细菌纤维素高产菌株超高压诱变选育及冻干保藏研究
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摘要
合成纤维素不是植物所特有的功能,某些动物以及少数微生物也可以合成纤维素。通常将由微生物合成的纤维素称为“细菌纤维素(bacterial cellulose)”。细菌纤维素是一种新型生物材料,它是已知天然纤维中最细、性能最好的纤维素,其微纤维直径约为10-100nm,但弹性模数却是普通纤维的数十倍。还由于其纯度高,结晶度高和重合度高,又有很强的亲水性和优良的生物可降解性,因此倍受国内外研究者的关注。
为了提高细菌纤维素的产量,并降低其生产成本,本研究从实验室长有丰厚凝胶状膜的荞麦醋中分离筛选出了一株性能优良的产细菌纤维素菌株J2,对其进行超高压处理后,获得了一株高产诱变菌株M438,并对其发酵工艺参数进行了优化研究,为工业化生产奠定了基础;同时对菌株M438的冷冻干燥保藏工艺,以及其代谢产物—细菌纤维素的性质、表观形态和超微结构进行了研究,为其进一步研究和应用提供了理论指导。本研究得到的主要结论如下:
(1)从荞麦醋中分离筛选出了一株性能优良的产细菌纤维素菌株J2,对菌株形态及生理生化特征进行研究后,初步确定菌株J2属于氧化葡糖杆菌。
(2)对菌株J2进行超高压处理,经过初筛和复筛,获得了一株诱变株M438,其产纤维素能力比J2有了很大提高且遗传性能稳定。静态培养7d后,诱变株M438的细菌纤维素产量达到15.7457g/100ml,是原菌株J2(10.76 g/100ml)的1.485倍。
(3)确立了适合诱变株M438静态条件下生产细菌纤维素的发酵培养基及发酵条件,发酵培养基配方为:碳源5%(葡萄糖/蔗糖=4:1),酵母浸出汁1.25%,CaCl20.15%,ZnS040.2%,K2HPO40.2%,MgSO40.93%,富马酸0.3%,无水乙醇0.5%,最适发酵条件为:种龄24h,接种量9%,发酵时间7d。在此培养条件下,获得的纤维素的产量为28.9878 g/100ml,是优化前基础发酵培养基的1.841倍,是原菌株J2产量的2.694倍。
(4)确定了最佳的冻干工艺条件:保护剂组成为12%脱脂牛奶+5%葡萄糖/蔗糖(1:2)、预冻温度为-80℃、预冻时间为3h、冻干时间为4d,在此条件下,冻干菌的存活率达到70%以上。通过与其它保藏方法比较得知,冷冻干燥是最佳的保藏方法,可使菌种存活2年左右。
(5)对诱变株M438代谢的细菌纤维素膜的主要成分进行了测定,并对其表观形态和超微结构进行了研究。研究结果表明,细菌纤维素湿膜的含水量为98.77%,干膜的复水率为80.53%,干膜中纤维素含量为89.24%,蛋白质含量为7.9586%,脂肪含量为1.63%;采用不同方式处理的纤维素膜的表观形态有所不同;利用扫描电镜观察不同干燥条件处理的细菌纤维素膜的超微结构,结果表明,与临界点干燥方法相比,真空冷冻干燥和热风干燥两种方法都会使纤维素结构发生一定的改变。
It’s not only plants that can synthesize cellulose, but some animals and micro-organisms also have this ability. The cellulose produced by micro-organisms is called“bacterial cellulose (BC)”. It was a new bio-material which has many characteristics, such as pure, high crystal degree and so on. BC is the thinnest cellulose in the world. Besides, owing to its high coincident degree, high hydrophilic property and better adaptive responses to environmental factors, BC has attracted most reaearchers’attention both at home and abroad.
