Lactobacillus reuteri胞外多醣最適化生產及生理活性之研究

Abstract

Lactobacillus reuteri DSM17938是一種源於人體的益生菌，具有改善嬰兒腹絞痛症狀、增加Helicobacter pylor根除率、牙齒保健及改善腸胃道症狀等功效。本研究針對L. reuteri DSM17938產生之胞外多醣抗氧化及抗發炎功效加以探討，進行抗氧化實驗，結果發現L. reuteri DSM17938發酵液經培養18小時後離心去除菌體，其發酵上清液有良好的抗氧化效果，對DPPH自由基清除力為84.28 ± 0.37%、對亞鐵離子螯合率為97.78 ± 0.32%、還原力236.53 ± 20.9 μg/L、總抗氧化力405.32 ± 8.17μM。利用反應曲面法 (response surface methodology, RSM)，選擇半乳糖濃度 (17 ± 5%)、溫度 (22.6 ± 5 ℃)、pH (6.8 ± 1) 三因子進行最適化實驗，結果顯示在半乳糖濃度17.12%、培養溫度22.9 ℃、起始pH 6.5時，L. reuteri DSM17938胞外多醣生合成產量可達到最高含量643.32 ± 10.7 mg/L，產量相較原先培養方法：葡萄糖濃度2%、溫度37 ℃、pH 6.4時，胞外多醣生合成產量220.92±0.94 mg/L，高出2.3倍。之後將發酵液中的胞外多醣以酒精沉澱法收集，分別觀察抗氧化及抗發炎效果，在DPPH自由基清除力、還原力及總抗氧化力實驗中，發現原發酵上清液之抗氧化力顯著大於胞外多醣；而亞鐵離子螯合力實驗中，胞外多醣的螯合率為 97.47 ± 0.47%，顯著高於原發酵上清液70.11 ± 0.53%，推測胞外多醣可干擾Fe2+-Ferrozine複合物形成，具有捕獲及螯合金屬離子能力。另外在抗發炎實驗中，以LPS誘導RAW264.7巨噬細胞發炎，其一氧化氮產生量為33.36 ± 2.12μM，加入胞外多醣200 mg/L則一氧化氮產生量顯著下降至12.96 ± 1.07μM。

Lactobacillus reuteri DSM17938 is a human-borne probiotic that has some beneficial to human health, such as improving infantile abdominal cramps, increasing Helicobacter pylori eradication, tooth care and improving gastrointestinal symptoms. In this study, we discuss the antioxidant and anti-inflammatory effects of extracellular polysaccharide produced by L. reuteri DSM17938. The results showed that L. reuteri DSM17938 fermentation broth was centrifuged after 18 hours of culture, and had the antioxidant effect of DPPH radical was 84.28 ± 0.37%, ferrous ion chelation rate was 97.78 ± 0.32%, reducring power was 236.53 ± 20.9 g/L, the trolox equivalent antioxidant capacity was 405.32 ± 8.17μM. The optimum experiment was carried out by using the response surface methodology (RSM) and choose three factors, which is galactose concentration (17 ± 5%), temperature (22.6 ± 5 ℃) and pH (6.8 ± 1). The results showed that the concentration of galactose 17.12%, culture temperature 22.9 ℃, pH 6.5, L. reuteri DSM17938 extracellular polysaccharide production reached the highest content of 643.32 ± 10.7 mg/L, the yield was higher than the original culture method 2.3 times. The original culture method: glucose concentration 2%, temperature 37 ℃ , PH 6.4, the extracellular polysaccharide production yield 220.92 ± 0.94 mg/L. The antioxidant and anti-inflammatory effects of the extracellular polysaccharides in the fermentation broth were collected by alcohol precipitation. The DPPH free radical scavenging power, reducing power and trolox equivalent antioxidant capacity were observed that the antioxidant capacity of the supernatant was significantly larger than that of extracellular polysaccharide, But the chelating rate of extracellular polysaccharide was 97.47 ± 0.47%, which was significantly higher than original fermentation supernatant 70.11 ± 0.53%. Extracellular polysaccharide was inferred the Fe2 + - Ferrozine complexes, have the ability to capture and chelate metal ions. In the anti-inflammatory experiment, using LPS induced RAW264.7 macrophage inflammation, the amount of nitric oxide production was 33.36 ± 2.12 μM, adding extracellular polysaccharide 200 mg/L, the amount of nitric oxide production decreased significantly to 12.96 ± 1.07 μM.