传统发酵牦牛奶中益生乳杆菌筛选及其免疫调节功能研究
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摘要
为了满足市场对营养保健需求的不断增加,获得具有益生保健功能产品,对益生菌的研究日益深入广泛,对益生菌类相关产品的开发越来越重要。乳酸菌发酵乳制品是具有益生性能乳杆菌菌株的潜在来源,本研究从甘肃牧区12个不同采样点采集发酵牦牛奶样品,以期分离获得具有优良益生和免疫调节功能的乳杆菌分离株。试验通过表型和快速鉴定分析甘肃牧区乳杆菌分离株物种多样性,并在此基础上采用体外筛选方法,分别从工业特性、益生特性和安全特性3方面进行具有优良益生性能乳杆菌分离株筛选,并对筛选获得的优良乳杆菌分离株进行免疫调节功能体内研究,评价其免提调节功能,丰富益生乳杆菌资源。主要研究结果如下:
1)通过采用菌落形态、菌体特征、生理生化检验和API快速鉴定技术,对甘肃牧区传统自制发酵牦牛奶中分离获得的71株乳杆菌分离株进行属种鉴定。结果表明,其中Lactobacillus. plantarum,23株,Lactobacillus. acidophilus,21株,Lactobacillus. casei,12株,Lactobacillus. fermentum,7株,Lactobacillus. brevis,3株,Lactobacillus. minor,3株和Lactobacillus. curvatus,2株。
2)采用益生菌体外筛选方法,从工业特性、益生特性和安全性能全面考察乳杆菌分离株益生性能,优选出遗传性状稳定,产酸、凝乳性能优良,耐酸、耐胆盐,能顺利通过模拟人体胃肠道,有效黏附小肠上层细胞,产生抑菌物质,以便发挥其益生功能,同时对30种抗生素中27种均表现敏感,安全性能高的乳杆菌分离株G12和E2。
3)采用16S rDNA分子鉴定技术,对优选的乳杆菌分离株G12和E2进行分子水平鉴定,通过同源性分析可知,G12与Lactobacillus. casei同源性最高,E2与Lactobacillus. acidophilus的同源性最高。Lactobacillus. casei G12和Lactobacillus. acidophilus E2的16S rDNA序列已收录于GenBank数据库,登录号分别为HM 598683和HM 598684。
4)采用BALB/c小鼠活体试验方法,研究发现Lactobacillus. casei G12和Lactobacillus. acidophilus E2菌液饲喂30天,G12和E2不同剂量对小鼠免疫器官,即胸腺、脾脏指数均没有显著影响(P>0.05)。
5)通过BALB/c小鼠体内试验结果表明,Lactobacillus. casei G12和Lactobacillus. acidophilus E2高、中剂量组可显著提高小鼠派伊尔结巨噬细胞吞噬能力(P<0.05),说明Lactobacillus. casei G12和Lactobacillus. acidophilus E2具有促进固有免疫调节功能作用。
6)通过BALB/c小鼠体内试验结果表明,Lactobacillus. casei G12和Lactobacillus. acidophilus E2可显著提高细胞因子IL-10、IL-12和IFN-γ分泌(P<0.05),其中Lactobacillus. casei G12高剂量组可以极显著提高细胞免疫因子的分泌(P<0.01),说明Lactobacillus. casei G12和Lactobacillus. acidophilus E2具有促进细胞免疫调节功能作用。
7)给BALB/c小鼠喂服不同剂量Lactobacillus. casei G12和Lactobacillus. acido-philus E2菌液后,Lactobacillus. casei G12高、中和低剂量组与Lactobacillus. acidophilus E2高、中剂量组均可显著刺激浆细胞分泌slgA(P<0.05,P<0.01),其中Lactobacillus. casei G12高剂量组较Lactobacillus. acidophilus E2高剂量组效果显著(P<0.05),sIgA分泌量与乳杆菌株黏附能力呈正相关。此外,Lactobacillus. casei G12和Lactobacillus. acidophilus E2菌液可有效提高小鼠血清半数溶血值,促进IgM分泌。说明Lactobacillus. casei G12和Lactobacillus. acidophilus E2具有体液免疫调节功能。
Probiotic strains require investigation to obtain functional products and to satisfy the in-creasing market demand for probiotics. Traditional fermented yak milk is a potential source of probiotic Lactobacillus strains. In our study, depended on evaluation of the potential probiotic properties and immune regulation function of Lactobacillus strains isolated from traditional fermented yak milk that were collected from the pasture region of Gansu, China. The domi-nant Lactobacillus strains were isolated, and their potential probiotic properties were eva-luated with respect to function, technology and safety in vitro. Finally, evaluation immune regulation function of Lactobacillus isolates that isolation in vivo. The fundamental results were as follows:
1)Lactobacillus isolates were identified on the basis of their physiological and bio-chemical characteristics. A total of 71 isolates of Lactobacillus were isolated from 12 samples of homemade fermented yak milk, which were collected from different individual households in the pasture region of Gansu, China. The isolates were identified as follows: Lactobacillus plantarum ( 23 strains ), Lactobacillus acidophilus ( 21 strains ), Lactobacillus casei ( 12 strains ), Lactobacillus fermentum ( 7 strains ), Lactobacillus brevis ( 3 strains ), Lactobacillus minor ( 3 strains ), and Lactobacillus curvatus ( 2 strains ).
