆菌发酵乳不仅可以明显缓解血压的升高而且作用较稳定,有助于阻止SHR高血压形成前过度期的发展,对动脉硬化和高血脂症也可能有作用。这一结果还需临床观察进一步验证。
本文还对瑞士乳杆菌及其发酵产物的增强免疫功力能进行了研究,给小鼠灌服瑞士乳杆菌(Lactobacillus helveticus)活菌液,细胞裂解液,发酵产物及普通酸乳和乳清后,对其免疫功能指标进行测定,发现瑞士乳杆菌活菌液能够促进小鼠的胸腺发育,显著提高巨噬细胞的吞噬功能,显著的激活B淋巴细胞活性,最佳作用活菌数为2×10~9cfu/mL,最佳作用剂量为20 mL/kg b.w;瑞士乳杆菌发酵乳清可以显著提高巨噬细胞的吞噬功能,显著增加血清中IgG的含量,最佳作用剂量为25 mL/kg b.w。这些结果表明,瑞士乳杆菌活菌液(活菌数大于2×10~8cfu/mL)对特异性细胞免疫功能和非特异性免疫功能有明显的促进作用,其发酵乳清高剂量组对特异性细胞免疫功能和体液免疫功能有明显的促进作用。二者的免疫活性均强于其细胞裂解液及普通酸乳。瑞士乳杆菌的免疫活性成分既包括菌体所含细胞壁成分,也包括其代谢产物。
总之,瑞士乳杆菌及其发酵产物,在动物实验中表现了降血压,调节血脂和增强免疫力活性的功能,是一种很有开发潜力的生物活性物质。
The key technology of concentrated Starter of Lactobacillus helveticus was studied in this paper,including filtration of the culture medium and fine freeze-dry protestant,Optimizing the condition of cell cultivation,concentration and the technology of lyophilization.The effects of Lactobacillus helveticus and its fermentation product on reductions in mean systolic blood pressure and immune function in mice were also studied, the biological mechanism of Lactobacillus helveticus was explored and the best dose-effect relationship was determined.
The optimum medium was prepared as:enzyme hydrolysed 11%RSM,0.5.0%lactose, 0.5%peptone,0.3%yeast extract and 0.1 mol/L KH_2PO_4-NaOH,culture time 14 h.The viable counts was 5.87×10~9,100 and 15.4 fold higher than fermented in 11%RSM and enzyme hydrolyzed 11%RSM.Fermented liquor of Lactobacillus helveticus was centrifuged(4000 r/min×20 min),the components and dosage of the cryoprotective agents were ascertained.The results indicated that the cryoprotective agents showed significant effect on increasing the survival rate of Lactobacillus helveticus.When using the combination of 10%trehalose and 4%sodium glutamate and 11%skim milk,the cryoprotective agents showed the most positive effect.Compared with spray drying,the effect of freeze drying was more obvious with the survival rate of 83%.
The effects of Lactobacillus helveticus fermented milk and its whey on blood pressure, heart rate and blood lipid were evaluated in spontaneously hypertensive rats(SHR).The SHR were each given Lactobacillus helveticus fermented milk and its whey by gastric intubations in six weeks.The cholesterol and triglyceride in serum were ameliorated and the systolic blood pressure of rats decreased since fourth week,Compared with control group,the systolic blood pressure of rats was decreased significantly:11.4±1.7 mmHg (P＜0.05).Th0s result testified that the Lactobacillus helveticus fermented milk and its whey containing bioactive peptides,in daily use,does have a blood pressure-lowering effect in hypertensive subjects and is thus a potential for the dietary treatment of hypertension,arteriosclerosis and hyperlipidemia.For its mechanism,it should be further studied in clinic.
The effects of Lactobacillus helveticus and its fermentation product on immune function in mice was also studied.It is discovered that L.helreticus-living bacteria liquid could improve the growth of thymus,promote functions of macrophage and the proliferation of B lymphocyte,the best number of active microorganism is 2×10~9cfu/mL,the best dosage is 20 mL/kg b.w;L.helreticus-whey could promote functions of macrophage and increase the levels of serum IgG,the best dosage is 25 mL/kg b.w.It may suggest that the L.helreticus-living bacteria liquid can remarkably enhance immune function in mice such as cellular immunity and non-specific immunity.L.helreticus-whey can more remarkably enhance immune function in mice such as cellular immunity and humoral immunity with high dose.Compared with L.helveticus cell lysis buffer and common yoghurt,the L.helveticus living bacteria liquid and its whey enhance immune function more remarkably.The immunoactive constituents of Lactobacillus helveticus include its cell wall constituents and metabolites.
In conclusion,Lactobacillus helveticus and its fermentation product had apparent effects on depressing blood pressure,ameliorating blood lipid and improving immunologic function.It is a potential novel bioactive substance.
