紫外线诱变选育富硒酵母及其培养条件的研究
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摘要
硒是生物体必需的微量元素,具有很多生物学功能。谷胱甘肽过氧化物酶催化谷胱甘肽参与过氧化反应,清除过氧化物的毒害,硒是保持谷胱甘肽过氧化物酶活性的必要成分。但是无机硒的吸收率低、毒性大。酵母菌对微量元素具有很强的吸附作用,可以作为微量元素良好的载体,将无机硒转化成有机硒,提高硒在机体内吸收利用率,降低其毒副作用,且酵母本身含丰富的蛋白质和B族维生素,是一种良好的微生物蛋白饲料营养源。所以,获得高产富硒酵母的关键就在于拥有一株好菌株并找到适合的培养条件。本试验就是利用紫外线照射诱变,进行抗性筛选高产突变株,并确定制备富硒酵母的最佳条件。
实验以热带假丝酵母(Candida tropicalis)1254为出发菌,进行紫外线照射诱变,酵母经紫外线诱变,其致死率曲线表现为“肩形”,即经小剂量辐射的酵母细胞致死率较低,而大剂量的辐射会使酵母细胞的致死率明显升高。随着辐射时间的累计,辐射剂量的增加,突变率逐渐提高,但照射到一定程度时,突变率会降低。筛选出20株生长快、菌落大的突变菌株,接种于含有不同[Se~(4+)]的平板培养基,进行梯度抗性筛选,得到耐高浓度硒的能力较强的菌株6株,进一步复筛得到突变株Y-5。结果显示:Y-5的生物量和硒含量分别由原来的0.619g/100mL、688mg/kg提高到0.779g/100mL、1252mg/kg。利用单因素试验摸索出了Y-5培养条件:11B~0的麦芽汁培养基为最适培养基,发酵培养时间为35h,培养温度为28℃,培养基含硒量20μg/ml,硒的添加时间为11h,接种量10%,装液量为250ml的锥形瓶中装60ml。然后选出对硒含量影响较大的3个因素进行正交试验,找到最优组合为培养基含硒量20μg/ml,发酵培养时间为35h,接种量10%。在最优条件下培养的突变株Y-5,生物量提高了58%,硒含量提高了74.87%,硒含量达到1343mg/kg。变异菌株Y-5经传5代培养,生产性能稳定。
本试验对富硒酵母中有机和无机硒进行测定,发现筛选菌株Y-5有机硒含量达93.6%,而出发菌的有机硒含量是80%。
Selenium is an essential trace element of animals,which has a great deal of biological effects.Selenium is an essential component to keep the activity of glutathione peroxidase which can catalyze glutathione participating peroxidation to get rid of the toxicity of peroxide.However the absorption ration of inorganic selenium is low but high toxicity.Yeast is a good carrier of trace element because of its strong adsorption to trace element.It can convert the inorganic selenium to organic selenium,which can improve the adsorption ration of selenium and lower its toxicity. Yeast is a favourable microprotein source,because it contains abundant protein and vitamin B.So the key to obtain high yield selenium-enriched yeast is a good strain and its suitable cultivate condition.The experiment purpose to obtain a high yield mutant strain utilizing uviolize mutagenesis and resistance screening,and search for optimal incubate condition of it.
The experiment take Candida tropicalis 1254 as original strains,through uviolize mutagenesis,the lethality rate curve of yeast shows "shoulder",videlicet the lethality rate of yeast is lower when the yeast undergo low dose radiation and higher when undergo high dose radiation.With duration of radiation increasing,the rate increase gradually.However until a certain degree the rate will cut down.20 mutation strains which grow rapidly and the colony of them are big,are inoculated in flat plate with different density of Se~(4+).6 strains whose ability to bear high density of Se is higher was obtained.Y-5 mutation strain was obtained,after advanced screening.Results demonstrated:biomass and content of Se increased from 0.619 g/100mL,688mg/kg to 0.779g/100mL,1252mg/kg separately.The cultivated condition of Y-5 was find out utilizing a single-module experiment:the optimized medium of 11B~0 is beer wort culture,fermented time is 35h,temperature of cultivate is 28℃,content of Se in medium is 20μg/ml,time for add Se is 11h,dose of inoculation is 10%,the volume of medium is 60ml.Orthogonal experiment was carried out,after 3 factors which affected the content of Se higher.Finding out that the optimized combination is that: content of Se in medium is 20μg/ml,fermented time is 35h,dose of inoculation is 10%.In this condition the biomass of Y-5 increased for 58%,content of Se 74.87% and reached 1343mg/kg.After cultivate for 5 generations,the performances of Y-5 becomes stable.