2)The dominant Lactobacillus strains were isolated, and their potential probiotic prop-erties were evaluated with respect to function, technology and safety. Two isolates from tradi-tional Gansu fermented yak milk, E2 and G12, possess all of the requirements of useful pro-biotic candidates, including acidifying and coagulating abilities, high tolerance to acid, bile salts and artificial gastrointestinal conditions, high antimicrobial activity against pathogenic bacteria and a high degree of hydrophobic potential for colonization and adherence to intes-tinal epithelium cells.
3)The two isolates was further identified by 16S rDNA gene sequencing analysis. Isolate E2 was identified as Lactobacillus acidophilus and G12 was most homologous to Lactobacil-lus casei. The 16S rDNA gene sequences were submitted to the NCBI GenBank, and the GenBank Accession numbers for the 16S rDNA gene sequences of isolates E2 and G12 are HM 598684 and HM 598683 respectively.
4)Taking the in vivo experiment, several little mice are orally taken the live bacillus made from L. casei G12 and L. acidophilus E2 in three dosages with high dose, middle dose and low dose and observed for 30 days. Measure the thymus index and spleen index of the mice, observe the function and ability of immune regulation function. The results showed G12 and E2 would not own the ability of immune regulation function.
5)Taking the mice for example, the high and middle dose group of L. casei G12 and L. acidophilus E2 can enhance the ratio of macrophage’s phagocytosis, compared with the nega-tive control group were significant differences ( P<0.05 ). The results indicated that L. casei G12 and L. acidophilus E2 may enhance non-specific immune.
6)Taking the in vivo experiment, test the content of IFN-γ, IL-12 and IL-10 in the blood serum in the method of ELISA. IFN-γ, IL-12 and IL-10 activity in the low, middle and high dose group of L. casei G12 and L. acidophilus E2 compared with the negative control group were significant differences ( P<0.05 ). The high dose group of L. casei G12 compared with the negative control group were highly significant differences ( P<0.01 ). The results indicated L. casei G12 and L. acidophilus E2 may enchance cell immune in mice.
7)sIgA content in the gut of the mice which were fed with high dose, middle dose and low dose L. casei G12 and L. acidophilus E2. The results showed high dose, middle dose and low dose L. casei G12 and high dose, middle dose L. acidophilus E2 were significantly in-creased compared with the mice which were not fed ( P<0.05,P<0.01 ). L. casei G12 and L. acidophilus E2 had a regulation effect on serum hemloysin consent in mice. It meaned serum hemolysin content increased continuously, both were higher than the control group signifi-cantly ( P<0.05 ). The concentration of serum IgM antibodies were significantly increase ( P<0.05 ). The result indicated L. casei G12和L. acidophilus E2 may enhance humoral im-mune in mice.
1)通过采用菌落形态、菌体特征、生理生化检验和API快速鉴定技术,对甘肃牧区传统自制发酵牦牛奶中分离获得的71株乳杆菌分离株进行属种鉴定。结果表明,其中Lactobacillus. plantarum,23株,Lactobacillus. acidophilus,21株,Lactobacillus. casei,12株,Lactobacillus. fermentum,7株,Lactobacillus. brevis,3株,Lactobacillus. minor,3株和Lactobacillus. curvatus,2株。
2)采用益生菌体外筛选方法,从工业特性、益生特性和安全性能全面考察乳杆菌分离株益生性能,优选出遗传性状稳定,产酸、凝乳性能优良,耐酸、耐胆盐,能顺利通过模拟人体胃肠道,有效黏附小肠上层细胞,产生抑菌物质,以便发挥其益生功能,同时对30种抗生素中27种均表现敏感,安全性能高的乳杆菌分离株G12和E2。
3)采用16S rDNA分子鉴定技术,对优选的乳杆菌分离株G12和E2进行分子水平鉴定,通过同源性分析可知,G12与Lactobacillus. casei同源性最高,E2与Lactobacillus. acidophilus的同源性最高。Lactobacillus. casei G12和Lactobacillus. acidophilus E2的16S rDNA序列已收录于GenBank数据库,登录号分别为HM 598683和HM 598684。
4)采用BALB/c小鼠活体试验方法,研究发现Lactobacillus. casei G12和Lactobacillus. acidophilus E2菌液饲喂30天,G12和E2不同剂量对小鼠免疫器官,即胸腺、脾脏指数均没有显著影响(P>0.05)。
5)通过BALB/c小鼠体内试验结果表明,Lactobacillus. casei G12和Lactobacillus. acidophilus E2高、中剂量组可显著提高小鼠派伊尔结巨噬细胞吞噬能力(P<0.05),说明Lactobacillus. casei G12和Lactobacillus. acidophilus E2具有促进固有免疫调节功能作用。
6)通过BALB/c小鼠体内试验结果表明,Lactobacillus. casei G12和Lactobacillus. acidophilus E2可显著提高细胞因子IL-10、IL-12和IFN-γ分泌(P<0.05),其中Lactobacillus. casei G12高剂量组可以极显著提高细胞免疫因子的分泌(P<0.01),说明Lactobacillus. casei G12和Lactobacillus. acidophilus E2具有促进细胞免疫调节功能作用。
7)给BALB/c小鼠喂服不同剂量Lactobacillus. casei G12和Lactobacillus. acido-philus E2菌液后,Lactobacillus. casei G12高、中和低剂量组与Lactobacillus. acidophilus E2高、中剂量组均可显著刺激浆细胞分泌slgA(P<0.05,P<0.01),其中Lactobacillus. casei G12高剂量组较Lactobacillus. acidophilus E2高剂量组效果显著(P<0.05),sIgA分泌量与乳杆菌株黏附能力呈正相关。此外,Lactobacillus. casei G12和Lactobacillus. acidophilus E2菌液可有效提高小鼠血清半数溶血值,促进IgM分泌。说明Lactobacillus. casei G12和Lactobacillus. acidophilus E2具有体液免疫调节功能。
