米曲霉菌株的诱变选育及其制剂的应用研究
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摘要
以米曲霉3.4383作为原始菌株,经紫外、EMS复合诱变,通过培养筛选,得到一株高产蛋白酶和纤维素酶的变异菌株UV-2’。其蛋白酶和纤维素酶活比原始菌株(蛋白酶2526U/g,纤维素酶987U/g)提高到3250U/g和1145U/g,传代培养20代,菌株遗传稳定性好。
确定复合诱变条件为:紫外线照射用30W紫外灯,距离为28cm,照射时间为60s,EMS浓度为0.8%。复合诱变之后,从中筛选出一株蛋白酶活力高的菌株UV-2’,其蛋白酶活和纤维素酶活较出发菌株分别提高29%和16%。
研究发酵培养条件对菌株产酶影响中发现:碳氮源、活性剂、培养基的料水比对菌株产酶都有影响;发酵温度、发酵时间、pH、无机盐等对菌株产酶也有影响。通过单因素试验确定的培养条件为:吐温-80含量0.24%,料水比为5/3,pH7.5,在40℃下,发酵84h,酶活可达3250U/g和1145U/g干曲。探索出了FeSO4能强烈抑制菌株产酶,加入KNO3菌株产酶大大提高;并进一步研究了不同氮源和碳源对米曲霉UV-2’产蛋白酶和纤维素酶的影响。确定了饲料酶制剂生产中最佳的氮源和碳源分别为豆粕和麸皮,进一步研究了发酵培养基中麸皮:豆粕=1:5。最后以上述各单因素试验结果做参照,设计正交试验来确定综合因素对菌株产酶的影响,通过正交试验结果可知,发酵培养物的最适合条件为:培养基在豆粕含量60%,麸皮含量10%,添加0.2%的KNO3,吐温-80含量在0.24%,料水比在5/3,pH7.5时产蛋白酶可达4572U/g、纤维素酶可达1923 U/g。
选择1日龄商品代AA肉鸡,研究分别在玉米-豆粕型和玉米-小麦-豆粕型日粮中添加粗酶制剂对肉鸡生产性能、肠道菌群及免疫器官发育的影响。结果表明,添加粗酶制剂显著降低了肉鸡的料肉比(P < 0.05),有提高肉鸡1-42日龄日增重和出栏体重的趋势(P < 0.10);促进了肉鸡免疫器官发育提高了免疫功能,并能够促进肉鸡肠道中乳酸杆菌的增殖,减少肠道大肠杆菌的数量,从而在一定程度上改善肉鸡的肠道微生态环境;在玉米-小麦-豆粕型日粮中添加粗酶制剂可以显著降低肉鸡食糜的黏度(P < 0.05);添加粗酶制剂显著提高了肉鸡食糜中蛋白酶活(P < 0.05),并有提高粗蛋白回肠表观消化率的趋势(P < 0.10),对肉鸡食糜中淀粉酶活和淀粉的回肠表观消化率影响不显著(P > 0.05)。本研究结果揭示,添加粗酶制剂可以改善肉鸡的生产性能。
The Aspergillus oryzae 3.4383 start strain mutated multiply by UV and EMS, a mutant strain UV-2’was obtained by separation, purification and cultivation.The mutant strains’activity of proteinase and cellulase was increased 3250 U/g and 1145 U/g to compare with the original (2526 U/g and 1145 U/g), the strain of UV-2’Aspergillus oryzae has good hereditary stability by inoculating 20 times.
The mutation conditions:the distance is 28 cm,the time of irradiation is 60s with 30W ultraviolet lamp,the concentration of lithium chloide solution was 0.8%. The strain of UV-2’Aspergillus Oryzae was screened with high proteinase and cellulase activity. the proteinase and cellulase activity activity have improved 29% and 16% than the original strain.
