【作者】 妥彦峰；

【导师】 张兰威； 杨富民；

【作者基本信息】 甘肃农业大学 ， 动物食品营养与工程， 2010， 博士

【摘要】 微生物对人类免疫系统发挥着重要的影响,具有免疫调节功能的益生菌株的研究和筛选是目前功能食品研究的一个热点。Lactobacillus属的一些菌株可以增强机体特异性和非特异性免疫功能,有助于维护机体健康。本研究采集甘肃、青海、新疆、西藏传统发酵食品及健康婴儿粪便,分离其中的乳酸菌,并采用体外研究方法从分离获得的菌株中筛选具有潜在免疫调节功能的Lactobacillus益生菌株。采用半选择性培养基,从传统发酵食品和婴儿粪便中分离得到具有产酸能力的168株革兰氏阳性杆菌、39株革兰氏阳性球菌,过氧化氢酶试验为阴性,杆菌经果糖-6-磷酸盐磷酸酮酶试验均为阴性,初步鉴定为乳酸杆菌,在传统发酵发酵食品分离过程中也发现有酵母存在。以分离得到的168株杆菌为筛选具有潜在免疫调节功能的目标菌株,与人外周血单核细胞（PBMCs）共同培养,采用酶联免疫法（ELISA）测定培养上清液中白细胞介素-12(IL-12, p⁷⁰)的浓度。从168株杆菌中筛选出刺激PBMCs产生IL-12 (p⁷⁰)浓度较高的菌株34株。并采用ELISA方法测定初筛获得的34株杆菌刺激PBMCs产生肿瘤坏死因子-α（TNF-α）、γ-干扰素（IFN-γ）的浓度,筛选出8株刺激PBMCs产生IL-12 (p⁷⁰)、IFN-γ和TNF-α3种细胞因子浓度较高的菌株,作为具有潜在免疫调节功能的菌株,分别编号为J23ANL、J5ANL、IN1ANL、SB5AL、SB31AL、M5AL、G15AL、T3AL。采用Biolog微生物鉴定系统将菌株J23ANL、M5AL和G15AL鉴定为Lactobacillus paracasei supsp. paracasei;菌株J5ANL、IN1ANL、SB5AL、SB31AL鉴定为Lactobacillus rhamnosus;T3AL鉴定为Lactobacillus coryniformis supsp. torquens。采用模拟胃、肠液研究获得的8株Lactobacillus菌株在人体胃肠道环境中的耐受性,在人结肠癌腺细胞系HT-29上的粘附能力以及对致病菌增殖的抑制能力。8株Lactobacillus菌株在模拟胃液中暴露1 h,仍能保持一定的活菌数,在模拟胃液中暴露3 h,活菌数进一步下降,只有菌株J23ANL、J5ANL、IN1ANL、SB5AL和M5AL仍可检出活菌,而G15AL、T3AL、SB31AL不能检出活菌。8株菌在模拟肠液中的耐受性较好,在模拟肠液中暴露4 h,仍能保持较高的活菌数。不同菌株在HT-29细胞上的粘附能力不同,其中菌株IN1ANL、M5AL和G15AL有较强的粘附性,其余菌株粘附性较差。8株Lactobacillus菌株对致病菌E.coli、S.typhimurium和S.sonnei繁殖有抑制作用。进一步研究筛选获得的8株Lactobacillus菌株活菌体、热灭活菌体、细胞壁及DNA对人PBMCs增殖、细胞因子(IL-12 (p⁷⁰)、IFN-γ和TNF-α)的产生及NK细胞杀伤活性的影响,说明不同菌体成分的免疫调节功能。结果表明菌株J23ANL、J5ANL、IN1ANL、SB5AL、SB31AL、M5AL、G15AL、T3AL活菌体细胞、热灭活菌体细胞及细胞壁均使人PBMCs增殖,且当完整菌体与细胞壁作用浓度增大时,对PBMCs增殖系数减小,而菌体DNA对PBMCs增殖的影响均小于完整菌体和细胞壁对PBMCs增殖的影响。菌株J23ANL、J5ANL、IN1ANL、SB5AL、SB31AL、M5AL、G15AL、T3AL活菌体细胞、菌体细胞壁均能显著增强NK细胞杀伤活性。除菌株SB31AL,其它菌株DNA均显著增强NK细胞杀伤活性。菌株增强NK细胞杀伤活性与菌株刺激免疫细胞产生细胞因子有关。菌株J23ANL、J5ANL、IN1ANL、SB5AL、SB31AL、M5AL、G15AL、T3AL活菌体细胞、热灭活菌体、细胞壁和DNA均能刺激人PBMCs产生细胞因子IL-12(p⁷⁰)、TNF-α和IFN-γ,但各菌株细胞壁、DNA刺激PBMCs产生IL-12和TNF-α的水平均低于活菌体刺激产生细胞因子的水平。采用3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT）法研究8株Lactobacillus菌株菌体成分（包括热灭活完整菌体细胞、菌体细胞壁和菌体DNA）对人结肠癌细胞系HT-29及人慢性髓原白血病细胞K562增生的抑制作用,并采用单细胞凝胶电泳（Single cell gel electropherosis,SCGE）研究菌体成分是否诱导肿瘤细胞凋亡。结果表明,筛选得到的8株Lactobacillus菌株热灭活菌体细胞、菌体细胞壁和菌体DNA均对HT-29细胞的增生产生显著抑制。其中,菌株Lactobacillus coryniformis ss torquens T3AL对HT-29细胞的抑制率显著高于其它菌株的抑制率。单细胞凝胶电泳结果表明菌株T3AL菌体各成分诱导HT-29细胞凋亡,从而抑制HT-29细胞的增生。各菌株热灭活菌体均抑制K562细胞增生;IN1ANL、SB5AL、SB31AL、M5AL、G15AL、T3AL菌株细胞壁对K562细胞的增生具有不同程度的抑制作用,而菌株J23ANL、J5ANL细胞壁对K562细胞增生无抑制作用;菌株G15AL、M5AL、SB31AL的菌体DNA抑制K562细胞增生,其它菌株DNA均对K562细胞的增生无抑制作用。体外试验结果表明,从传统发酵食品和健康婴儿粪便中筛选获得的8株Lactobacillus菌株具有潜在的免疫调节功能,在胃、肠道环境有一定耐受性,能够抑制病原菌生长以及抑制肿瘤细胞的增生。但它们在益生功能方面表现出不同差别,需要针对这些特点进一步深入研究其免疫调节等益生功能。更多还原

