压力对酵母菌及其产海藻糖的影响
摘要
高压条件下，酵母菌细胞受到刺激后发生应激性反应。海藻糖是很多生物体受环境胁迫所产生的重要的应激性代谢产物之一，它是由两个葡萄糖分子结合组成的非还原性双糖，由于它对生物大分子具有非特异性保护作用，从而在食品、医药、生物化学等领域得到了广泛的应用。本文研究主要内容为压力条件下酵母菌积累海藻糖最佳条件的确立、不同酵母菌积累胞内海藻糖随处理时间的变化和相应合成酶系活力的研究、不同酵母菌种及其他条件下细胞的变化等。
首先本文以面包酵母为研究菌株，初步研究了压力条件下不同压力值、处理时间、反应温度、pH值、升降压速率、处理前培养时间、培养基等因素对菌体积累海藻糖所产生的影响。确定了压力为0.5～1.0MPa，反应处理时间为3h，反应温度为34℃，发酵液pH值调整为6.0，升降压速率为0.05～0.10MPa／min，选用复合培养基为酵母菌积累胞内海藻糖的最适条件。
其次研究了六种不同酵母菌在1.0MPa的压力条件下，菌体积累海藻糖(可能还有其他低分子量的应激性代谢产物)与高压处理时间的关系，说明不同酵母菌种对压力具有不同的敏感性；它们之间的不同说明海藻糖的合成途径可能有多种方式，即便是同一种生物中，也有可能存在一种以上的合成途径。同时确立了一种测定海藻糖合成酶系活力的方法，以2％甲苯和0.2％EDTA溶液作为细胞渗透剂处理酵母细胞，以1.0％的葡萄糖溶液作为酶促反应的底物，采用生物传感仪—硫酸蒽酮显色法测定了相应的海藻糖合成酶系的活力。
最后以扫描式电子显微镜拍摄图像，说明经过高压处理后酵母菌体细胞发生变形，胞质和内含物聚集两端，呈现纺锤形，细胞表面有收缩褶皱和塌陷。同时还有活细胞数减少、存活率的降低、菌体量减小以及pH值略微升高等各种现象，均与海藻糖含量高低有关系。还分别以CO_2和N_2作为加压气源，以面包酵母WR—5作为研究菌株，以溶液电导率值的变化来表示细胞的渗透性，分别研究了不同压力、升降压速率、处理时间和处理温度对细胞渗透性变化的影响。
Under the condition of high pressure, the irritative reaction occurred after being stimulated for the Yeast cells. Trehalose is one of the important irritative metabolic productions of plenty of microorganism , during they were intimidated by the environment, it is a nonreducing disaccharide with two glucose residues. Because trehalose exhibit protective action against damage of high molecular substances, it achieve widespread application in a variety of fields including foods , pharmaceutics and biochemistry etc. In this paper, the main research contents include establishing the optimum conditions of Yeast thalli accumulating trehalose in the high pressure circumstance, intracellular trehalose accumulated by different sorts of Yeasts varying from the precessing time and corresponding trehalose synthase system activity, the change of some parameter of different Yeast cells under other conditions.
At first, by using Bakers Yeast (saccharomyces cerevisiae) as initial strain, the factors affecting the intracecellular trehalose production were studied in this paper, containing different pressure value, processing time, reaction temperature, pH value, pressing and decompressing rates, culture time before processing, culture medium and many factors. The optimum conditions of Yeast thalli accumulating trehalose in the high pressure circumstance was established , as they are, pressure value0.5~1.0MPa, processing time 3h, reaction temperature 34 C, pH6.0, pressing and depressing rates0.05~0.10MPa/min, selecting composite culture medium.
Secondly, three sorts of Saccharomyces cerevisiae, six strains(Beer yeast GZH and USA, Baker yeast Saf and WR-5, Alcohol yeast AY-3 and AY-11) wre selected as our study objects under the condition of 1.0MPa. Thalli accumulating intracecellular trehalose varying from processing time wre studied. The results showed different sorts of yeasts had various sensitiveity for the high pressure, and further explained the synthesis path of intracecellular trehalose had possibly many kinds, as if the same sort had also probably more than one paths. Simultaneously the new method of determining trehalose synthase system activity was established. Yeast cells wre penetrated by 2% toluene and 0.2% EDTA solution as infiltration reagent, 1.0% glucose solution was used enzyme catalysis substrates. Corresponding trehalose synthase system activity was determined by
using biosensor instrument-sulfate anthrone colorimetric.
Finally the cell electron micrograph were taken by using scan electron microscope. The results showed Yeast cells distortion after high pressure processing, There were many shrinkage, drapes, pleats and dents on the cell surfaces, presented spindle shape. Simultaneously concomitant characters were the active cell quantity and survival rate deceasing, biomass reducing and pH appreciably rising etc. All of them had a relation with the intracecellular trehalose content. In addition, CO2 and N2 wre used as pressing gas and Baker's Yeast WR-5 was selected research strain, the cell penetrability was denoted by the conductance value variety of the solution. The factors affecting the cell penetrability including different pressure value, pressing and decompressing rates, processing time, reaction temperature wre studied in this paper.
