大豆糖蜜发酵生产乙醇关键技术研究
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摘要
大豆糖蜜是醇法生产大豆浓缩蛋白时所产生的副产品，目前主要只作为价格低廉的初级产品出售，利用于饲料加工当中，产品的附加值非常低。将大豆糖蜜用来发酵生产乙醇，将进一步提高其利用价值，增加经济收益。因此，本论文基于扩大大豆糖蜜的利用范围，以其为原料，分离筛选高产乙醇的酵母菌种并对其进行鉴定、发酵培养基和发酵条件的优化研究，为工业化利用大豆糖蜜生产乙醇提供理论基础。
1对大豆糖蜜中的野生酵母菌进行分离，得到1380株；采用TTC平板法、乙醇耐性测定以及产乙醇性能测定筛选共三级筛选，最终筛得一株可耐受20%(v/v)乙醇、初始发酵的乙醇产量可达9.07%的菌株P14。通过形态、生理生化及26S rDNA D1/D2区序列分析将其鉴定为酿酒酵母菌（Saccharomycescerevisiae）。
2以发酵液中的菌体浓度为指标，通过单因素试验考察了糖浓度，接种量，生长温度，pH以及转速对发酵液中的Saccharomyces cerevisiae P14菌体浓度的影响。实验结果表明，P14最适生长条件为：糖浓度12%，接种量4%，生长温度26℃，pH6.0，转速140r/min。在此条件下，摇瓶培养16h获得最佳的发酵种子培养液。
3以单因素试验为基础，采用四元二次通用旋转组合实验，以乙醇产量为响应值作响应面设计，对Saccharomyces cerevisiae P14的发酵培养基进行了优化。确定大豆糖蜜高产乙醇的最佳发酵培养基为：大豆糖蜜浓度为40%，蛋白胨添加量为1.2g/L，MgSO_4添加量为0.34g/L，Na_2HPO_4添加量为1.87g/L，FeSO_4添加量为0.025g/L，生长因子添加量为2.78g/L。在此条件下乙醇产量可达12.43%。
4以乙醇产量为指标，通过单因素试验和正交试验，对大豆糖蜜高产乙醇Saccharomyces cerevisiaeP14的发酵条件进行优化，确定大豆糖蜜高产乙醇的最佳发酵条件为：初始pH6.5、发酵时间84h、发酵温度26℃和接种量8%，在此条件下乙醇产量可达13.56%。
The soybean molasses is a byproduct from the process of product soybean protein concentrate by ethanol.The soybean molasses is mainly sold as cheap raw product at present, used in feed processing. Its added valueis very low. Using the soybean molasses as raw material to ferment ethanol will enhance its use value andincrease economically viable of factory. This research is aimed to expand the use range of soybean molasses,to isolate, select and identify high-alcohol-yield yeast from the soybean molasses. In addition, this researchwill also optimize the fermentation medium and fermentation conditions so that provide theoretical basis forindustrial producing of ethanol with soybean molasses.
1380yeast strains had been isolated from soybean molasses after enrichment and cultivating from the wildyeast in the soybean molasses. After three-grade screening including the TTC plate method screening, theethanol tolerance determinating and the ethanol production performance measuring, the yeast strain P14,which had the ethanol tolerance of20%(v/v) and the initial fermentation ethanal yields up to9.07%, wasfinally obtained. By the morphological characteristics, physiological and biochemical tests and the molecularmethod of the sequence analysis in the26S rDNA D1/D2area, the strain P14was identified as Saccharomycescerevisiae.
Taking cell concentration as index, the effects of sugar concentration, inoculum concentration, growthtemperature, pH value and the rotate-speed in the fermentation on the growth of Saccharomyces cerevisiae P14were investigated by single factor experiment. The experimental results show that the optimum growthconditions are sugar concentration of12%, inoculum concentration of4%, growth temperature of26℃, pHvalue of6.0, rotate-speed of140r/min. The best seed culture was obtained by shaking flask culture for16h onthe optimum growth conditions.
On the basis of the results of the single factor experiments, the fermentation medium of soy malesses foralcohol fermentation by Saccharomyces cerevisiae P14with the high-alcohol-yield was optimized by theresponse surface method using the design of four element Second power general rotation combinationexperiment with SAS system9.2. The optimum fermentation medium composition of the high-alchol-yieldyeast Saccharomyces cerevisiae P14in the soybean molasses were sugar concentration of40%, the peptoneadding amount of1.2g/L, MgSO_4adding amount of0.34g/L, Na2HPO_4adding amount of1.87g/L, FeSO_4adding amount of0.025g/L and the growth factor adding amount of2.78g/L. The ethanol production rate canachieve to12.43%under the optimum fermentation medium compositions.
