多菌株乳酸菌組合在飼料添加物及保健食品之應用- 口服乳酸菌組合以降低肉雞之沙門氏菌感染及發炎之研究 曾浩洋1、 陳志遠2、林峻立1、 莊笠岑1、 余碧3、 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 3國立中興大學 動物科學系 連絡電話：04-26318652#5080 E-mail：hytsen@sunrise.hk.edu.tw 計畫名稱 Conclusions Effect of multistrain probiotics are associated with changes in cytokines expression, such as IL-1β, IL-6, IFN-γ and IL-10, in the gut-associated lymphoid tissues of the chicken, and this correlates with the protection against both the visera invasion and gut colonization by S. Typhimurium. Therefore, in preparation of the probiotics for protection of chicks against Salmonella infection, selection of LAB strains with high immunomodulatory activity and able to adhere to chicken crop epithelium cells is important. 101農科-6.1.1-科-aB開發微生物及植物性產品取代含藥物飼料添加物 中文摘要 一日齡之雞隻易受沙門氏桿菌之感染，由於報告顯示多重菌株之組合具有加成或協同作用，比單菌株生菌劑更為有效，在本研究中，我們以小鼠巨噬細胞 RAW 264.7 之 TNF-α 產生能力，挑選 34株具有免疫活性之乳酸菌株，並組合為生劑 (MSFA)；此 MSFA 組合對於沙門氏菌 (Salmonella Typhimurium)，對一日齡肉雞隻之沙門氏菌腸道定殖、內臟侵入，以及發炎作用等具有拮抗作用。一日齡之肉雞，於雞齡1至3天期間，餵食乳酸酸後，於4天，再口投感染沙門氏菌 (200μL, 106 CFU/mL)，經3天及6天後，分析小雞之肝臟、脾臟及腸道中之沙門氏菌之侵入量。進一步分析之雞隻腸道之發炎，我們發現其促發炎細胞激素，例如 interleukin (IL)-β、 IL-6、 Interferon (IFN-γ) 可因餵食 MSFA 之組合，而降低。其發炎激素 IL-10，則是提升現象 ；而經挑選之乳酸菌組合：比任意組合之生菌劑，更可以降低其發炎症狀，本研究因此認為商業化之乳酸菌組合使用菌株，宜排選具有免疫力及吸附能力之乳酸菌加以組合。 Table 1 用於 real-time PCR 之引子及序列 Material and methods (1) Bacteria strains, their growth conditions and the preparation of multistrain formula A (MSFA) and MSFB Table 2 口投兩種乳酸菌組合配方減少沙門氏菌細胞在肉雞的肝臟、脾臟及盲腸之效果 Four different LAB strains including: three Lactobacillus spp. and one Enterococcus spp. selected with high TNF-α inducing activity were combined to a multistrain formula termed as MSFA. Another four randomly selected strains of Lactobacillus spp, were also combined to a multistrain formula (MSFB) (2) Experimental design Forty chicks were used in this study. Chicks were randomly divided into four groups, Group I: ie, MSFA plus Salmonella group; in which 10 chicks were fed by gavage with MSFA (0.2 ml/ chick, 109 CFU/ml) once every day for 3 days followed by challenge with S. Typhimurium on day 4; Group II, ie, the MSFB plus Salmonella group, Group III: the Salmonella group, Group IV, the control group. For all groups, on day 3 and day 6 post infection, chicks were euthanized with Forane® (Aesica Queenborough Ltd) and their livers, spleens and cecal content were collected, and subsequently analyzed for the viable counts of Salmonella cells in these samples. (3) Measurement of oral feeding of MSFA on the inflammation of broiler chicks due to Salmonella infection by reverse transcription quantitative polymerase chain reaction (RT-QPCR) (4) Statistical analysis 感染後 3 天 感染後 6 天 Fig. 1 (A)乳酸菌組合抑制沙門氏菌感染仔雞之效果。(B)(C)(D)改善沙門氏菌引起之發炎反應及提升抗發炎功能。 Results One day old chicks were fed with MSFA from day 1 to day 3 and on day 4, chicks were challenged with S. Typhimurium (200μL, 106 CFU/ml). The livers, spleens and cecal tonsils of chicks were then removed on day 3 and 6 post-infection (dpi). Comparing with the multistrain formula B (MSFB) which consists of LAB strains selected at random, the efficacy of MSFA to reduce the Salmonella counts recovered from the cecal tonsils, spleens and livers of chicks, were significantly higher (Table 2). When the levels of proinflammatory cytokines, such as interleukin (IL)-1β, IL-6, interferon (IFN)-γ and anti-inflmmatory cytokine, ie, IL-10, in cecal tonsils were measured by reverse transcription real-time quantitative PCR (RT-Q PCR), it was found that chicks fed with MSFA for 3 days had lower levels of IL-1β, IL-6, IFN-γ and higher level of IL-10 in the cecal tonsils of chicks as compared with those of the chicks fed with MSFB or without LAB (Figure 1).