In order to improve the yield of BC and reduce its cost, a strain (J2) with high yield of BC was initially selected from buckwheat vinegar on whose surface grew much thicker membrane and the characteristics of the strain J2 was researched, and then the high-yield mutant M438 was breeded by the techniques of high-pressure, finally the parameters of fermentation technology were optimized which established the basis for industry. The property of the cellulose membrane was studied and its apparent structure and ultra-micro-structure were observed which gave some direction to its application. The results of this research were as follows:
(1) A strain(J2) with ability to produce high yield of BC was selected from buckwheat vinegar. After the configuration and physiological and biochemical property were studied, the strain was primarily ascertained to belong to Gluconobacter.
(2) Taking strain J2 as the original strain, through the ultra-high pressure treatment mutagenesis, primary screening and the second screening, mutation strain M438 with high-yield property was selected. And the genetic stability of M438 was better than the original strain J2. After 7d cultivation, BC yield improved up to 15.7457g/100ml, which was as 1.485 times as that of pre-mutagenic strain (10.76 g/100ml).
(3) The stationary fermentation medium and fermentation conditions were confirmed which were apt to yield BC for Mutant M438. The components of medium were as follows: carbon source5 %( glucose: sucrose =4:1), yeast juice 1.25%, CaCl2 0.15%, ZnS04 0.2%, K2HPO4 0.2%, MgSO4 0.93%, fumaric acid 0.3%, ethanol 0.5%. The parameters of fermentation conditions were as follows: seed age 24h, inoculums amount 9%, fermentation time 7d. Under this fermentation medium, the yield of BC was 28.9878 g/100ml which was as 1.841 times as that under initial fermentation medium and 2.694 times as that of the unmutant strain J2.
(4) The optimal Freeze-drying conditions were as follows: protectant consisting of 12% skim milk and 5% glucose / sucrose (1:2), pre-freezing temperature of -80℃, pre-freezing time for 3h, freeze-drying time for 4d. Under these conditions, the survival rate of freeze-dried bacteria added up to more than 70%, and through comparison with other preservation methods, freeze-drying was the best method of preservation which could make the bacteria to survive about 2 years.
(5) The main components of the cellulose membrane produced by mutant M438 was mensurated, and the apparent structure and ultra-micro-structure were observed. The water content in wet membrane was 98.77%; the water content in dry membrane after absorbed water was 80.53%; cellulose content in dry membrane was 89.24%, protein content was 7.9586%, and fat content was 1.63%. The apparent structure of both wet and dry membrane were different under different disposing methods; the results of ultra-micro-structure observed with SEM was that vacuum freeze-drying way and air drying way could change the ultra-micro-structure of membrane.
为了提高细菌纤维素的产量,并降低其生产成本,本研究从实验室长有丰厚凝胶状膜的荞麦醋中分离筛选出了一株性能优良的产细菌纤维素菌株J2,对其进行超高压处理后,获得了一株高产诱变菌株M438,并对其发酵工艺参数进行了优化研究,为工业化生产奠定了基础;同时对菌株M438的冷冻干燥保藏工艺,以及其代谢产物—细菌纤维素的性质、表观形态和超微结构进行了研究,为其进一步研究和应用提供了理论指导。本研究得到的主要结论如下:
(1)从荞麦醋中分离筛选出了一株性能优良的产细菌纤维素菌株J2,对菌株形态及生理生化特征进行研究后,初步确定菌株J2属于氧化葡糖杆菌。
(2)对菌株J2进行超高压处理,经过初筛和复筛,获得了一株诱变株M438,其产纤维素能力比J2有了很大提高且遗传性能稳定。静态培养7d后,诱变株M438的细菌纤维素产量达到15.7457g/100ml,是原菌株J2(10.76 g/100ml)的1.485倍。
(3)确立了适合诱变株M438静态条件下生产细菌纤维素的发酵培养基及发酵条件,发酵培养基配方为:碳源5%(葡萄糖/蔗糖=4:1),酵母浸出汁1.25%,CaCl20.15%,ZnS040.2%,K2HPO40.2%,MgSO40.93%,富马酸0.3%,无水乙醇0.5%,最适发酵条件为:种龄24h,接种量9%,发酵时间7d。在此培养条件下,获得的纤维素的产量为28.9878 g/100ml,是优化前基础发酵培养基的1.841倍,是原菌株J2产量的2.694倍。
(4)确定了最佳的冻干工艺条件:保护剂组成为12%脱脂牛奶+5%葡萄糖/蔗糖(1:2)、预冻温度为-80℃、预冻时间为3h、冻干时间为4d,在此条件下,冻干菌的存活率达到70%以上。通过与其它保藏方法比较得知,冷冻干燥是最佳的保藏方法,可使菌种存活2年左右。