This research assess the content of Se of Se-enriched yeast,results demonstrate that content of organic Se of Y-5 reach up to 93.6%,while that content of organic Se of original strains is 80%.
实验以热带假丝酵母(Candida tropicalis)1254为出发菌,进行紫外线照射诱变,酵母经紫外线诱变,其致死率曲线表现为“肩形”,即经小剂量辐射的酵母细胞致死率较低,而大剂量的辐射会使酵母细胞的致死率明显升高。随着辐射时间的累计,辐射剂量的增加,突变率逐渐提高,但照射到一定程度时,突变率会降低。筛选出20株生长快、菌落大的突变菌株,接种于含有不同[Se~(4+)]的平板培养基,进行梯度抗性筛选,得到耐高浓度硒的能力较强的菌株6株,进一步复筛得到突变株Y-5。结果显示:Y-5的生物量和硒含量分别由原来的0.619g/100mL、688mg/kg提高到0.779g/100mL、1252mg/kg。利用单因素试验摸索出了Y-5培养条件:11B~0的麦芽汁培养基为最适培养基,发酵培养时间为35h,培养温度为28℃,培养基含硒量20μg/ml,硒的添加时间为11h,接种量10%,装液量为250ml的锥形瓶中装60ml。然后选出对硒含量影响较大的3个因素进行正交试验,找到最优组合为培养基含硒量20μg/ml,发酵培养时间为35h,接种量10%。在最优条件下培养的突变株Y-5,生物量提高了58%,硒含量提高了74.87%,硒含量达到1343mg/kg。变异菌株Y-5经传5代培养,生产性能稳定。
本试验对富硒酵母中有机和无机硒进行测定,发现筛选菌株Y-5有机硒含量达93.6%,而出发菌的有机硒含量是80%。
Selenium is an essential trace element of animals,which has a great deal of biological effects.Selenium is an essential component to keep the activity of glutathione peroxidase which can catalyze glutathione participating peroxidation to get rid of the toxicity of peroxide.However the absorption ration of inorganic selenium is low but high toxicity.Yeast is a good carrier of trace element because of its strong adsorption to trace element.It can convert the inorganic selenium to organic selenium,which can improve the adsorption ration of selenium and lower its toxicity. Yeast is a favourable microprotein source,because it contains abundant protein and vitamin B.So the key to obtain high yield selenium-enriched yeast is a good strain and its suitable cultivate condition.The experiment purpose to obtain a high yield mutant strain utilizing uviolize mutagenesis and resistance screening,and search for optimal incubate condition of it.
The experiment take Candida tropicalis 1254 as original strains,through uviolize mutagenesis,the lethality rate curve of yeast shows "shoulder",videlicet the lethality rate of yeast is lower when the yeast undergo low dose radiation and higher when undergo high dose radiation.With duration of radiation increasing,the rate increase gradually.However until a certain degree the rate will cut down.20 mutation strains which grow rapidly and the colony of them are big,are inoculated in flat plate with different density of Se~(4+).6 strains whose ability to bear high density of Se is higher was obtained.Y-5 mutation strain was obtained,after advanced screening.Results demonstrated:biomass and content of Se increased from 0.619 g/100mL,688mg/kg to 0.779g/100mL,1252mg/kg separately.The cultivated condition of Y-5 was find out utilizing a single-module experiment:the optimized medium of 11B~0 is beer wort culture,fermented time is 35h,temperature of cultivate is 28℃,content of Se in medium is 20μg/ml,time for add Se is 11h,dose of inoculation is 10%,the volume of medium is 60ml.Orthogonal experiment was carried out,after 3 factors which affected the content of Se higher.Finding out that the optimized combination is that: content of Se in medium is 20μg/ml,fermented time is 35h,dose of inoculation is 10%.In this condition the biomass of Y-5 increased for 58%,content of Se 74.87% and reached 1343mg/kg.After cultivate for 5 generations,the performances of Y-5 becomes stable.
This research assess the content of Se of Se-enriched yeast,results demonstrate that content of organic Se of Y-5 reach up to 93.6%,while that content of organic Se of original strains is 80%.
引文
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