高品质生化培养基用酵母抽提物制备的研究
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摘要
本研究以啤酒废酵母为原料,首先对制备高品质生化培养基用酵母抽提物的工艺条件进行了研究,然后对酵母抽提液的超滤处理工艺条件进行了研究,并对得到的酵母抽提物的品质进行了理化和生物学检测,最后对酶促自溶后的胞壁残渣中葡聚糖的提取工艺进行了研究,主要研究结果如下:
1、利用低温储存3天以内的啤酒废酵母为原料,采用自溶与添加外源酶相结合的方法制备酵母抽提物。以氨基氮得率和固形物得率为主要指标考察了对酵母自溶影响关键的几个因素,包括外加蛋白酶的种类及添加量、酵母悬浮液浓度、自溶时间、酶解时间,并对酵母酶联自溶的相关促进剂进行正交试验优选。随后,优化选择了后续抽提物生产的旋转蒸发及喷雾干燥工艺的参数。确定的最佳工艺条件为:1%(干基)破壁酶和1%(干基)酵母抽提酶,悬浮液浓度7.5%,自溶促进剂1%乙醇、5%磷酸氢二钾和5%葡萄糖,自溶时间6h,酶解时间28h。
2、利用超滤去除酵母抽提液中的未水解蛋白、大分子物质、多酚类色素物质和盐分,实验结果表明最佳的超滤操作条件为操作压力0.2Mpa,料液浓度2%~4%,操作温度25～30℃,超滤时间120min,在此条件下酵母抽提液的脱色率达到75.0%,氯化物去除率达53.3%,氨基氮损失率为13.1%。
3、通过3种苛养菌的灵敏度实验,发现本实验室自制酵母抽提物是含有对3种苛养菌生长所必需的营养(维生素和其他生长因子)的。菌落计数实验,表明本实验室自制酵母抽提物在对3种苛养菌生长所必需的营养物质的含量上,基本达到了英国OXOID公司酵母粉的水平。
4、通过对比添加2种酵母抽提物对啤酒酵母发酵性能的影响,发现添加2种酵母抽提物是可以提高啤酒酵母的起发速度、发酵效率和存活率的,并同时增强所制啤酒的缓冲能力、风味稳定性和抗氧化能力。但是,添加2种酵母抽提物也会增加不利呈味物质双乙酰和α-AN的含量,增幅不如有益呈味物质(高级醇酯等)。通过进一步量化分析,发现添加自制酵母抽提物0.5%的发酵栓酿制的初啤酒,是唯一一个满足高级醇与酯的含量之比为2~3∶1范围内的样品,对比添加OXOID0.5%的样品,其缓冲能力、风味稳定性和抗氧化能力与添加OXOID0.5%的相当,发酵后酵母细胞死亡率比OXOID低24.39%,风味也比添加OXOID0.5%的提高14.97%,实际发酵效率也与添加OXOID0.5%的相当,皆比空白提高了约6%的水平。
5、采用外加碱液和碱性蛋白酶共同作用的方法,制备碱不溶性β-1,3-D-葡聚糖并降低其他杂质的含量以提高成品纯度。以2个指标(杂多糖干重和多糖纯度)评价啤酒酵母自溶后胞壁残渣碱溶效果,通过单因素实验及正交实验,对酶联自溶制备酵母抽提物后的胞壁残渣碱溶工艺进行了优化,确定了最终的啤酒酵母自溶制备酵母抽提物后胞壁残渣碱溶的工艺条件。采用优化后碱溶工艺,制取的杂多糖干重降低了7.27%,而多糖纯度提高了14.04%,最终产物中多糖提取率提高了10.41%。
With waste beer yeast as material,yeast extract were prepared.Then Ultrafiltration was studied and thel quality of yeast extract was inspected.Furthmore the alkaline-enzymatic process of extractβ-1,3-D-glucan was studied.The main results were follows:
1.At first,With waste brewer’s yeast as raw material, the singer factor test, such as the category and quantity of the proteases, the concentration of yeast solution , autolysis time and enzymolysis time, together with the orthogonal test were carried out to optimize the conditions of the autolysis promoter related to the reaction. And then , the parameter of evaporation and spray desiccation had been tested and chose . The results showed that the autolysis combined with enzyme hydrolysis is an ideal method to produce yeast extract, and the yields of amino nitrogen and solid reached 3.937%and 59.24%, respectively.