实验确定了瑞士乳杆菌的最佳增殖培养基组成为:酶水解11%脱脂乳中加0.5%乳糖,0.5%胰蛋白胨,0.3%酵母膏,体积分数为1%KH_2PO_4-NaOH,40℃培养14 h,此培养基中活菌数为5.87×10~9cfu/mL,分别比在11%脱脂乳培养基和酶解脱脂乳培养基中提高100倍和15.4倍。将瑞士乳杆菌发酵液离心,选择最佳离心条件:转速为4000r/min,离心时间为20 min,添加复合保护剂10%海藻糖、11%脱脂乳、4%谷氨酸钠制成菌悬液,经过真空冷冻干燥,冻干存活率高达83%。
在制备了浓缩型发酵剂的基础上,利用8周龄先天性高血压大鼠(SHR)观察瑞士乳杆菌发酵乳及乳清对尾部动脉血压和心率,血脂可能产生的影响。发现瑞士乳杆菌发酵乳及乳清可以改善血清中总胆固醇和甘油三酯的含量;灌胃6周发酵乳的SHR大鼠血压在第四周与对照组比有明显下降(P＜0.05),收缩压最多下降了11.4±1.7 mmHg,且随灌胃时间的延长,与对照组间的差值缓缓增加,这些结果表明,长期服用瑞士乳杆菌发酵乳不仅可以明显缓解血压的升高而且作用较稳定,有助于阻止SHR高血压形成前过度期的发展,对动脉硬化和高血脂症也可能有作用。这一结果还需临床观察进一步验证。
本文还对瑞士乳杆菌及其发酵产物的增强免疫功力能进行了研究,给小鼠灌服瑞士乳杆菌(Lactobacillus helveticus)活菌液,细胞裂解液,发酵产物及普通酸乳和乳清后,对其免疫功能指标进行测定,发现瑞士乳杆菌活菌液能够促进小鼠的胸腺发育,显著提高巨噬细胞的吞噬功能,显著的激活B淋巴细胞活性,最佳作用活菌数为2×10~9cfu/mL,最佳作用剂量为20 mL/kg b.w;瑞士乳杆菌发酵乳清可以显著提高巨噬细胞的吞噬功能,显著增加血清中IgG的含量,最佳作用剂量为25 mL/kg b.w。这些结果表明,瑞士乳杆菌活菌液(活菌数大于2×10~8cfu/mL)对特异性细胞免疫功能和非特异性免疫功能有明显的促进作用,其发酵乳清高剂量组对特异性细胞免疫功能和体液免疫功能有明显的促进作用。二者的免疫活性均强于其细胞裂解液及普通酸乳。瑞士乳杆菌的免疫活性成分既包括菌体所含细胞壁成分,也包括其代谢产物。
总之,瑞士乳杆菌及其发酵产物,在动物实验中表现了降血压,调节血脂和增强免疫力活性的功能,是一种很有开发潜力的生物活性物质。
The key technology of concentrated Starter of Lactobacillus helveticus was studied in this paper,including filtration of the culture medium and fine freeze-dry protestant,Optimizing the condition of cell cultivation,concentration and the technology of lyophilization.The effects of Lactobacillus helveticus and its fermentation product on reductions in mean systolic blood pressure and immune function in mice were also studied, the biological mechanism of Lactobacillus helveticus was explored and the best dose-effect relationship was determined.
The optimum medium was prepared as:enzyme hydrolysed 11%RSM,0.5.0%lactose, 0.5%peptone,0.3%yeast extract and 0.1 mol/L KH_2PO_4-NaOH,culture time 14 h.The viable counts was 5.87×10~9,100 and 15.4 fold higher than fermented in 11%RSM and enzyme hydrolyzed 11%RSM.Fermented liquor of Lactobacillus helveticus was centrifuged(4000 r/min×20 min),the components and dosage of the cryoprotective agents were ascertained.The results indicated that the cryoprotective agents showed significant effect on increasing the survival rate of Lactobacillus helveticus.When using the combination of 10%trehalose and 4%sodium glutamate and 11%skim milk,the cryoprotective agents showed the most positive effect.Compared with spray drying,the effect of freeze drying was more obvious with the survival rate of 83%.
The effects of Lactobacillus helveticus fermented milk and its whey on blood pressure, heart rate and blood lipid were evaluated in spontaneously hypertensive rats(SHR).The SHR were each given Lactobacillus helveticus fermented milk and its whey by gastric intubations in six weeks.The cholesterol and triglyceride in serum were ameliorated and the systolic blood pressure of rats decreased since fourth week,Compared with control group,the systolic blood pressure of rats was decreased significantly:11.4±1.7 mmHg (P＜0.05).Th0s result testified that the Lactobacillus helveticus fermented milk and its whey containing bioactive peptides,in daily use,does have a blood pressure-lowering effect in hypertensive subjects and is thus a potential for the dietary treatment of hypertension,arteriosclerosis and hyperlipidemia.For its mechanism,it should be further studied in clinic.
The effects of Lactobacillus helveticus and its fermentation product on immune function in mice was also studied.It is discovered that L.helreticus-living bacteria liquid could improve the growth of thymus,promote functions of macrophage and the proliferation of B lymphocyte,the best number of active microorganism is 2×10~9cfu/mL,the best dosage is 20 mL/kg b.w;L.helreticus-whey could promote functions of macrophage and increase the levels of serum IgG,the best dosage is 25 mL/kg b.w.It may suggest that the L.helreticus-living bacteria liquid can remarkably enhance immune function in mice such as cellular immunity and non-specific immunity.L.helreticus-whey can more remarkably enhance immune function in mice such as cellular immunity and humoral immunity with high dose.Compared with L.helveticus cell lysis buffer and common yoghurt,the L.helveticus living bacteria liquid and its whey enhance immune function more remarkably.The immunoactive constituents of Lactobacillus helveticus include its cell wall constituents and metabolites.
In conclusion,Lactobacillus helveticus and its fermentation product had apparent effects on depressing blood pressure,ameliorating blood lipid and improving immunologic function.It is a potential novel bioactive substance.
引文
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