壓力適應對Liquorilactobacillus mali APS1於冷凍和噴霧乾燥後之生存及其生理特性影響之探討

Abstract

Liquorilactobacillus mali APS1 (Lactobacillus mali APS1) 為一株具有免疫調節、抗結腸炎及抗肥胖功效的益生菌。本研究之目的主要探討壓力適應對L. mali APS1於冷凍和噴霧乾後之生存及對其生理特性影響。 本實驗所使用冷、熱、酸及膽鹽之次致死適應條件分別為，10°C、42°C、pH 3.5及0.1%膽鹽，次致死時間為1小時。首先，實驗先探討經過次致死壓力適應後之L. mali APS1，經乾燥(冷凍及噴霧乾燥法)、貯存及暴露於模擬腸胃液環境下菌株之存活表現。結果顯示，在未適應和次致死壓力適應組別中，經冷適應之L. mali APS1，冷凍乾燥前後菌數無顯著差異，而未適應、冷及熱適應之菌體於噴霧乾燥後，亦可維持菌數，代表部分次致死適應可以改善乾燥後L. mali APS1的存活率；冷凍乾燥及噴霧乾燥L. mali APS1之菌粉貯存於30°C菌數顯著下降及存活率最低，其次為4及-20°C，然而，在任何貯存溫度下，次致死適應及未適應之L. mali APS1菌粉間之存活率皆無顯著差異；膽鹽適應之L. mali APS1乾燥菌粉經模擬胃液 (pH 2.0) 及腸液(2%膽鹽)暴露後，相較於未適應、酸、冷及熱適應處理組，可維持較高的存活表現。綜合上述結果，四種不同次致死適應中，以膽鹽次致死適應為最具有潛力作為提升L. mali APS1於壓力環境中耐受性。 此外，本研究亦藉由氣相層析儀及二維膠體電泳分析次致死壓力適應之L. mali APS1脂肪酸組成及蛋白質表現量。脂肪酸分析結果指出，酸及熱適應可提升L. mali APS不飽和脂肪酸含量，減少飽和脂肪酸含量。蛋白質分析顯示酸、膽鹽、冷及熱適應處理可分別改變菌體9、14、16及15種蛋白質之表現量，這些蛋白質功能被鑑定出主要與壓力反應、醣類、能量及核甘酸代謝、細胞壁及肽聚醣生合成相關。 最後，探討次致死壓力適應之L. mali APS1繼代後之壓力耐受性、乳酸產量及3T3-L1前脂肪細胞分化之影響。結果顯示，次致死適應之L. mali APS1經繼代培養後，其存活率與未適應之菌體並無顯著差異。膽鹽適應L. mali APS1與未適應處理菌體之乳酸產量相似。此外，在3T3-L1脂肪細胞分化之過程，觀察到未適應及膽鹽適應處理組間油滴累積無顯著差異。

Liquorilactobacillus mali APS1 (Lactobacillus mali APS1), a potential probiotic strain, has been demonstrated to possess immune-regulatory, anti-colitis and anti-obesity effects in vitro and in vivo experiments. The objective of this study was to investigate the effects of stress adaptation on the viability of L. mali APS1 after freeze drying and spray drying and its physiological characteristics. The sublethal condition of cold, heat, and bile salt for L. mali APS1 were 10°C, 42°C, pH 3.5 and 0.1% bile salts, respectively, for one hour. First, the viability of stress-adapted L. mali APS1 after drying (freeze drying and spray drying), storage, and exposure to simulated gastric and intestinal fluids was investigated. The results showed that cold-adapted L. mali APS1 remained high viable counts after freeze drying. For spray drying, non-, cold- and heat-adapted L. mali APS1 had similarly viable counts as before drying. L. mali APS1 in freeze- and spray‐dried powders exhibited the highest population reduction and the lowest survival rate during storage at 30°C, followed by storage at 4 and -20°C. However, there was no significant difference in bacterial counts between stress-adapted and non-adapted L. mali APS1 during storage under same temperature. Bile-adapted L. mali APS1 in freeze- and spray‐dried powders maintained higher values of cell viability after exposure to simulated gastric (pH 2.0) and intestinal (2% bile) fluids than non-, acid-, cold- and heat-adapted cells. According to above findings, bile-adaptation demonstrates a potential to protect L. mali APS1 against environmental stress. Next, gas chromatography (GC) and two dimensional gel electrophoresis (2-DE) were performed to analyze the fatty acid composition and protein expression to investigate the physiological changes of L. mali APS1 after stress adaptation. An increased unsaturated fatty acid and a decreased saturated fatty acid were observed in acid- and heat- adapted L. mali APS1 cells. Proteomic analysis revealed that the protein expression levels of 9, 14, 16, and 15 protein spots in cells of L. mali APS1 were altered by acid, bile salts, cold and heat adaptation, respectively. These proteins were identified and can be classified into different categories, including stress response, carbohydrate, energy and nucleotide metabolism, and cell wall and peptidoglycan biosynthesis. Additonally, the effects of stress adaptation on the stress tolerance after subculture and functionalities of L. mali APS1 were also investigated. The survival under stress challenges was not significantly different between non-adapted and stress-adapted L. mali APS1 after 3-time subculture, indicating the changes of L. mali APS1 after stress adaptation is not heredity. Furthermore, bile-adapted L. mali APS1 showed no effect on lactate production and in-vitro preventing lipid accumulation during differentiation of 3T3-L1 pre-adipocytes.