To determine the effect of the fermentation conditions and medium ingredinents on enzyme production,flask solid-state fermentation experiments were carried out.It was found that C/N source,activator,powder and water retio of the medium,fermentation temperature,fermentation time,pH,inorganic salt etc affected the enzymes formation in various degrees.The optimal medium compositionand fermentation conditions on proteinase and cellulase production were determined as: tween-80 0.24%, compounding ratio of solid state and water 5:3、pH7.5,as a result,high enzyme activity of 4572 U/g and 1923U/g was obtained after 84h culture at 40℃. Have probed out FeSO4 being able to restrain bacterial strain intensely producing enzyme , and KNO3 bacterial strain produce an enzyme improve greatly the effects of different nitrogen source and carbon source on the activity of proteinase and cellulose produced by Aspergillus oryzae UV-2 has been studied.Definited the best nitrogen source and carbon source were beancake and wheat bran,moreover the ratio of wheat bran:beancake in culture medium was 1:5 has been studied. Finally, test result does the effect coming to ascertain the synthetical factor producing an enzyme to bacterial strain the experiment consulting , designing orthogonal experiment with as said or narrated above respectively single factor. From this orthogonal experiment result we konow the most suit condition of the fermentation culture: beancake 60%,wheat bran 10%,KNO3 0.2%,tween-80 0.24%, compounding ratio of solid state and water 5:3、pH7.5,as a result,high enzyme activity of 4572 U/g and 1923U/g was obtained after 84h culture at 40℃.
An experiment was conducted to study the effects of crude enzyme preparation supplementation on the performance of broilers fed corn-soybean diets and corn-wheat-soybean diets. Crude enzyme preparation supplementation reduced feed conversion rate (FCR) (P < 0.05). Average daily gain (ADG) and body weight (BW) wuggested that crude enere numerically increased (P < 0.10) for broilers fed the diet supplemented with Crude enzyme preparation. The digesta viscosity of corn-wheat-soybean diets decreased with the addition of crude enzyme preparation (P<0.05). Crude enzyme preparation supplementation increased the activity of protease significantly in intestinal content of broilers (P < 0.05), and the apparent ileal digestibility of protein tended to increase (P<0.10). Crude enzyme preparation supplementation had no effect on the activity of amylase in intestinal content of broilers and the apparent ileal digestibility of starch (P > 0.05).The results szyme preparation can improve growth performance in broilers.
确定复合诱变条件为:紫外线照射用30W紫外灯,距离为28cm,照射时间为60s,EMS浓度为0.8%。复合诱变之后,从中筛选出一株蛋白酶活力高的菌株UV-2’,其蛋白酶活和纤维素酶活较出发菌株分别提高29%和16%。
研究发酵培养条件对菌株产酶影响中发现:碳氮源、活性剂、培养基的料水比对菌株产酶都有影响;发酵温度、发酵时间、pH、无机盐等对菌株产酶也有影响。通过单因素试验确定的培养条件为:吐温-80含量0.24%,料水比为5/3,pH7.5,在40℃下,发酵84h,酶活可达3250U/g和1145U/g干曲。探索出了FeSO4能强烈抑制菌株产酶,加入KNO3菌株产酶大大提高;并进一步研究了不同氮源和碳源对米曲霉UV-2’产蛋白酶和纤维素酶的影响。确定了饲料酶制剂生产中最佳的氮源和碳源分别为豆粕和麸皮,进一步研究了发酵培养基中麸皮:豆粕=1:5。最后以上述各单因素试验结果做参照,设计正交试验来确定综合因素对菌株产酶的影响,通过正交试验结果可知,发酵培养物的最适合条件为:培养基在豆粕含量60%,麸皮含量10%,添加0.2%的KNO3,吐温-80含量在0.24%,料水比在5/3,pH7.5时产蛋白酶可达4572U/g、纤维素酶可达1923 U/g。
选择1日龄商品代AA肉鸡,研究分别在玉米-豆粕型和玉米-小麦-豆粕型日粮中添加粗酶制剂对肉鸡生产性能、肠道菌群及免疫器官发育的影响。结果表明,添加粗酶制剂显著降低了肉鸡的料肉比(P < 0.05),有提高肉鸡1-42日龄日增重和出栏体重的趋势(P < 0.10);促进了肉鸡免疫器官发育提高了免疫功能,并能够促进肉鸡肠道中乳酸杆菌的增殖,减少肠道大肠杆菌的数量,从而在一定程度上改善肉鸡的肠道微生态环境;在玉米-小麦-豆粕型日粮中添加粗酶制剂可以显著降低肉鸡食糜的黏度(P < 0.05);添加粗酶制剂显著提高了肉鸡食糜中蛋白酶活(P < 0.05),并有提高粗蛋白回肠表观消化率的趋势(P < 0.10),对肉鸡食糜中淀粉酶活和淀粉的回肠表观消化率影响不显著(P > 0.05)。本研究结果揭示,添加粗酶制剂可以改善肉鸡的生产性能。
The Aspergillus oryzae 3.4383 start strain mutated multiply by UV and EMS, a mutant strain UV-2’was obtained by separation, purification and cultivation.The mutant strains’activity of proteinase and cellulase was increased 3250 U/g and 1145 U/g to compare with the original (2526 U/g and 1145 U/g), the strain of UV-2’Aspergillus oryzae has good hereditary stability by inoculating 20 times.