(5)对诱变株M438代谢的细菌纤维素膜的主要成分进行了测定,并对其表观形态和超微结构进行了研究。研究结果表明,细菌纤维素湿膜的含水量为98.77%,干膜的复水率为80.53%,干膜中纤维素含量为89.24%,蛋白质含量为7.9586%,脂肪含量为1.63%;采用不同方式处理的纤维素膜的表观形态有所不同;利用扫描电镜观察不同干燥条件处理的细菌纤维素膜的超微结构,结果表明,与临界点干燥方法相比,真空冷冻干燥和热风干燥两种方法都会使纤维素结构发生一定的改变。
It’s not only plants that can synthesize cellulose, but some animals and micro-organisms also have this ability. The cellulose produced by micro-organisms is called“bacterial cellulose (BC)”. It was a new bio-material which has many characteristics, such as pure, high crystal degree and so on. BC is the thinnest cellulose in the world. Besides, owing to its high coincident degree, high hydrophilic property and better adaptive responses to environmental factors, BC has attracted most reaearchers’attention both at home and abroad.
In order to improve the yield of BC and reduce its cost, a strain (J2) with high yield of BC was initially selected from buckwheat vinegar on whose surface grew much thicker membrane and the characteristics of the strain J2 was researched, and then the high-yield mutant M438 was breeded by the techniques of high-pressure, finally the parameters of fermentation technology were optimized which established the basis for industry. The property of the cellulose membrane was studied and its apparent structure and ultra-micro-structure were observed which gave some direction to its application. The results of this research were as follows:
(1) A strain(J2) with ability to produce high yield of BC was selected from buckwheat vinegar. After the configuration and physiological and biochemical property were studied, the strain was primarily ascertained to belong to Gluconobacter.
(2) Taking strain J2 as the original strain, through the ultra-high pressure treatment mutagenesis, primary screening and the second screening, mutation strain M438 with high-yield property was selected. And the genetic stability of M438 was better than the original strain J2. After 7d cultivation, BC yield improved up to 15.7457g/100ml, which was as 1.485 times as that of pre-mutagenic strain (10.76 g/100ml).
(3) The stationary fermentation medium and fermentation conditions were confirmed which were apt to yield BC for Mutant M438. The components of medium were as follows: carbon source5 %( glucose: sucrose =4:1), yeast juice 1.25%, CaCl2 0.15%, ZnS04 0.2%, K2HPO4 0.2%, MgSO4 0.93%, fumaric acid 0.3%, ethanol 0.5%. The parameters of fermentation conditions were as follows: seed age 24h, inoculums amount 9%, fermentation time 7d. Under this fermentation medium, the yield of BC was 28.9878 g/100ml which was as 1.841 times as that under initial fermentation medium and 2.694 times as that of the unmutant strain J2.
(4) The optimal Freeze-drying conditions were as follows: protectant consisting of 12% skim milk and 5% glucose / sucrose (1:2), pre-freezing temperature of -80℃, pre-freezing time for 3h, freeze-drying time for 4d. Under these conditions, the survival rate of freeze-dried bacteria added up to more than 70%, and through comparison with other preservation methods, freeze-drying was the best method of preservation which could make the bacteria to survive about 2 years.
(5) The main components of the cellulose membrane produced by mutant M438 was mensurated, and the apparent structure and ultra-micro-structure were observed. The water content in wet membrane was 98.77%; the water content in dry membrane after absorbed water was 80.53%; cellulose content in dry membrane was 89.24%, protein content was 7.9586%, and fat content was 1.63%. The apparent structure of both wet and dry membrane were different under different disposing methods; the results of ultra-micro-structure observed with SEM was that vacuum freeze-drying way and air drying way could change the ultra-micro-structure of membrane.
引文
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