應用熱致死乳酸菌組合在小鼠模式下抑制沙門氏菌感染誘導發炎之研究 熱處理乳酸菌產品之免疫與抗發炎功能及其應用- 應用熱致死乳酸菌組合在小鼠模式下抑制沙門氏菌感染誘導發炎之研究 曾浩洋1、 陳志遠2、林峻立1、 蔣育錚1、莊笠岑1、 陳錦樹2, 1弘光科技大學 食品科技學系 2國立中興大學 食品暨應用生物科技學系 連絡電話：04-26318652#5080 E-mail：hytsen@sunrise.hk.edu.tw . 計畫名稱：NSC101-2324-B-241-001生菌劑之量產組合及其熱致死菌產品之製備、功能性機制探討與豬飼料添加物之應用-總計劃暨子計畫一-不同乳酸菌株之功能性成分及熱致死菌與乳酸菌質之豬飼料添加物作用機制探討 Abstract 熱致死乳酸菌相較於活的乳酸菌有其容易保存及易於運輸等優點。在本研究中以兩種條件來製備熱致死乳酸菌，分別為100℃加熱30分鐘以及121℃加熱15分鐘，並評估其菌株及菌株組合之吸附能力及免疫調節活性。試驗乳酸菌包含3株Lactobacillus屬及1株Enterococcus屬，此4菌株是藉由140多株乳酸菌中篩選出其較佳之腸道吸附能力及免疫調節活性，並在小鼠動物實驗中證實其菌株能有效改善沙門氏菌侵入之現象。4株乳酸菌熱致死處理後在Caco-2細胞吸附能力試驗結果並無明顯差異性，但其中2株乳酸菌及乳酸菌組合在誘導刺激小鼠巨噬細胞產生腫瘤壞死因子(TNF-α)及細胞激素(IL-12)表現方面，顯現出較高的誘導程度。此外，在餵食熱致死乳酸菌組合下，能有效抑制沙門氏菌侵入小鼠臟器，且在小鼠血清中促發炎細胞激素(TNF-α及IL-6)的表現程度也明顯降低，並同時提高了抗發炎細胞激素(IL-10)的表現。綜上所述，此熱致死乳酸菌組合對於小鼠及或許人類在抑制沙門氏菌感染誘導發炎方面有其應用之潛力。 Table 1 乳酸菌及其熱致死 LAB 對腸道細胞之吸附 Materials and methods (1) Bacteria strains and conditions of heat treatment on LAB strains (2) Adhesion of heat-killed LAB to Caco-2 cells (3) Effect of viable, heat-killed LAB cells on the expression of TNF-α and IL-12 in mouse macrophage RAW 264.7 cells Aliquots of 100 μL of each of the viable and heat killed LAB strains, MF, HKMFs (106 cells mL-1 cell counts), or cell walls of MF were pipetted to stimulate the macrophages. Lipopolysaccharide (20 μg mL-1) was used as a positive control. The macrophage cells were co-incubated with LAB or LPS at 37°C for 20 hrs. The levels of cytokines produced in these culture supernatants were analyzed. Cytokines of TNF-α and IL-12 were measured using enzyme-linked immunosorbent assay (ELISA) kit. (4) Oral feeding of mice with HKMF and Salmonella challenge Each mouse was given a single 200 μL dose of HKMF preparation daily by oral administration for 7 consecutive days. Mice in the control group were given 200 μL dose of sterile normal saline. After 7 days, on day 8, each mouse was challenged with S. Typhimurium I50 by oral administration of a single 200 μL dose of Salmonella Typhimurium I50 with a cell count of 3.0×107 CFU mL-1.6 (5) Assessment of TNF-α, IL-6 and IL-10 in mouse serum Fig.1 乳酸菌組合及其熱致死產品誘導巨噬細胞產生細胞激素之結果 Results (1) The levels of TNF-α and IL-12 expression induced with MF and HKMF were higher than those of their individual cells (Fig. 1). (2) The LAB strains treated at 100˚C for 30 min showed close adherent capability as their live strains did. However, for strains heated under 121˚C for 15 min, different but significant effects on the adherent properties of these strains were observed (Table 1). Fig.2乳酸菌組合及其熱致死產品之抑制沙門氏菌侵入小鼠肝臟、脾臟 (3) Significant reduction of Salmonella counts were observed 3 and 6 days post Salmonella challenge (Fig. 2). (4) Mice fed with HKMF showed lower levels of TNF-α and IL-6 in their serum as compared with those of the challenged mice without HKMF feeding (Fig. 3A, B). For the anti-inflammatory cytokine, ie, IL-10, HKMF prior to Salmonella challenge showed higher levels of serum IL-10 as compared with those of the challenged mice without HKMF treatment (Fig.3C). Conclusion Oral feeding the mice with these two HKMF preparations prior to Salmonella challenge could reduce the Salmonella invasion of mice and the induced inflammation. These HKMFs reported here may have potential to be used as an effective probiotics to reduce the Salmonella infections and the induced inflammation of human and animals. Fig.3 乳酸菌組合及其熱致死產品之抑制沙門氏菌引起之發炎及效應