酸適應處理對腸炎弧菌耐酸性與蛋白質表現之影響

Abstract

本研究之目的乃測試酸適應處理、生長時期及菌株對腸炎弧菌在後續酸性壓力下存活之影響，研究亦探討酸適應對腸炎弧菌蛋白質表現之影響。於實驗中，腸炎弧菌菌體分別以pH 5.0、5.5和6.0進行30、60 和90分鐘不同之酸適應處理。 結果顯示，酸適應處理之pH值和時間長短會影響腸炎弧菌之耐酸性。當腸炎弧菌690以pH 5.5和6.0進行酸適應處理，菌體暴露於pH 4.5下4小時後之存活率大致隨酸適應處理時間之延長而增加。在不同酸適應之處理條件中，腸炎弧菌690以pH 5.5進行90分鐘之酸適應處理可展現最高之耐酸性。酸適應處理之對數期末期腸炎弧菌690菌體所提昇耐酸性之程度高於酸適應處理之對數期中期和靜止期菌體。 此外，研究亦發現酸適應處理之腸炎弧菌菌體所引發之耐酸性可因菌株（405、556、690、BCRC 13023、BCRC 13025、BCRC 12863及BCRC 12864）和所試有機酸（乳酸、醋酸、檸檬酸和丙酸）不同而有所差異。此外，酸適應處理會增加暴露於3% NaCl磷酸緩衝液中三株腸炎弧菌（690、BCRC 13023和BCRC 13025）菌體之耐熱性（47℃），但不影響菌體之耐冷性（4和-18℃）。然而，此酸適應效應並未在牡蠣均質液中觀察到，暴露於牡蠣均質液中之酸適應處理菌體在高溫及低溫下之存活率均與控制組相當。 迷你和二維電泳分析顯示，於腸炎弧菌690、BCRC 13023和BCRC 13025菌體中，某些蛋白質會共同地或單獨地被酸適應處理誘導增加或減少其表現量。於本研究中，這些壓力蛋白質之誘導應與酸適應處理之腸炎弧菌增加對酸和熱壓力之耐受性有關。酸適應處理所誘導產生之壓力蛋白質需進一步於研究中鑑定。

The objective of this study was conducted to examine the effect of acid adaptation treatment, growth phase and bacterial strain on the survival of Vibrio parahaemolyticus under environmental stresses. The protein expression of V. parahaemolyticus influenced by acid adaptation was also investigated. In the experiments, cells of V. parahaemolyticus were subjected to various acid adaptation treatments at pH 5.0, 5.5 and 6.0 for 30, 60 and 90 min, respectively. Results showed that the pH value and length of acid adaptation treatment affected the acid tolerance of V. parahaemolyticus. When cells acid-adapted at pH 5.5 and 6.0, the survival of V. parahaemolyticus 690 after exposure at pH 4.5 for 4 h generally increased as the length of acid adaptation treatment extended. Among the various acid adaptation treatments examined, cells of V. parahaemolyticus 690 subjected to acid adaptation at pH 5.5 for 90 min exhibited the highest acid tolerance. The acid-adapted late-exponential phase cells of V. parahaemolyticus 690 had a higher magnitude of increased acid tolerance than the cells acid-adapted in mid-exponential and stationary phases. Additionally, it was found that the extent of induced acid tolerance on the acid-adapted cells of V. parahaemolyticus varied with the bacterial strain （405, 556, 690, BCRC 13023, BCRC 13025, BCRC 12863 and BCRC 12864） and organic acid （lactic acid, acetic acid, citric acid and propanoic acid） tested. Furthermore, acid adaptation increased the thermal tolerance （47℃） of three V. parahaemolyticus strains （690, BCRC 13023 and BCRC 13025）, while did not affect their cold tolerance （4 and -20℃）, when cells exposed to PBS-3% NaCl. However, the acid-adapted response was not observed in oyster homogenates, the survival of acid-adapted cells exposed to oyster homogenates was similar to that of control cells at high and low temperatures. Analysis with mini SDS-PAGE and two-dimensional electrophoresis revealed that some proteins were jointly or individually enhanced or even reduced by acid adaptation in cells of V. parahaemolyticus 690, BCRC 13023 and BCRC 13025. In the present study, the induction of these stress proteins might relate to increased tolerance of acid-adapted V. parahaemolyticus to acid and heat stresses. Identification of the stress proteins induced by acid adaptation requires in further studies.