The mutation conditions:the distance is 28 cm,the time of irradiation is 60s with 30W ultraviolet lamp,the concentration of lithium chloide solution was 0.8%. The strain of UV-2’Aspergillus Oryzae was screened with high proteinase and cellulase activity. the proteinase and cellulase activity activity have improved 29% and 16% than the original strain.
To determine the effect of the fermentation conditions and medium ingredinents on enzyme production,flask solid-state fermentation experiments were carried out.It was found that C/N source,activator,powder and water retio of the medium,fermentation temperature,fermentation time,pH,inorganic salt etc affected the enzymes formation in various degrees.The optimal medium compositionand fermentation conditions on proteinase and cellulase production were determined as: tween-80 0.24%, compounding ratio of solid state and water 5:3、pH7.5,as a result,high enzyme activity of 4572 U/g and 1923U/g was obtained after 84h culture at 40℃. Have probed out FeSO4 being able to restrain bacterial strain intensely producing enzyme , and KNO3 bacterial strain produce an enzyme improve greatly the effects of different nitrogen source and carbon source on the activity of proteinase and cellulose produced by Aspergillus oryzae UV-2 has been studied.Definited the best nitrogen source and carbon source were beancake and wheat bran,moreover the ratio of wheat bran:beancake in culture medium was 1:5 has been studied. Finally, test result does the effect coming to ascertain the synthetical factor producing an enzyme to bacterial strain the experiment consulting , designing orthogonal experiment with as said or narrated above respectively single factor. From this orthogonal experiment result we konow the most suit condition of the fermentation culture: beancake 60%,wheat bran 10%,KNO3 0.2%,tween-80 0.24%, compounding ratio of solid state and water 5:3、pH7.5,as a result,high enzyme activity of 4572 U/g and 1923U/g was obtained after 84h culture at 40℃.
An experiment was conducted to study the effects of crude enzyme preparation supplementation on the performance of broilers fed corn-soybean diets and corn-wheat-soybean diets. Crude enzyme preparation supplementation reduced feed conversion rate (FCR) (P < 0.05). Average daily gain (ADG) and body weight (BW) wuggested that crude enere numerically increased (P < 0.10) for broilers fed the diet supplemented with Crude enzyme preparation. The digesta viscosity of corn-wheat-soybean diets decreased with the addition of crude enzyme preparation (P<0.05). Crude enzyme preparation supplementation increased the activity of protease significantly in intestinal content of broilers (P < 0.05), and the apparent ileal digestibility of protein tended to increase (P<0.10). Crude enzyme preparation supplementation had no effect on the activity of amylase in intestinal content of broilers and the apparent ileal digestibility of starch (P > 0.05).The results szyme preparation can improve growth performance in broilers.
引文
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Zanella I, Sakomura N K, Silversides F G, et al. Effect of enzyme supplementation of broiler diets based on corn and soybeans[J]. Poultry Science, 1999, 78(4): 561- 568.
Zurdo J, Guijarro J I, Jimenez J L, etal. Dependence on solution conditions of aggregation and amyloid formation by an SH3 domain[J]. Mol. Biol, 2001, 311: 325- 340.