The fermentation conditions for alcohol fermentation by Saccharomyces cerevisiae P14in the soybeanmolasses were optimized by the single factor and orthogonal experiments. Tthe optimum fermentationconditions were determined as the inoculum concentration of10%, initial pH of6.5, fermentation temperatureof26°C and the fermentation time of84h. The ethanol production rate can achieve to13.56%under theoptimum fermentation conditions.
1对大豆糖蜜中的野生酵母菌进行分离，得到1380株；采用TTC平板法、乙醇耐性测定以及产乙醇性能测定筛选共三级筛选，最终筛得一株可耐受20%(v/v)乙醇、初始发酵的乙醇产量可达9.07%的菌株P14。通过形态、生理生化及26S rDNA D1/D2区序列分析将其鉴定为酿酒酵母菌（Saccharomycescerevisiae）。
2以发酵液中的菌体浓度为指标，通过单因素试验考察了糖浓度，接种量，生长温度，pH以及转速对发酵液中的Saccharomyces cerevisiae P14菌体浓度的影响。实验结果表明，P14最适生长条件为：糖浓度12%，接种量4%，生长温度26℃，pH6.0，转速140r/min。在此条件下，摇瓶培养16h获得最佳的发酵种子培养液。
3以单因素试验为基础，采用四元二次通用旋转组合实验，以乙醇产量为响应值作响应面设计，对Saccharomyces cerevisiae P14的发酵培养基进行了优化。确定大豆糖蜜高产乙醇的最佳发酵培养基为：大豆糖蜜浓度为40%，蛋白胨添加量为1.2g/L，MgSO_4添加量为0.34g/L，Na_2HPO_4添加量为1.87g/L，FeSO_4添加量为0.025g/L，生长因子添加量为2.78g/L。在此条件下乙醇产量可达12.43%。
4以乙醇产量为指标，通过单因素试验和正交试验，对大豆糖蜜高产乙醇Saccharomyces cerevisiaeP14的发酵条件进行优化，确定大豆糖蜜高产乙醇的最佳发酵条件为：初始pH6.5、发酵时间84h、发酵温度26℃和接种量8%，在此条件下乙醇产量可达13.56%。
The soybean molasses is a byproduct from the process of product soybean protein concentrate by ethanol.The soybean molasses is mainly sold as cheap raw product at present, used in feed processing. Its added valueis very low. Using the soybean molasses as raw material to ferment ethanol will enhance its use value andincrease economically viable of factory. This research is aimed to expand the use range of soybean molasses,to isolate, select and identify high-alcohol-yield yeast from the soybean molasses. In addition, this researchwill also optimize the fermentation medium and fermentation conditions so that provide theoretical basis forindustrial producing of ethanol with soybean molasses.
1380yeast strains had been isolated from soybean molasses after enrichment and cultivating from the wildyeast in the soybean molasses. After three-grade screening including the TTC plate method screening, theethanol tolerance determinating and the ethanol production performance measuring, the yeast strain P14,which had the ethanol tolerance of20%(v/v) and the initial fermentation ethanal yields up to9.07%, wasfinally obtained. By the morphological characteristics, physiological and biochemical tests and the molecularmethod of the sequence analysis in the26S rDNA D1/D2area, the strain P14was identified as Saccharomycescerevisiae.
Taking cell concentration as index, the effects of sugar concentration, inoculum concentration, growthtemperature, pH value and the rotate-speed in the fermentation on the growth of Saccharomyces cerevisiae P14were investigated by single factor experiment. The experimental results show that the optimum growthconditions are sugar concentration of12%, inoculum concentration of4%, growth temperature of26℃, pHvalue of6.0, rotate-speed of140r/min. The best seed culture was obtained by shaking flask culture for16h onthe optimum growth conditions.
On the basis of the results of the single factor experiments, the fermentation medium of soy malesses foralcohol fermentation by Saccharomyces cerevisiae P14with the high-alcohol-yield was optimized by theresponse surface method using the design of four element Second power general rotation combinationexperiment with SAS system9.2. The optimum fermentation medium composition of the high-alchol-yieldyeast Saccharomyces cerevisiae P14in the soybean molasses were sugar concentration of40%, the peptoneadding amount of1.2g/L, MgSO_4adding amount of0.34g/L, Na2HPO_4adding amount of1.87g/L, FeSO_4adding amount of0.025g/L and the growth factor adding amount of2.78g/L. The ethanol production rate canachieve to12.43%under the optimum fermentation medium compositions.
The fermentation conditions for alcohol fermentation by Saccharomyces cerevisiae P14in the soybeanmolasses were optimized by the single factor and orthogonal experiments. Tthe optimum fermentationconditions were determined as the inoculum concentration of10%, initial pH of6.5, fermentation temperatureof26°C and the fermentation time of84h. The ethanol production rate can achieve to13.56%under theoptimum fermentation conditions.
引文
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