纤维素酶产生菌的筛选及其在菌糠开发中的利用
摘要
纤维素是自然界中存在最广泛的一类碳水化合物,是地球上最丰富且取之不尽的天然可再生资源。自然界中纤维素只有一小部分得到了利用,绝大多数纤维素不仅被白白浪费,而且还由于使用不当,造成环境污染。
菌糠是食用菌培养料收获子实体后的下脚料,未处理菌糠的粗蛋白含量较低,纤维素、木质素等碳水化合物较高。
为了探索利用微生物生产纤维素酶,将纤维素转化为人类急需的能源、食物和化工原料的方法,解决解决环境污染、食物短缺和能源危机问题,课题通过对来自土壤、牲畜粪便等处的微生物进行大量的筛选工作,分离出了能够高效利用菌糠纤维素的菌株,并通过筛选、对最佳发酵条件的探索以及混合发酵等手段,获得由菌糠出发,生产单细胞蛋白的材料与工艺。
首先通过以纤维素为唯一碳源的固体培养基筛选,得到40株菌株,进而通过纤维素刚果红培养基筛选,获得8株菌株。最后通过菌糠培养基中筛选,获得A、B、E、F、G五株菌株。之后进行的纤维素分解菌的连续发酵,兼有筛选和确定发酵条件两个目的。这一步确定了F菌株为木屑菌糠最佳利用菌株,G菌株为棉籽壳菌糠最佳利用菌株,并且确定它们的最佳发酵时间为24h。通过载片培养法观察菌株形态,初步判断F菌株为毛霉,G菌株为根霉。
F菌株和G菌株在30℃时,它们产生的发酵液酶活力最高。F菌株在pH=7时,产生的发酵液酶活力最高,而菌密度则是pH=6时最高;G菌株在pH=6时,产生的发酵液酶活力最高,菌密度也最高。实验中使用的各种金属离子使发酵液中的菌密度有了不同程度的下降,相应地,Co~(2+)、Mn~(2+)、Fe~(3+)、Zn~(2+)对应的酶的活力也随之下降;而Fe~(2+)虽然对菌株生长不利,但这种金属离子对纤维素酶有很强的激活作用,使得酶活力反而上升。F菌株与G菌株在2倍浓度营养液中有最高生长水平,并使发酵液有最高酶活力;两种菌株对于尿素、氯化铵不敏感。酶的提取实验证明,F菌株和G菌株产生的酶活力不次于购买得到的对照菌株。
在确定了F、G菌株的最佳发酵产糖条件之后,通过在培养体系中使用F、G菌种与酵母菌的混合培养,获得了较高水平产蛋白的初步方法。与酵母菌的液态混合发酵使F菌株产蛋白水平提高了一倍左右,而使G菌株的产蛋白水平提高了二倍以上。F菌株与酵母混合发酵产蛋白峰值为5.46 g/L,是液态发酵的6.42倍;G菌株与酵母混合发酵产蛋白峰值为5.75 g/L,是液态发酵的6.05倍。
As the carbohydrate most broadly existing in the world, cellulose is a kind of abundant and sustainable resource. However, only a small part of the cellulose in the nature is used, with a large amount wasted, and even brings pollution to the world because of improper usage.
The waste material from fungal culture (WMFC) is waste material after fruiting body harvest with remnant of edible fungi and carbohydrate such as cellulose, hemicellulose and lignin degraded by the edible fungi. Before desposal, WMFC has low level of raw protein, but high level of carbohydrate such as celloluse and lignin.
In order to find a method to produce cellulase by microorganism, get energy resource, food and chemical material from cellulose, and solve problems such as the pollution to the environment, the lack of food and energy resource crisis, microorganisms from the soil are screened. After getting the strain that can make use of WMFC in high effect, the study of optimal fermentation condition and mixed culture fermentation are launched to get the technic producing single cell protein form WMFC.
First, 40 strains are gained by the screening of medium with cellulose as only carbon resource, then, 8 strains a gotten from cellulose-Gango red medium. Finally, strains A, B, E, F, G are gotten from WMFC medium. By glass slide culture and observation, F is judged as Mucor, while G Rhizopus. Continuous fermentation with double purposes both screening and ascertaining the fermentation condition is launched later. Strain F is ascertained as the best strain for using wood bits WMFC, while strain G the best for cottonseed hull. The best fermention time is 24 hours.
Both strains F and G have the highest enzyme activity and population at 30℃. Strain F has the best enzyme activity at pH=7, but highest population at pH=6; strain G has highest enzyme activity and population at pH=6. Every kind of metal ions used in the study brings bad effect to the strains’growth, accordingly, Co~(2+)、Mn~(2+)、Fe~(3+)、Zn~(2+) have the enzyme activity declined. However, Fe~(2+) is harmful to strains’growth, but it has strong activation to the enzyme activity, and makes it higher. Both strains F and G grow best in 2×nutrition concentration, and show highest enzyme activity. Over high concentration is not good for the growth of strain and fermention because of high filter pressure. Neither of the strains is sensitive to carbamide and ammonium chloride. The extract of enzyme proves that the enzyme activity of strains F and G are not lower than the strain bought.
After the ascertaining of optimal sugar producing condition of strains F and G, how we can heighten the protein produce capability is to be studied. By mixed culture fermentation of strains F and G with yeast, we got the way to produce protein in higher level. The mixed culture fermentation with yeast hightened the production of protein of strain F by one time, while more than twice of strain G. The peak of protein production by solid mixed culture fermentation of strain F and yeast is 5.46 g/L, 6.42 times of liquid, while strain G and yeast 5.75 g/L, and 6.05 times.
