冷凍乾燥對標準雙叉桿菌及其篩選菌株生理特性影響之研究

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Title:	冷凍乾燥對標準雙叉桿菌及其篩選菌株生理特性影響之研究
Other Titles:	Study of the Effect of Freeze-Drying on the Physiological Characteristics of the Standard and the Selected Bifidobacteria
Authors:	張贊岩 Chang, Chan-Yen
Contributors:	施宗雄 Chung-Hsung Shih 東海大學畜產與生物科技學系
Keywords:	冷凍乾燥;雙叉桿菌;耐氧性 Freeze-Drying;Bifidobacteria;oxygen-tolerant
Date:	1999
Issue Date:	2011-05-25T09:15:14Z (UTC)
Abstract:	本試驗先篩選乳酸菌中的雙叉桿菌（Bifidobacteria）之耐氧性菌株再與標準菌株進行冷凍乾燥處理，藉由其生理及生化特性之測定，以了解冷凍乾燥對雙叉桿菌及所篩選菌株之影響。本次試驗選用Bifidobacterium breve CCRC 14601、B. infantis CCRC 14604、B. longum CCRC 14605、B. longum CCRC 11847、B. bifidum CCRC 14146與B. bifidum CCRC 11844作為試驗標準菌株。試驗一為採用純系選種（Pure culture selection）方式，以標準菌株為基礎，進行選育在有氧環境下能穩定生長的雙叉桿菌，選出後之菌株再與標準菌株比較其生理特性上的差異，包括乳凝固時間、菌數、滴定酸度、β-半乳糖甘脢（β-galactosidase）及α- 半乳糖甘脢(α-galactosidase)活性。試驗二乃將標準菌株與所篩選出之菌株共10株，在冷凍乾燥處理前後，進行各項生理生化試驗，以比較冷凍乾燥對雙叉桿菌的影響，期能篩選出耐氧之雙叉桿菌，以提供乳業界實際之應用。試驗一結果︰六株標準雙叉桿菌在有氧狀態下經15次繼代培養並加以篩選後，發現除B. longum CCRC 14605與B. bifidum CCRC 11844之外其餘四株雙叉桿菌可明顯在選育後生長於有氧環境下。不過篩選菌株與標準菌株相互比較，發現乳凝固時間顯著比標準菌株長（p<0.05），且產生的凝乳並不完全呈半凝狀態，同時篩選菌株比標準菌株之菌數明顯減少了將近1∼2 log CFU/ml（p<0.05）。在酸度上亦顯著較標準菌株低（p<0.05），可能與各篩選菌株之β-galactosidase活性顯著比標準菌株低（p<0.05）有關。篩選菌株中除B. bifidum 14146的α-galactosidase活性顯著高於其標準菌株外（p<0.05），其餘皆顯著低於標準菌株的活性（p<0.05）。試驗二結果︰冷凍乾燥後標準菌株之存活率以B. bifidum 14146降低最顯著（p<0.05），而篩選出之雙叉桿菌B. infantis 14604對冷凍乾燥的耐受性最高，其菌數與乾燥前無顯著差異（p>0.05），B. breve 14601與B. bifidum 14146之菌數則降至6 log10/ml以下。乾燥菌體在經溶菌酵素（lysozyme）、牛膽汁(oxgall)及氯化鈉（NaCl）處理後發現，不論標準雙叉桿菌或篩選菌株皆顯著增加對這些物質的敏感性（p<0.05）。另外，冷凍乾燥後的標準菌株及篩選菌株，經培養於含2％乳糖的MRS液態培養基後發現，B. breve 14601及B. infantis 14604在厭氧與有氧的狀態下，β-galactosidase及α-galactosidase活性有顯著增加的現象（p<0.05）。各培養菌液經離心後所得上清液部份，不論標準菌株或篩選菌株皆顯著測得乳糖酵素的存在（p<0.05），代表細胞有通透性改變的現象而造成酵素從菌體內洩漏至細胞外。細胞萃取物方面，從聚丙烯凝膠電泳的結果發現，冷凍乾燥之菌體不論是標準菌株及篩選菌株，經0.2％ SDS處理後，明顯比未乾燥菌體多出了一分子量約30 Kd的蛋白質，這可能是菌體受冷凍乾燥的影響，使得在細胞壁上鍵結的蛋白質變得不穩定，故易被萃取出來。 The aims of this study were to select the oxygen-tolerant bifidobacteria first and then investigate the effect of freeze drying on the standard and the selected bifidobacteria which were subjected to this treatment. Six bifidobacteria including Bifidobacterium. breve CCRC 14601, B. infantis CCRC 14604, B. longum CCRC 14605, B. longum CCRC 11847, B. bifidum CCRC 14146, and B. bifidum CCRC 11844 were studied. In the first part of this study, pure culture selection was adopted to select the bifidobacteria that could survive normally under aerobic environment and the physiological characteristics which included milk coagulation time, total plate counts of bifidobacteria, titration acidity, activity of β-galactosidase and α-galactosidase for the standard and the selected bifidobacteria were compared. In the second part, the effect of freeze drying on ten standard and selected bifidobacteria in total was further investigated by conducting physiological and biochemical experiments before and after freeze drying. The results of the first part study indicated that four out of six bifidobacteria under aerobic condition, except B. longum CCRC 14605 and B. longum CCRC 11847, could normally survive through 15 times transfer cultivation and selection. It was found that milk coagulation time for the selected bifidobacteria was generally longer than that for the standards (p<0.05) and also the coagulated milk was not completed in the state of semi-coagulation. Moreover, the population of the selected bifidobacteria was obviously less than that of the standards by nearly 1~2 log CFU/ml (p<0.01). The selected bifidobacteria were also markedly lower than the standards in acidity (p <0.05). It was suggested that it might be related to the activity of β-galactosidase for the selected bifidobacteria which was significantly lower than the standards (p<0.01). The yield of lactic acid was indirectly affected and , the acidity was limited for ascending. The selected bifidobacteria were significantly weaker than the standards for the activity of α-galactosidase (p<0.01), except for B. bifidum CCRC 14146 that was much stronger than standards (p<0.01). The results of the second study were indicated that the survival percentage of the standard B. bifidum CCRC14146 significantly declined after freeze drying (p<0.01) and also the population for the two out of four selected bifidobacteria sharply dropped to less than 6 log10/ml. However the selected B. infantis 14604 showed the highest tolerance to the freeze drying, so its population was not obviously changed by freeze drying (p>0.05). Both the standard and the selected bifidobacteria which treated with lysozyme, oxygall and NaCl after freeze drying had significantly increased their sensitivity to these materials (p<0.01). It suggested that freeze drying would affect the stability of cell wall and the permeability of cell membrane for bifidobacteria. In addition, the standard, B. breve CCRC 14601, and the selected B. infantis CCRC 14604 incubated in MRS broth with 2% lactose after freeze drying had apparently increased the activity of β-galactosidase and α-galactosidase under anaerobic and naerobic conditions. The supernatant for the standard and the selected bifidobacteria were detected obviously to have the above two enzymes after centrifuging (p<0.01), which indicated that the permeability of bifidobacteria cell membrane were changed, therefore the enzymes could flow and leak in outside of the cell. The SDS-PAGE for the cell extract revealed that both the standard and the selected bifidobacteria treated with 0.2% SDS after freeze drying had apparently about 30Kd molecular weight of protein as compared to that without freeze drying. It suggested that freeze drying had made the linkage of protein on the cell wall of bifidobacteria unstable, so that the protein could be readily extracted.
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