2.Ultrafiltration was employed to illuminated the unhydrolysated protein, polypeptides of high molecular weight, pigment and salts. The results show that the optimum conditions are concentration of of 2%~4%, pressure of 0.2Mpa, temperature of 25～30℃, time of 120min. After ultrafiltration of this condition, the decoloration rate was 75%, the desalination rate was 58%, the lossratio of aminonitrogen contents was 13%.
3.It is sure that there are nutrient in self-made yeast extract through experimental results on sensitivity.Furthmore it is know that self-made yeast extract’s vitamine and grow factor are the same level of OXOID’s yeast extract.This paper inspect biological quality of yeast extract which is made through autolysis coupled with enzyme addition. The results show that there is no difference between yeast extract’s vitamine and grow factor of self-made and OXOID.
4.The following experiment is to detect the effects of yeast extract on the beer fermentation.The results showed that the addition of self-made and OXOID yeast extract both improved beer yeast’s initiating speed, fermentation efficiency,survival rate, and enhance the beer’s bufferring ability,flavour stability, antioxidant activity.But it can aslo raise the dimethylglyoxal andα-AN which amplification are less than higher alcohols and ester.
The results show that the addition of 0.5% self-made yeast extract is the only sample satisfy higher alcohols to ester is 2~3 to 1.It can enhance the beer’s bufferring ability,flavour stability, antioxidant activity like OXOID yeast extract. The yeast’s death rate after fermentation reduce 24.39% and the flavour increase 14.97%.This experiment prove the nutrient of self-made yeast extract.
5.The waste beer yeast was used to produce high-quality yeast extract through autolysis coupled with enzyme addition, and there was a great deal ofβ-1,3-D-glucan left in the cell wall of the waste beer yeast. The alkaline-enzymatic process was employed to extractβ-1,3-D-glucan in this paper. The factors influencing extraction of glucan including alkaline concentration, reaction time, reaction temperature and the addition quantity of alkaline protease, were investigated with the dry weight and purity of glucan as main indicators. Besides, the factors related to alkaline process was optimized by orthogonal experiments. Then, the acids used for neutralization and eluting process were optimized. After optimizing , the dry weight of glucan reduced by 7.27%, purity of glucan increased by 14.04%, and glucan yield rate increased by 10.41%.
1、利用低温储存3天以内的啤酒废酵母为原料,采用自溶与添加外源酶相结合的方法制备酵母抽提物。以氨基氮得率和固形物得率为主要指标考察了对酵母自溶影响关键的几个因素,包括外加蛋白酶的种类及添加量、酵母悬浮液浓度、自溶时间、酶解时间,并对酵母酶联自溶的相关促进剂进行正交试验优选。随后,优化选择了后续抽提物生产的旋转蒸发及喷雾干燥工艺的参数。确定的最佳工艺条件为:1%(干基)破壁酶和1%(干基)酵母抽提酶,悬浮液浓度7.5%,自溶促进剂1%乙醇、5%磷酸氢二钾和5%葡萄糖,自溶时间6h,酶解时间28h。
2、利用超滤去除酵母抽提液中的未水解蛋白、大分子物质、多酚类色素物质和盐分,实验结果表明最佳的超滤操作条件为操作压力0.2Mpa,料液浓度2%~4%,操作温度25～30℃,超滤时间120min,在此条件下酵母抽提液的脱色率达到75.0%,氯化物去除率达53.3%,氨基氮损失率为13.1%。
3、通过3种苛养菌的灵敏度实验,发现本实验室自制酵母抽提物是含有对3种苛养菌生长所必需的营养(维生素和其他生长因子)的。菌落计数实验,表明本实验室自制酵母抽提物在对3种苛养菌生长所必需的营养物质的含量上,基本达到了英国OXOID公司酵母粉的水平。
4、通过对比添加2种酵母抽提物对啤酒酵母发酵性能的影响,发现添加2种酵母抽提物是可以提高啤酒酵母的起发速度、发酵效率和存活率的,并同时增强所制啤酒的缓冲能力、风味稳定性和抗氧化能力。但是,添加2种酵母抽提物也会增加不利呈味物质双乙酰和α-AN的含量,增幅不如有益呈味物质(高级醇酯等)。通过进一步量化分析,发现添加自制酵母抽提物0.5%的发酵栓酿制的初啤酒,是唯一一个满足高级醇与酯的含量之比为2~3∶1范围内的样品,对比添加OXOID0.5%的样品,其缓冲能力、风味稳定性和抗氧化能力与添加OXOID0.5%的相当,发酵后酵母细胞死亡率比OXOID低24.39%,风味也比添加OXOID0.5%的提高14.97%,实际发酵效率也与添加OXOID0.5%的相当,皆比空白提高了约6%的水平。
5、采用外加碱液和碱性蛋白酶共同作用的方法,制备碱不溶性β-1,3-D-葡聚糖并降低其他杂质的含量以提高成品纯度。以2个指标(杂多糖干重和多糖纯度)评价啤酒酵母自溶后胞壁残渣碱溶效果,通过单因素实验及正交实验,对酶联自溶制备酵母抽提物后的胞壁残渣碱溶工艺进行了优化,确定了最终的啤酒酵母自溶制备酵母抽提物后胞壁残渣碱溶的工艺条件。采用优化后碱溶工艺,制取的杂多糖干重降低了7.27%,而多糖纯度提高了14.04%,最终产物中多糖提取率提高了10.41%。
