高富集生物活性硒啤酒酵母的研究
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摘要
微量元素硒是维持人体正常生命活动所必需的元素之一,缺硒会导致多种疾病的发生。富硒酵母作为人类营养的最重要的补硒形式已成为近几年研究的热点。本课题以本实验室提供的啤酒酵母菌为出发菌株,对其进行了硒抗性的筛选和富硒培养条件的优化,并且采用了复合酶(蜗牛酶)处理细胞壁、超声波处理细胞膜以及在加压的条件下培养等一系列方法,使啤酒酵母的富硒能力大大增强,为进一步应用于工业生产打下了良好的基础。
采用平板菌落计数法对三株啤酒酵母菌株进行了抗性筛选,通过对其在含有亚硒酸钠溶液的培养基中生长后所得到的生物量和硒的摄入率的检测,筛选出一株生物量较高和对亚硒酸钠抗性较高的酵母菌株362k;且通过单因素实验和正交实验对酵母中硒的检测方法——亚甲蓝还原法进行了实验条件的改进,得到在弱光照下,反应温度为32℃、甲醛的添加量为1mL时反应速度适中,利于计时,线性良好,灵敏度较高。对啤酒酵母菌株362k的富硒培养条件进行了优化,并通过单因素实验和正交实验确定了其最适培养条件:在温度为28℃下,亚硒酸钠添加浓度为20μg/mL且采用分批添加的方式、接种量为10%(v/v)、转速为200r/min、摇床培养48h时,啤酒酵母362k的生长状况、生物量和富集硒元素的能力最好。在此最优条件下,硒酵母生物量(干重)达到8.486g/L,细胞的总硒含量达到706.423μg/g干菌体,其中有机硒含量达到了642.916μg/g干菌体,转化率达到91.01%。
通过对富硒酵母培养条件的优化可知,啤酒酵母转化无机硒为有机硒的能力很强,均达到80%以上,因此细胞对硒的摄入率成为其富硒量的关键因素,本课题采用了三种方法:用复合酶处理细胞壁、超声波处理细胞膜以及加压培养的方式,增大啤酒酵母细胞的硒摄入率,从而提高其总硒含量和有机硒含量。结果显示:在36h时加入酶液效果最好,比不加酶液时总硒含量和有机硒含量有了明显的增加,增幅分别在10.4%和12.8%;在36h时用超声波处理效果最好,比不用超声波处理时总硒含量和有机硒含量分别增长了约12.4%和15.4%,而生物量基本不变;加压培养时,在12h封盖时总硒含量和有机硒含量分别达到了820.84和746.22ug/g干菌体,涨幅分别为19.25%和24.50%。
Selenium is one of the necessary elements to maintain the normal life activity of human’s body. The use of selenized yeast as enriched selenium supplements in human nutrition has become a topic of increasing interest over the last decade. This subject used the beer yeast of laboratory as the original strains, strains were filtered for the resistance to the Selenium and optimized the culture conditions of the Se-rich , and by a series of training methods made the ability of selenium-riched had a great increase ,such as handled of the cell wall using a complex enzyme (snail-enzyme), ultrasound treatment in the cell membrane and increased pressure in the culture process, and it laid a good foundation for the further applied to industrial production.
The three brewers yeast strains were filtered for the resistance to the Selenium with the method of flat-panel colony counting. Through detected their biomass and selenium intake rate in the medium containing the sodium selenite solution, and the strain 362k had higher biomass and resistance to the sodium selenite. The experimental conditions of the method for detecting the selenium——the reduction of methylene blue was improved through the single factor and orthogonal experiment, and the result showed: As the light was weak, the reaction temperature was 32℃and formaldehyde added quantity was 1mL, the reaction speed was appropriate to time, linear good, and sensitivity was high. The Se-riched culture conditions for the strain 362k were optimized through single factor and orthogonal experiments and confirmed its optimal culture conditions: the temperature was 28℃, the concentration of sodium selenite added was batch to 20μg/mL, the vaccination was 10% (v/v), running speed was 200r/min, and cultured for 48h, the 362 k’s growth conditions, biomass and Se-enrichment capacity was best. In this optimal conditions, selenium yeast biomass (dry weight)could up to 8.486g/L, the total selenium content reached 706.423μg/g dry cell, including organic selenium was 642.916μg/g dry cell, the conversion rate reached 91.01 percent.
It could be showed that the beer yeast’s ability of transforming the inorganic selenium to organic selinum was strong through the optimization of culture conditions, it had reached more than 80 percent. So the selenium intake became a critical factor of selenium-enriched rate. In this issue used the three methods to increase the intake of selenium following: the treatment cell wall with compound enzyme, ultrasound treatment and compression culture, thus increased its total selenium content and content of organic selenium. The results showed that: when cultured for 36h, adding the enzyme was the best, the total selenium content and content of organic selenium had increased significantly, an increase of 10.4 and 12.8 percent, respectively; when cultured for 36h, dealing with the cell by ultrasonic was effective, the total and organic selenium content had increase about 12.4% and 15.4% than no ultrasound treatment, and biomass remained basically unchanged. The total and organic selenium content reached 820.84 and 746.22μg/g dry cell when decked in 12h with pressurized culture, the increase rate reached 19.25 and 24.50 percent respectively.