【Abstract】 The study of probiotic bacteria, especially the probiotic bacteria having the immunostimulating ability is promising and has gained most interesting. Some Lactobacillus strains could exerted their immunity enhancing effects by augmenting both non-specific and specific host immune responses.The purpose of this study was to isolate Lactobacillus strains from traditional fermented foods made by locals in Gansu, Qinghai, Xinjiang and Tibet and the feces of healthy infants, and to screen potential probiotics having immunostimulating activity from the isolates.The total of 168 bacilli strains and 39 coccus strains were isolated from the traditional foods and infant feces. The bacilli and coccus strains were gram-positive, catalase-negative and F6PPK-negative, so they were presumedly considered as lactic acid bacteria. Yeasts were also isolated from the traditional foods.The isolated bacilli strains were co-cultured with human peripheral blood mononuclear cells （PBMCs） and the interleukin 12 (IL-12, p⁷⁰) concentration of the culture supernate were determined by ELISA method. The total of 34 bacilli strains, which stimulated PBMCs to produce IL-12(p⁷⁰) at higher concentration, were screened from 168 strains of isolated bacilli. The concentrations of interferon gamma （IFN-γ）and tumor necrosis factor alpha （TNF-α）, which were produced by PBMCs co-cultured with the 34 strains, were determined by ELISA method. Among the 34 bacilli strains, eight bacilli strains stimulated PBMCs to produce IL-12(p⁷⁰), IFN-γand TNF-αat higher concentrations compared to rest strains. The eight bacilli strains, J23ANL, J5ANL, IN1ANL, SB5AL, SB31AL, M5AL, G15AL and T3AL were screened as potential probiotics having immunostimulating activity. Using Biolog MicroLog2 system, the strains J23ANL, M5AL and G15AL were identified as Lactobacillus paracasei supsp. paracasei, J5ANL, IN1ANL, SB5AL and SB31AL as Lactobacillus rhamnosus, T3AL as Lactobacillus coryniformis supsp. torquens.The eight Lactobacillus strains were examined in vitro for resistance to simulated gastro and intestinal juice, adhesion to HT-29 cells, and antagonistic activity against pathogens. All eight strains retained their viability after 1 h of exposure to simulated stomach juice with some reduction of the log of viable colony counts （1.94³.48）; after 3 h of exposure to simulated stomach juice, the viability of the strains was lost markedly and the strain G15AL, T3AL and SB31AL didn’t maintain viability. All eight strains retained their viability even after 4 h of exposure to simulated small intestinal juice just with slightly reduction of the log of viable cology counts （0.18¹.64）. The strains IN1ANL, M5AL and G15AL had adhesive ability with more than 40 bacteria per 100 HT-29 cells; the rest strains didn’t have adhesive ability with fewer 40 bacteria per 100 HT-29 cells. All the eight strains had antagonistic activity against E.coli, S.typhimurium and S.sonnei.The effects of live