首先本文以面包酵母为研究菌株，初步研究了压力条件下不同压力值、处理时间、反应温度、pH值、升降压速率、处理前培养时间、培养基等因素对菌体积累海藻糖所产生的影响。确定了压力为0.5～1.0MPa，反应处理时间为3h，反应温度为34℃，发酵液pH值调整为6.0，升降压速率为0.05～0.10MPa／min，选用复合培养基为酵母菌积累胞内海藻糖的最适条件。
其次研究了六种不同酵母菌在1.0MPa的压力条件下，菌体积累海藻糖(可能还有其他低分子量的应激性代谢产物)与高压处理时间的关系，说明不同酵母菌种对压力具有不同的敏感性；它们之间的不同说明海藻糖的合成途径可能有多种方式，即便是同一种生物中，也有可能存在一种以上的合成途径。同时确立了一种测定海藻糖合成酶系活力的方法，以2％甲苯和0.2％EDTA溶液作为细胞渗透剂处理酵母细胞，以1.0％的葡萄糖溶液作为酶促反应的底物，采用生物传感仪—硫酸蒽酮显色法测定了相应的海藻糖合成酶系的活力。
最后以扫描式电子显微镜拍摄图像，说明经过高压处理后酵母菌体细胞发生变形，胞质和内含物聚集两端，呈现纺锤形，细胞表面有收缩褶皱和塌陷。同时还有活细胞数减少、存活率的降低、菌体量减小以及pH值略微升高等各种现象，均与海藻糖含量高低有关系。还分别以CO_2和N_2作为加压气源，以面包酵母WR—5作为研究菌株，以溶液电导率值的变化来表示细胞的渗透性，分别研究了不同压力、升降压速率、处理时间和处理温度对细胞渗透性变化的影响。
Under the condition of high pressure, the irritative reaction occurred after being stimulated for the Yeast cells. Trehalose is one of the important irritative metabolic productions of plenty of microorganism , during they were intimidated by the environment, it is a nonreducing disaccharide with two glucose residues. Because trehalose exhibit protective action against damage of high molecular substances, it achieve widespread application in a variety of fields including foods , pharmaceutics and biochemistry etc. In this paper, the main research contents include establishing the optimum conditions of Yeast thalli accumulating trehalose in the high pressure circumstance, intracellular trehalose accumulated by different sorts of Yeasts varying from the precessing time and corresponding trehalose synthase system activity, the change of some parameter of different Yeast cells under other conditions.
At first, by using Bakers Yeast (saccharomyces cerevisiae) as initial strain, the factors affecting the intracecellular trehalose production were studied in this paper, containing different pressure value, processing time, reaction temperature, pH value, pressing and decompressing rates, culture time before processing, culture medium and many factors. The optimum conditions of Yeast thalli accumulating trehalose in the high pressure circumstance was established , as they are, pressure value0.5~1.0MPa, processing time 3h, reaction temperature 34 C, pH6.0, pressing and depressing rates0.05~0.10MPa/min, selecting composite culture medium.
Secondly, three sorts of Saccharomyces cerevisiae, six strains(Beer yeast GZH and USA, Baker yeast Saf and WR-5, Alcohol yeast AY-3 and AY-11) wre selected as our study objects under the condition of 1.0MPa. Thalli accumulating intracecellular trehalose varying from processing time wre studied. The results showed different sorts of yeasts had various sensitiveity for the high pressure, and further explained the synthesis path of intracecellular trehalose had possibly many kinds, as if the same sort had also probably more than one paths. Simultaneously the new method of determining trehalose synthase system activity was established. Yeast cells wre penetrated by 2% toluene and 0.2% EDTA solution as infiltration reagent, 1.0% glucose solution was used enzyme catalysis substrates. Corresponding trehalose synthase system activity was determined by
using biosensor instrument-sulfate anthrone colorimetric.
Finally the cell electron micrograph were taken by using scan electron microscope. The results showed Yeast cells distortion after high pressure processing, There were many shrinkage, drapes, pleats and dents on the cell surfaces, presented spindle shape. Simultaneously concomitant characters were the active cell quantity and survival rate deceasing, biomass reducing and pH appreciably rising etc. All of them had a relation with the intracecellular trehalose content. In addition, CO2 and N2 wre used as pressing gas and Baker's Yeast WR-5 was selected research strain, the cell penetrability was denoted by the conductance value variety of the solution. The factors affecting the cell penetrability including different pressure value, pressing and decompressing rates, processing time, reaction temperature wre studied in this paper.
引文
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