采用平板菌落计数法对三株啤酒酵母菌株进行了抗性筛选,通过对其在含有亚硒酸钠溶液的培养基中生长后所得到的生物量和硒的摄入率的检测,筛选出一株生物量较高和对亚硒酸钠抗性较高的酵母菌株362k;且通过单因素实验和正交实验对酵母中硒的检测方法——亚甲蓝还原法进行了实验条件的改进,得到在弱光照下,反应温度为32℃、甲醛的添加量为1mL时反应速度适中,利于计时,线性良好,灵敏度较高。对啤酒酵母菌株362k的富硒培养条件进行了优化,并通过单因素实验和正交实验确定了其最适培养条件:在温度为28℃下,亚硒酸钠添加浓度为20μg/mL且采用分批添加的方式、接种量为10%(v/v)、转速为200r/min、摇床培养48h时,啤酒酵母362k的生长状况、生物量和富集硒元素的能力最好。在此最优条件下,硒酵母生物量(干重)达到8.486g/L,细胞的总硒含量达到706.423μg/g干菌体,其中有机硒含量达到了642.916μg/g干菌体,转化率达到91.01%。
通过对富硒酵母培养条件的优化可知,啤酒酵母转化无机硒为有机硒的能力很强,均达到80%以上,因此细胞对硒的摄入率成为其富硒量的关键因素,本课题采用了三种方法:用复合酶处理细胞壁、超声波处理细胞膜以及加压培养的方式,增大啤酒酵母细胞的硒摄入率,从而提高其总硒含量和有机硒含量。结果显示:在36h时加入酶液效果最好,比不加酶液时总硒含量和有机硒含量有了明显的增加,增幅分别在10.4%和12.8%;在36h时用超声波处理效果最好,比不用超声波处理时总硒含量和有机硒含量分别增长了约12.4%和15.4%,而生物量基本不变;加压培养时,在12h封盖时总硒含量和有机硒含量分别达到了820.84和746.22ug/g干菌体,涨幅分别为19.25%和24.50%。
Selenium is one of the necessary elements to maintain the normal life activity of human’s body. The use of selenized yeast as enriched selenium supplements in human nutrition has become a topic of increasing interest over the last decade. This subject used the beer yeast of laboratory as the original strains, strains were filtered for the resistance to the Selenium and optimized the culture conditions of the Se-rich , and by a series of training methods made the ability of selenium-riched had a great increase ,such as handled of the cell wall using a complex enzyme (snail-enzyme), ultrasound treatment in the cell membrane and increased pressure in the culture process, and it laid a good foundation for the further applied to industrial production.
The three brewers yeast strains were filtered for the resistance to the Selenium with the method of flat-panel colony counting. Through detected their biomass and selenium intake rate in the medium containing the sodium selenite solution, and the strain 362k had higher biomass and resistance to the sodium selenite. The experimental conditions of the method for detecting the selenium——the reduction of methylene blue was improved through the single factor and orthogonal experiment, and the result showed: As the light was weak, the reaction temperature was 32℃and formaldehyde added quantity was 1mL, the reaction speed was appropriate to time, linear good, and sensitivity was high. The Se-riched culture conditions for the strain 362k were optimized through single factor and orthogonal experiments and confirmed its optimal culture conditions: the temperature was 28℃, the concentration of sodium selenite added was batch to 20μg/mL, the vaccination was 10% (v/v), running speed was 200r/min, and cultured for 48h, the 362 k’s growth conditions, biomass and Se-enrichment capacity was best. In this optimal conditions, selenium yeast biomass (dry weight)could up to 8.486g/L, the total selenium content reached 706.423μg/g dry cell, including organic selenium was 642.916μg/g dry cell, the conversion rate reached 91.01 percent.
It could be showed that the beer yeast’s ability of transforming the inorganic selenium to organic selinum was strong through the optimization of culture conditions, it had reached more than 80 percent. So the selenium intake became a critical factor of selenium-enriched rate. In this issue used the three methods to increase the intake of selenium following: the treatment cell wall with compound enzyme, ultrasound treatment and compression culture, thus increased its total selenium content and content of organic selenium. The results showed that: when cultured for 36h, adding the enzyme was the best, the total selenium content and content of organic selenium had increased significantly, an increase of 10.4 and 12.8 percent, respectively; when cultured for 36h, dealing with the cell by ultrasonic was effective, the total and organic selenium content had increase about 12.4% and 15.4% than no ultrasound treatment, and biomass remained basically unchanged. The total and organic selenium content reached 820.84 and 746.22μg/g dry cell when decked in 12h with pressurized culture, the increase rate reached 19.25 and 24.50 percent respectively.
引文
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