Sugahara Takuya, Onda Hiroyuki, Shinohara Yusuke, Horii Mayumi, Akiyama Koichi, Nakamoto Katsunori, Hara Kazushi
Faculty of Agriculture, Ehime University, 3-5-7, Tarumi, Matsuyama, Ehime 790-8566, Japan.
Biochim Biophys Acta. 2005 Sep 15;1725(2):233-40. doi: 10.1016/j.bbagen.2005.05.008.
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-gamma production of human PBL was also accelerated 6.0-fold by 50 microg/ml of the fermented milk. However, interleukin-4 production of PBL was not affected, and tumor necrosis factor-alpha production was suppressed. The activity was enhanced 2.5-fold by the thermal treatment for 30 min at 65 degrees C 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.
由12种乳酸菌和酵母发酵的脱脂牛乳，在无血清培养基中可使人类杂交瘤HB4C5细胞的单克隆抗体产量提高2.8倍。体外培养时，人外周血淋巴细胞（PBL）的免疫球蛋白生成也受到刺激。人PBL的IgM和IgG生成分别加快至2.8倍和5.4倍。50微克/毫升的发酵乳还使人PBL的干扰素-γ生成加快6.0倍。然而，PBL的白细胞介素-4生成未受影响，肿瘤坏死因子-α生成受到抑制。65℃热处理30分钟后，活性增强2.5倍，经胰蛋白酶消化则完全丧失活性。这些发现表明，发酵乳中的活性物质是热稳定蛋白。凝胶过滤和SDS-PAGE分析显示，活性物质的分子量估计为19.0 kDa，在发酵前的脱脂牛乳中未检测到。19.0 kDa蛋白的N端氨基酸序列与脉胨组分3（PP3）高度同源。由于PP3的分子量为28 kDa，因此表明19.0 kDa蛋白源自脱脂乳发酵过程中PP3的降解。此外，从脱脂乳中纯化的PP3也可提高HB4C5细胞的IgM生成。
