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Studies on a novel immunostimulation factor in fermented milk.

发酵乳中新型免疫刺激因子的研究。

基本信息

批准号：
16580102
负责人：
SUGAHARA Takuya
金额：
$ 2.05万
依托单位：
Ehime University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

项目摘要

Fat-free bovine milk fermented by 12 kinds of lactic acid bacteria and yeast enhanced monoclonal antibody production of human hybridoma HB4C5 cells 2.8-fold in serum-free medium. Immunoglobulin production of human peripheral blood lymphocytes (PBL) was also stimulated in vitro. IgM and IgG production of human PBL was accelerated up to 2.8-fold and 5.4-fold, respectively. Interferon-y production of human PBL was also accelerated 6.0-fold by 50 μg/ml of the fermented milk. However, interleukin-4 production of PBL was not affected, and tumor necrosis factor-a production was suppressed. The activity was enhanced 2.5-fold by the thermal treatment for 30 min at 65 ℃, and was completely lost by trypsin digestion. The findings suggested that the active substance in the fermented milk was heat stable protein. Gel-filtration and the SDS-PAGE analysis revealed that the molecular weight of the active substance was estimated as 19.0 kDa, which was not detected in fat-free bovine milk before fermentation. N-terminal amino acid sequence of the 19.0 kDa protein was highly homologous to proteose-peptone component 3 (PP3). Since molecular weight of PP3 is 28 kDa, it is suggested that the 19.0 kDa protein is derived from degradation of PP3 during fermentation of fat-free milk. Moreover, PP3 purified from fat-free milk also enhanced IgM production of HB4C5 cells.PP3 did not enhanced IgM-mRNA transcription level in HB4C5 cells. However, PP3 stimulated IgM production of HB4C5 cells treated with Actinomycin D, transcription inhibitor.Moreover, PP from fermented milk suppressed IgE production by human myeloma U266 cells. As mentioned above, PP3 enhance IFN-y production. These facts suggest that PP may suppress allergic reaction.

12种乳酸菌和酵母菌发酵的脱脂牛乳在无血清条件下可使人杂交瘤HB4C5细胞产生的单抗增加2.8倍。体外刺激人外周血淋巴细胞(PBL)产生免疫球蛋白。人外周血免疫球蛋白IGM和Ig G的产生分别提高了2.8倍和5.4倍。发酵乳浓度为50μg/ml时，人外周血中干扰素-γ的产生也增加了6倍。但不影响PBL产生IL-4，抑制肿瘤坏死因子-α的产生。65℃热处理30min，酶活提高2.5倍，经胰酶消化后，酶活完全丧失。结果表明，发酵乳中的活性物质为热稳定蛋白。凝胶过滤和SDS-PAGE分析表明，该活性物质的相对分子质量为19.0 kDa，在发酵前脱脂牛乳中未检测到该活性物质。该19.0 kDa蛋白的N端氨基酸序列与蛋白质组分3(PP3)高度同源。由于PP3的相对分子质量为28 kDa，推测19.0 kDa的蛋白质来源于脱脂牛奶发酵过程中PP3的降解。此外，从脱脂牛奶中提纯的PP3还能促进HB4C5细胞产生IgM，但不能提高HB4C5细胞中IgM-mRNA的转录水平。然而，PP3能刺激转录抑制剂放线菌素D处理的HB4C5细胞产生IgM，而且发酵乳中的PP能抑制人骨髓瘤U266细胞产生IgE。如上所述，PP3可促进干扰素-γ的产生。这些事实表明，PP可以抑制过敏反应。