Lactobacillus mali APS1於不同壓力下存活與適應性反應之探討

Abstract

Lactobacillus mali APS1為自糖液克弗爾粒中分離出，一具潛力的益生菌株，其已被證實具有免疫調節、抗結腸炎和抗肥胖等諸多機能性。而益生菌之益生特質的發揮與其活菌數息息相關，然而益生菌產品生產及使用過程中，菌體將面臨各式各樣的環境壓力，如冷、熱、酸和膽鹽，這些環境壓力都將造成菌體菌數大幅減少，而影響機能性的發揮。因此，微生物為了抵抗環境壓力的威脅，演化出一套防禦系統，幫助菌體適應這些致死或次致死的環境，而本研究即為探Lactobacillus mali APS1於不同壓力下，如冷、熱、酸和膽鹽等環境中之適應性反應的表現和存活率變化。 首先在L. mali APS1壓力敏感性結果顯示， L. mali APS1之冷、熱、酸和膽鹽之次致死和致死環境分別為10°C和-20°C、42°C和52°C、pH 3.5和pH 2.0以及0.1% 和2.0%膽鹽濃度。冷、熱和膽鹽適應處理可誘導L. mali APS1之同源壓力耐受性，而冷和膽鹽適應可對L. mali APS1於熱致死挑戰下提供交叉保護的效果，而冷和熱適應亦可提供L. mali APS1於膽鹽致死挑戰下一定交叉保護作用，另酸和膽鹽適應可增加L. mali APS1於冷致死挑戰下之存活率。此外，酸和冷適應之L. mali APS1經冷凍乾燥處理後存活率顯著高於熱及膽鹽適應組 (p < 0.05) 。胞外多醣體產量各適應組與未適應組間無顯著差異 (p > 0.05) ，而經後續致死挑戰後存活率較高的適應處理組別，在掃描式電子顯微鏡觀察下可觀察到較少細胞損壞的情形，而在蛋白質體分析部分，各處理組間和未適應組以迷你膠體電泳分析之蛋白質條帶無發現明顯差異，但透過基質輔助雷射脫附離子化飛行時間質譜儀 (matrix-assisted laser desorption/ionization time-of-flight mass spectrometer, MALDI-TOF MS) 之分析，可觀察到各處理組間部份訊號變化，而各組進一步以主成分分析 (Principal Component Analysis, PCA) ，發現可將適應組和未適應組進行分群。 綜上所述，本研究發現次致死適應可作為提升L. mali APS1於致死環境壓力下存活表現之可能策略，此外，根據菌體形態和蛋白質體分析結果，發現經適應之L. mali APS1可被誘導壓力適應反應，幫助菌體維持生存能力和細胞完整性。

Lactobacillus mali APS1, a potential probiotic strain isolated from sugar kefir grains, has been proven its functionalities about immune-regulation, anti-colitis, and anti-obesity. Live probiotics with certain amount of number are essential for healthy beneficial. However, probiotics encounter critical environmental stress such as cold, heat, acid and bile salts, which dramatically decrease the survival and functionality of probiotics. Therefore, bacteria evolve a system defensing to help them adapting to lethal or sublethal condition. Thus, the objective of this study was to investigate the adaptive responses and viability of L. mali APS1 to various environmental stresses, such as cold, heat, acid, and bile salts. The stress susceptibility results showed that the sublethal and lethal condition of acid, bile salt, heat and cold for L. mali APS1 were 10°C and -20°C, 42°C and 52°C, pH 3.5 and pH 2.0, and 0.1% and 2%, respectively. The subsequent lethal challenge survival results showed that sublethal adaptation of L. mali APS1 to cold, heat and bile salts could induce homologous tolerance. Adaptation to cold and bile provided cross-protection against heat challenge (52°C). Adaptation to cold and heat improved the resistance to bile salt stress (2%). Adaptation to acid and bile increased survival of L. mali APS1 under cold lethal challenge. Besides, after freeze drying, survival of cold and acid adapted cells was significantly higher than heat and bile adapted cells (p < 0.05). Exopolysaccharides production of all stress-adapted and non-adapted cells have no significant difference (p > 0.05). Adapted cells with higher survival were observed minor cell damage after subsequent lethal challenge by scaning electron microscopy (SEM). For proteomics analysis, no significant difference was observed in Mini SDS-PAGE results, but signal change was detected from matrix-assisted laser desorption/ionization time-of-flight mass spectrometer(MALDI-TOF MS) between adapted and non-adapted cells and further grouped by principle component analysis (PCA). In conclusion, this study revealed that sublethal adaptation could be a possible strategy to improve survival of L. mali APS1 in lethal stress condition. Besides, according to morphological and proteomics analysis results, it was found that some adaptive response could be induced to maintain survival and integrity of adapted L. mali APS1.