bacteria, heat-killed bacteria, cell wall and genomic DNA of the eight Lactobacillus strains on PBMCs proliferation, cytokine secretion and NK cell activity were determined to study the immunostimulating activities of the bacteria cellular components. The live bacteria, heat-killed bacteria and cell walls of the eight strains exerted proliferative effect on PBMCs and the stimulate index of PBMCs declined with the increase of concentrations of the bacteria and cell walls. The stimulate index of PBMCs exerted by genomic DNA of the strains were little than those of PBMCs exerted by live bacteria, heat-killed bacteria and cell walls. The live bacteria of all the eight strains could significantly augment natural killer cell （NK） activity and the effect varied with different strain and dose. The cell wall of the strains could significantly augment NK cell activity the effect varied with different strain and dose. Except the DNA of strain SB31AL, other strains’DNA could enhance NK cell activity significantly. The enhancement of NK cell activity by the strains was related with the PBMCs cytokine secretion induced by the strains. The live bacteria, heat-killed bacteria, cell wall and genomic DNA of the eight strains stimulated PBMCs to produce IL-12(p⁷⁰), IFN-γand TNF-α.The concentrations of IL-12(p⁷⁰) and TNF-αstimulated by bacterial cell wall and DNA of the eight strains was lower than those stimulated by live bacteria of the strains.The antiproliferative effects of the heat-killed bacteria, bacterial cell wall and genomic DNA of the eight strains on HT-29 and K562 cancer cells were examined by using 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） method. The heat-killed bacteria, bacterial cell wall and genomic DNA of the eight strains all inhibited the growth of HT-29 cells significantly. The heat-killed bacteria of the eight strains inhibited the growth of K562 cells; except J23ANL and J5ANL, the cell wall of the other strains inhibited the growth of K562 cells; the DAN of the strain G15AL, M5AL and SB31AL inhibited the growth of K562 cells. Among of the eight stains, the inhibition rates of HT-29 by Lactobacillus coryniformis ss torquens T3AL were significantly higher than those by the rest strains. The results of Single cell gel electropherosis assay showed that t he strain T3AL exerted markedly antiproliferative effect on HT-29 cell through the induction of cell apoptosis.The results of in vitro assays showed that the eight Lactobacillus strains had immunostimulating activities, could be tolerant to gastrointestinal conditions, exerted antagonistic activity against pathogens and inhibited the growth of cancer cells. The probiotic potential of the eight Lactobacillus strains will be further studied in vivo.更多还原