Probiotic strains require investigation to obtain functional products and to satisfy the in-creasing market demand for probiotics. Traditional fermented yak milk is a potential source of probiotic Lactobacillus strains. In our study, depended on evaluation of the potential probiotic properties and immune regulation function of Lactobacillus strains isolated from traditional fermented yak milk that were collected from the pasture region of Gansu, China. The domi-nant Lactobacillus strains were isolated, and their potential probiotic properties were eva-luated with respect to function, technology and safety in vitro. Finally, evaluation immune regulation function of Lactobacillus isolates that isolation in vivo. The fundamental results were as follows:
1)Lactobacillus isolates were identified on the basis of their physiological and bio-chemical characteristics. A total of 71 isolates of Lactobacillus were isolated from 12 samples of homemade fermented yak milk, which were collected from different individual households in the pasture region of Gansu, China. The isolates were identified as follows: Lactobacillus plantarum ( 23 strains ), Lactobacillus acidophilus ( 21 strains ), Lactobacillus casei ( 12 strains ), Lactobacillus fermentum ( 7 strains ), Lactobacillus brevis ( 3 strains ), Lactobacillus minor ( 3 strains ), and Lactobacillus curvatus ( 2 strains ).
2)The dominant Lactobacillus strains were isolated, and their potential probiotic prop-erties were evaluated with respect to function, technology and safety. Two isolates from tradi-tional Gansu fermented yak milk, E2 and G12, possess all of the requirements of useful pro-biotic candidates, including acidifying and coagulating abilities, high tolerance to acid, bile salts and artificial gastrointestinal conditions, high antimicrobial activity against pathogenic bacteria and a high degree of hydrophobic potential for colonization and adherence to intes-tinal epithelium cells.
3)The two isolates was further identified by 16S rDNA gene sequencing analysis. Isolate E2 was identified as Lactobacillus acidophilus and G12 was most homologous to Lactobacil-lus casei. The 16S rDNA gene sequences were submitted to the NCBI GenBank, and the GenBank Accession numbers for the 16S rDNA gene sequences of isolates E2 and G12 are HM 598684 and HM 598683 respectively.
4)Taking the in vivo experiment, several little mice are orally taken the live bacillus made from L. casei G12 and L. acidophilus E2 in three dosages with high dose, middle dose and low dose and observed for 30 days. Measure the thymus index and spleen index of the mice, observe the function and ability of immune regulation function. The results showed G12 and E2 would not own the ability of immune regulation function.
5)Taking the mice for example, the high and middle dose group of L. casei G12 and L. acidophilus E2 can enhance the ratio of macrophage’s phagocytosis, compared with the nega-tive control group were significant differences ( P<0.05 ). The results indicated that L. casei G12 and L. acidophilus E2 may enhance non-specific immune.
6)Taking the in vivo experiment, test the content of IFN-γ, IL-12 and IL-10 in the blood serum in the method of ELISA. IFN-γ, IL-12 and IL-10 activity in the low, middle and high dose group of L. casei G12 and L. acidophilus E2 compared with the negative control group were significant differences ( P<0.05 ). The high dose group of L. casei G12 compared with the negative control group were highly significant differences ( P<0.01 ). The results indicated L. casei G12 and L. acidophilus E2 may enchance cell immune in mice.
7)sIgA content in the gut of the mice which were fed with high dose, middle dose and low dose L. casei G12 and L. acidophilus E2. The results showed high dose, middle dose and low dose L. casei G12 and high dose, middle dose L. acidophilus E2 were significantly in-creased compared with the mice which were not fed ( P<0.05,P<0.01 ). L. casei G12 and L. acidophilus E2 had a regulation effect on serum hemloysin consent in mice. It meaned serum hemolysin content increased continuously, both were higher than the control group signifi-cantly ( P<0.05 ). The concentration of serum IgM antibodies were significantly increase ( P<0.05 ). The result indicated L. casei G12和L. acidophilus E2 may enhance humoral im-mune in mice.
引文
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