With waste beer yeast as material,yeast extract were prepared.Then Ultrafiltration was studied and thel quality of yeast extract was inspected.Furthmore the alkaline-enzymatic process of extractβ-1,3-D-glucan was studied.The main results were follows:
1.At first,With waste brewer’s yeast as raw material, the singer factor test, such as the category and quantity of the proteases, the concentration of yeast solution , autolysis time and enzymolysis time, together with the orthogonal test were carried out to optimize the conditions of the autolysis promoter related to the reaction. And then , the parameter of evaporation and spray desiccation had been tested and chose . The results showed that the autolysis combined with enzyme hydrolysis is an ideal method to produce yeast extract, and the yields of amino nitrogen and solid reached 3.937%and 59.24%, respectively.
2.Ultrafiltration was employed to illuminated the unhydrolysated protein, polypeptides of high molecular weight, pigment and salts. The results show that the optimum conditions are concentration of of 2%~4%, pressure of 0.2Mpa, temperature of 25～30℃, time of 120min. After ultrafiltration of this condition, the decoloration rate was 75%, the desalination rate was 58%, the lossratio of aminonitrogen contents was 13%.
3.It is sure that there are nutrient in self-made yeast extract through experimental results on sensitivity.Furthmore it is know that self-made yeast extract’s vitamine and grow factor are the same level of OXOID’s yeast extract.This paper inspect biological quality of yeast extract which is made through autolysis coupled with enzyme addition. The results show that there is no difference between yeast extract’s vitamine and grow factor of self-made and OXOID.
4.The following experiment is to detect the effects of yeast extract on the beer fermentation.The results showed that the addition of self-made and OXOID yeast extract both improved beer yeast’s initiating speed, fermentation efficiency,survival rate, and enhance the beer’s bufferring ability,flavour stability, antioxidant activity.But it can aslo raise the dimethylglyoxal andα-AN which amplification are less than higher alcohols and ester.
The results show that the addition of 0.5% self-made yeast extract is the only sample satisfy higher alcohols to ester is 2~3 to 1.It can enhance the beer’s bufferring ability,flavour stability, antioxidant activity like OXOID yeast extract. The yeast’s death rate after fermentation reduce 24.39% and the flavour increase 14.97%.This experiment prove the nutrient of self-made yeast extract.
5.The waste beer yeast was used to produce high-quality yeast extract through autolysis coupled with enzyme addition, and there was a great deal ofβ-1,3-D-glucan left in the cell wall of the waste beer yeast. The alkaline-enzymatic process was employed to extractβ-1,3-D-glucan in this paper. The factors influencing extraction of glucan including alkaline concentration, reaction time, reaction temperature and the addition quantity of alkaline protease, were investigated with the dry weight and purity of glucan as main indicators. Besides, the factors related to alkaline process was optimized by orthogonal experiments. Then, the acids used for neutralization and eluting process were optimized. After optimizing , the dry weight of glucan reduced by 7.27%, purity of glucan increased by 14.04%, and glucan yield rate increased by 10.41%.
引文
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