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|Title: ||Pediococcus pentosaceus ACCEL 所產細菌素之純化、作用機制及應用之探討|
Purification, mode of action and application of the bacteriocin from Pediococcus pentosaceus ACCEL
|Authors: ||Chien-Wei Wu|
|Contributors: ||NTOU:Department of Food Science|
|Issue Date: ||2011-06-30T07:40:05Z
|Abstract: ||Pediococcus pentosaceus ACCEL所產細菌素可經由菌體吸附釋放法和Superose 12TM高效能液相層析法(Fast Performance Liquid Chromatography, FPLC)純化出。純化的細菌素分子量大小約為17.5 kDa，N-端序列為–KYYGNGVTXGKHSXXVDXG−，屬乳酸菌細菌素IIa屬型，因此命名為pediocin ACCEL。純化後的細菌素易受不同蛋白質分解酵素作用而失活。在100□C與pH 2.0-6.0範圍下具安定性。而pH 2.0-4.0的範圍內，經121□C加熱15分鐘仍保有80%的活性。Pediocin ACCEL可抑制許多的革蘭氏陽性菌但無法抑制革蘭氏陰性菌。儲存安定性的研究結果顯示，純化的細菌素在小於pH 6.0的低溫環境下具有較佳的安定性，而凍乾粉末經一個月的室溫儲存，活性變化情形並無明顯的變異。為了研究pediocin ACCEL的抑菌機制，利用carboxyfluorescein diacetate (cFDA)染色之Listeria monocytogenes BCRC 14845做為指示菌株，進一步與nisin相互比較來完成。實驗結果顯示隨著細菌素濃度提升至12.5 □g/mL，染色菌體中的螢光強度會減少並且流出的螢光會增加。當細菌素濃度超過12.5 □g/mL，螢光強度和菌數趨於緩和。物質的外漏情形可由菌體內紫外光吸收物質的釋出再度證明。在比較螢光的漏出時間時，nisin處理1分鐘的菌體和pediocin ACCEL處理40分鐘的菌體，其cFDA強度上相當接近。就cFDA、紫外光吸收物質的外漏、cFDA染色菌體在菌數上的改變以及其恢復能力而言，nisin與pediocin ACCEL對於L. monocytogenes的抑菌作用主要是造成細胞膜上的孔洞化所引起的，其中存在著靜菌和殺菌作用。 為了比較pediocin ACCEL與nisin的生物防腐效果，各自將生魚片浸泡在不同濃度pediocin ACCEL、nisin或者兩者混合的溶液中，接著分別儲藏在4□C和0□C下。除了1500 IU/mL的pediocin ACCEL處理組外，在4□C儲藏2天後這些經細菌素處理後的樣品其總生菌數會小於102 CFU/g。Nisin處理組在4□C儲藏2天後其好氧平板計數(APC)會超過2.0 log CFU/g，然而pediocin ACCEL處理組則在1天時會超過。Pediocin ACCEL與nisin分別在4□C儲藏2週期與1週期間可抑制L. monocytogenes的生長。在2週4□C的儲藏下pediocin ACCEL對L. monocytogenes的抑制能力較nisin好。|
The Pediococcus pentosaceus ACCEL bacteriocin was purified to electrophoretical homogeneity by cells-adsorption-desorption and Superose 12TM Fast Performance Liquid Chromatography (FPLC). The purified bacteriocin, with a Mw of 17.5 kDa and N-terminal sequence of -KYYGNGVTXGKHSXXVDXG-, was belonged to Class IIa and designated as pediocin ACCEL. It was inactivated by various proteases and stable at pH 2.0-6.0, <100□C. More than 80% activity was left even after 15 min heating at 121□C, pH 2.0–4.0. G (+) food-borne pathogens were inhibited by this bacteriocin, but G (−) was not. According to storage stability study, the purified pediocin ACCEL was stable at pH < 6.0 and low temperature. No significant change in bactericidal activity was observed after freeze-drying and subsequent 1-month storage at room temperature. In order to study the antimicrobial mechanism of pediocin ACCEL, effects of pediocin ACCEL and nisin on carboxyfluorescein diacetate (cFDA) stained Listeria monocytogenes BCRC 14845 was carried out. Decrease of fluorescence intensity in stained cells and increase in efflux were observed with the increase in bacteriocin concentrations up to 12.5 □g/mL. No further decreases in fluorescence intensity and cell numbers was obtained when the concentration of bacteriocins was over 12.5 □g/mL. The leakage of efflux was further confirmed by the release of intracellular ultraviolet-absorbing substances. Comparing the leaking time of fluorescence, the intensity of cFDA in nisin-treated cells within 1 min incubation was almost comparable to that in pediocin ACCEL-treated samples after 40 min incubation. Considering the leakage of cFDA, UV-absorbing substances, changes in cell numbers during incubation of cFDA stained cells, and restoration ability, nisin and pediocin ACCEL seemed to reveal inhihibiton effect on L. monocytogenes were mainly resulted from the pores formation on the cell membrane. They revealed both bacteriostatic and bactericidic effects. To compare the biopreservative effectiveness of pediocin ACCEL with nisin, fresh fish fillets were immersed in different concentrations of pediocin ACCEL, nisin or their combination and stored at 4□C and 0□C, respectively. The Aerobic Plate Counts (APC) of those with bacteriocins was <102 CFU/g after 2 days storage at 0□C, except that with 1,500 IU/mL of pediocin ACCEL. The APC of samples with nisin was >102 CFU/g after 2 d storage at 4oC, while that with pediocin ACCEL occurred after 1 d storage. Pediocin ACCEL and nisin could suppress L. monocytogenes during 2 and 1 wk storage at 4□C, respectively. It seemed to be better than nisin in the inhibition of L. monocytogenes during 2 wks storage at 4□C.
|Appears in Collections:||[食品科學系] 博碩士論文|
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