高产纤维素酶枯草芽孢杆菌的筛选、应用及其产酶条件研究
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摘要
膳食纤维(Dietary fiber, DF)具有多种生理功能,被誉为人体的“第七大营养素”。其中的可溶性成分SDF (Soluble Dietary fiber, SDF),拥有比不溶性成分IDF (Insoluble Dietary fiber, IDF)更优的生理活性,具有较高应用价值和重要的研究意义。豆渣在我国产量巨大,其富含的纤维素类物质是SDF的理想来源。但是豆渣中天然的SDF较少,因此,必须进行一定的技术处理,以提高豆渣SDF的含量与得率。当前,以发酵技术和酶技术为代表的生物技术,被认为是最有发展潜力的技术手段之一,而菌种和酶类的选择与确定,则尤为重要。枯草芽孢杆菌(Bacillus subtilis)的诸多优点,使其成为产酶和应用菌种的首选。
为此,本论文研究了高产纤维素酶枯草芽孢杆菌菌株的筛选、发酵豆渣制备SDF的应用及其产纤维素酶的条件,研究结果如下：
1、从自然界筛选获得一株产纤维素活力较高的枯草芽孢杆菌,并根据实验中的编号顺序,命名为BS-05。在筛选的自然条件下,该菌株产纤维素酶活力较高,羧甲基纤维素酶活力(Sodium carboxymethylcellulose activity, CMCA)和滤纸酶活力(Filter Paper Activity, FPA)分别为57.27 U/ml和58.97 U/ml。
2、应用菌株BS-05发酵豆渣,并联用均质技术,制备了高含量的豆渣SDF。豆渣经发酵与均质处理后,SDF的得率提高约4倍。并且,与未发酵的豆渣SDF相比,发酵获得的SDF颗粒变小,结构变松散,其溶解性与持水力等理化性质都有不同程度的改善。
3、研究获得了菌株BS-05产纤维素酶的最佳条件。其产酶培养基组成是：CMC-Na 2%、蛋白胨2%、酵母粉2%、NaCl 0.5%, K2HPO4 0.1%, MgSO4·7H2O0.02%；最优的培养条件是：培养温度37℃、培养基初始pH7、接种量5%、培养时间48h和产酶培养基装液量为60/250ml。在上述条件下,该菌株发酵产纤维素酶的活力最高,CMCA与FPA分别达195.46 U/ml和174.52 U/ml,分别为优化前的3.41倍和2.96倍。
4、研究获得了菌株BS-05所产纤维素酶的部分酶学特性。研究结果显示,该酶的最适pH值为pH6-7,在该pH范围内保持60min,酶活稳定；最适温度为55℃,在65℃以下保持30min,仍有较高酶活。这为该酶的推广提供了应用参数。
Owing to a variety of physiological functions, Dietary fiber (DF) was praised as "the seventh largest nutrient" of the human body, one of its composition the soluble components (Soluble Dietary fiber, SDF), has a better physiological function than the other composition the insoluble components (Insoluble Dietary fiber, IDF), and has more important applications and research significance. Soybean dregs with its rich cellulose materials and large output in our country, become an ideal source of SDF. However, it has fewer natural SDF, therefore, a certain degree of technical processing in order to improve the SDF content and yield rate must be taken. At present, biotechnology represented by the fermentation technology and enzyme technology is considered one of the most potential technologies, while the choice and identification of bacteria and enzymes is particularly important. Bacillus subtilis, with a number of advantages, making it becomes the first choice of enzyme production and application strain.
For above reasons, this paper studied the screening methods of the high-yielding cellulose Bacillus subtilis strain, and its applications of fermentation in preparing soybean dregs SDF,as well as this strain's cellulase production conditions, the results are as follows:
1. From the natural, obtained a Bacillus subtilis strain with high cellulose-producing activity, and named as BS-05 according to the order of experimental number. In the natural selection conditions, the strain has high produced cellulase activity, Sodium Carboxymethylcellulose Activity (CMCA) and the Filter Paper Activity (FPA) were 57.27 U/ml and 58.97 U/ml.
2. Used the BS-05 strains in fermented soybean dregs, and in conjunction with the homogenization technique, obtained the soybean dregs SDF. After fermentation and homogenization, the yield of the soybean dregs SDF was increased to nearly 4-fold, and the particles become smaller, structure become looser, solubility and water holding capacity and other physical and chemical properties have different degrees of improvement.
3. Obtained the best conditions for cellulase production of strain BS-05. Its enzyme production medium composition is:CMC-Na 2%, peptone 2%, yeast extract 2%, NaCl 0.5%, K2HPO4 0.1%, MgSO4·7H2O 0.02%; The optimal culture conditions are:culture temperature 37℃, culture base the initial pH7, inoculum size 5%, incubation time 48h and fermentation medium of liquid volume of 60ml. In the above-mentioned conditions, the strain has highest produced cellulase activity CMCA and the FPA respectively reached at 195.46 U/ml and 174.52 U/ml, is 3.41-fold and 2.96-fold before optimization.