菌糠是食用菌培养料收获子实体后的下脚料,未处理菌糠的粗蛋白含量较低,纤维素、木质素等碳水化合物较高。
为了探索利用微生物生产纤维素酶,将纤维素转化为人类急需的能源、食物和化工原料的方法,解决解决环境污染、食物短缺和能源危机问题,课题通过对来自土壤、牲畜粪便等处的微生物进行大量的筛选工作,分离出了能够高效利用菌糠纤维素的菌株,并通过筛选、对最佳发酵条件的探索以及混合发酵等手段,获得由菌糠出发,生产单细胞蛋白的材料与工艺。
首先通过以纤维素为唯一碳源的固体培养基筛选,得到40株菌株,进而通过纤维素刚果红培养基筛选,获得8株菌株。最后通过菌糠培养基中筛选,获得A、B、E、F、G五株菌株。之后进行的纤维素分解菌的连续发酵,兼有筛选和确定发酵条件两个目的。这一步确定了F菌株为木屑菌糠最佳利用菌株,G菌株为棉籽壳菌糠最佳利用菌株,并且确定它们的最佳发酵时间为24h。通过载片培养法观察菌株形态,初步判断F菌株为毛霉,G菌株为根霉。
F菌株和G菌株在30℃时,它们产生的发酵液酶活力最高。F菌株在pH=7时,产生的发酵液酶活力最高,而菌密度则是pH=6时最高;G菌株在pH=6时,产生的发酵液酶活力最高,菌密度也最高。实验中使用的各种金属离子使发酵液中的菌密度有了不同程度的下降,相应地,Co~(2+)、Mn~(2+)、Fe~(3+)、Zn~(2+)对应的酶的活力也随之下降;而Fe~(2+)虽然对菌株生长不利,但这种金属离子对纤维素酶有很强的激活作用,使得酶活力反而上升。F菌株与G菌株在2倍浓度营养液中有最高生长水平,并使发酵液有最高酶活力;两种菌株对于尿素、氯化铵不敏感。酶的提取实验证明,F菌株和G菌株产生的酶活力不次于购买得到的对照菌株。
在确定了F、G菌株的最佳发酵产糖条件之后,通过在培养体系中使用F、G菌种与酵母菌的混合培养,获得了较高水平产蛋白的初步方法。与酵母菌的液态混合发酵使F菌株产蛋白水平提高了一倍左右,而使G菌株的产蛋白水平提高了二倍以上。F菌株与酵母混合发酵产蛋白峰值为5.46 g/L,是液态发酵的6.42倍;G菌株与酵母混合发酵产蛋白峰值为5.75 g/L,是液态发酵的6.05倍。
As the carbohydrate most broadly existing in the world, cellulose is a kind of abundant and sustainable resource. However, only a small part of the cellulose in the nature is used, with a large amount wasted, and even brings pollution to the world because of improper usage.
The waste material from fungal culture (WMFC) is waste material after fruiting body harvest with remnant of edible fungi and carbohydrate such as cellulose, hemicellulose and lignin degraded by the edible fungi. Before desposal, WMFC has low level of raw protein, but high level of carbohydrate such as celloluse and lignin.
In order to find a method to produce cellulase by microorganism, get energy resource, food and chemical material from cellulose, and solve problems such as the pollution to the environment, the lack of food and energy resource crisis, microorganisms from the soil are screened. After getting the strain that can make use of WMFC in high effect, the study of optimal fermentation condition and mixed culture fermentation are launched to get the technic producing single cell protein form WMFC.
First, 40 strains are gained by the screening of medium with cellulose as only carbon resource, then, 8 strains a gotten from cellulose-Gango red medium. Finally, strains A, B, E, F, G are gotten from WMFC medium. By glass slide culture and observation, F is judged as Mucor, while G Rhizopus. Continuous fermentation with double purposes both screening and ascertaining the fermentation condition is launched later. Strain F is ascertained as the best strain for using wood bits WMFC, while strain G the best for cottonseed hull. The best fermention time is 24 hours.
Both strains F and G have the highest enzyme activity and population at 30℃. Strain F has the best enzyme activity at pH=7, but highest population at pH=6; strain G has highest enzyme activity and population at pH=6. Every kind of metal ions used in the study brings bad effect to the strains’growth, accordingly, Co~(2+)、Mn~(2+)、Fe~(3+)、Zn~(2+) have the enzyme activity declined. However, Fe~(2+) is harmful to strains’growth, but it has strong activation to the enzyme activity, and makes it higher. Both strains F and G grow best in 2×nutrition concentration, and show highest enzyme activity. Over high concentration is not good for the growth of strain and fermention because of high filter pressure. Neither of the strains is sensitive to carbamide and ammonium chloride. The extract of enzyme proves that the enzyme activity of strains F and G are not lower than the strain bought.
After the ascertaining of optimal sugar producing condition of strains F and G, how we can heighten the protein produce capability is to be studied. By mixed culture fermentation of strains F and G with yeast, we got the way to produce protein in higher level. The mixed culture fermentation with yeast hightened the production of protein of strain F by one time, while more than twice of strain G. The peak of protein production by solid mixed culture fermentation of strain F and yeast is 5.46 g/L, 6.42 times of liquid, while strain G and yeast 5.75 g/L, and 6.05 times.
引文
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