4. Obtained some enzyme properties of the cellulase produced by this strain were studied. The result is that:the optimum pH value of the cellulose is 6-7, the activity was stable after maintain 60min under this pH, and the optimum temperature is 55℃, there is still a high activity after maintain 30min at below 65℃. This research may provides parameters for the extension and application of the cellulose produced by BS-05.
为此,本论文研究了高产纤维素酶枯草芽孢杆菌菌株的筛选、发酵豆渣制备SDF的应用及其产纤维素酶的条件,研究结果如下：
1、从自然界筛选获得一株产纤维素活力较高的枯草芽孢杆菌,并根据实验中的编号顺序,命名为BS-05。在筛选的自然条件下,该菌株产纤维素酶活力较高,羧甲基纤维素酶活力(Sodium carboxymethylcellulose activity, CMCA)和滤纸酶活力(Filter Paper Activity, FPA)分别为57.27 U/ml和58.97 U/ml。
2、应用菌株BS-05发酵豆渣,并联用均质技术,制备了高含量的豆渣SDF。豆渣经发酵与均质处理后,SDF的得率提高约4倍。并且,与未发酵的豆渣SDF相比,发酵获得的SDF颗粒变小,结构变松散,其溶解性与持水力等理化性质都有不同程度的改善。
3、研究获得了菌株BS-05产纤维素酶的最佳条件。其产酶培养基组成是：CMC-Na 2%、蛋白胨2%、酵母粉2%、NaCl 0.5%, K2HPO4 0.1%, MgSO4·7H2O0.02%；最优的培养条件是：培养温度37℃、培养基初始pH7、接种量5%、培养时间48h和产酶培养基装液量为60/250ml。在上述条件下,该菌株发酵产纤维素酶的活力最高,CMCA与FPA分别达195.46 U/ml和174.52 U/ml,分别为优化前的3.41倍和2.96倍。
4、研究获得了菌株BS-05所产纤维素酶的部分酶学特性。研究结果显示,该酶的最适pH值为pH6-7,在该pH范围内保持60min,酶活稳定；最适温度为55℃,在65℃以下保持30min,仍有较高酶活。这为该酶的推广提供了应用参数。
Owing to a variety of physiological functions, Dietary fiber (DF) was praised as "the seventh largest nutrient" of the human body, one of its composition the soluble components (Soluble Dietary fiber, SDF), has a better physiological function than the other composition the insoluble components (Insoluble Dietary fiber, IDF), and has more important applications and research significance. Soybean dregs with its rich cellulose materials and large output in our country, become an ideal source of SDF. However, it has fewer natural SDF, therefore, a certain degree of technical processing in order to improve the SDF content and yield rate must be taken. At present, biotechnology represented by the fermentation technology and enzyme technology is considered one of the most potential technologies, while the choice and identification of bacteria and enzymes is particularly important. Bacillus subtilis, with a number of advantages, making it becomes the first choice of enzyme production and application strain.
For above reasons, this paper studied the screening methods of the high-yielding cellulose Bacillus subtilis strain, and its applications of fermentation in preparing soybean dregs SDF,as well as this strain's cellulase production conditions, the results are as follows:
1. From the natural, obtained a Bacillus subtilis strain with high cellulose-producing activity, and named as BS-05 according to the order of experimental number. In the natural selection conditions, the strain has high produced cellulase activity, Sodium Carboxymethylcellulose Activity (CMCA) and the Filter Paper Activity (FPA) were 57.27 U/ml and 58.97 U/ml.
2. Used the BS-05 strains in fermented soybean dregs, and in conjunction with the homogenization technique, obtained the soybean dregs SDF. After fermentation and homogenization, the yield of the soybean dregs SDF was increased to nearly 4-fold, and the particles become smaller, structure become looser, solubility and water holding capacity and other physical and chemical properties have different degrees of improvement.
3. Obtained the best conditions for cellulase production of strain BS-05. Its enzyme production medium composition is:CMC-Na 2%, peptone 2%, yeast extract 2%, NaCl 0.5%, K2HPO4 0.1%, MgSO4·7H2O 0.02%; The optimal culture conditions are:culture temperature 37℃, culture base the initial pH7, inoculum size 5%, incubation time 48h and fermentation medium of liquid volume of 60ml. In the above-mentioned conditions, the strain has highest produced cellulase activity CMCA and the FPA respectively reached at 195.46 U/ml and 174.52 U/ml, is 3.41-fold and 2.96-fold before optimization.
4. Obtained some enzyme properties of the cellulase produced by this strain were studied. The result is that:the optimum pH value of the cellulose is 6-7, the activity was stable after maintain 60min under this pH, and the optimum temperature is 55℃, there is still a high activity after maintain 30min at below 65℃. This research may provides parameters for the extension and application of the cellulose produced by BS-05.
引文
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