阅读说明：本技术 一种提高金霉素发酵罐原料利用率的方法 (Method for improving utilization rate of aureomycin fermentation tank raw materials ) 是由 远万里 吕向云 曹华伟 李学成 屈燕芳 孙庆峰 张欢 于 2021-01-15 设计创作，主要内容包括：本发明属于金霉素发酵技术领域,具体公开一种提高金霉素发酵罐原料利用率的方法,金霉素发酵包括一级发酵、二级发酵和三级发酵,在三级发酵阶段添加复合蛋白酶,所述复合蛋白酶为木瓜蛋白酶、中性蛋白酶、酸性蛋白酶和胰蛋白酶按照4:2:2:1比例混合配制而成,且复合蛋白酶的添加量为三级发酵阶段培养基中氮源重量的0.1-0.5%,复合蛋白酶活性为20万U/g,通过添加复合蛋白酶使迟效氮源减少10%,发酵效价提升6%。(The invention belongs to the technical field of aureomycin fermentation, and particularly discloses a method for improving the utilization rate of raw materials of an aureomycin fermentation tank, wherein the aureomycin fermentation comprises primary fermentation, secondary fermentation and tertiary fermentation, composite protease is added in the tertiary fermentation stage and is prepared by mixing papain, neutral protease, acid protease and trypsin according to the proportion of 4:2:2:1, the addition amount of the composite protease is 0.1-0.5% of the weight of a nitrogen source in a culture medium in the tertiary fermentation stage, the activity of the composite protease is 20 ten thousand U/g, the slow-acting nitrogen source is reduced by 10% by adding the composite protease, and the fermentation titer is improved by 6%.)

1. A method for improving the utilization rate of raw materials of a chlortetracycline fermentation tank is characterized by comprising the following steps: the fermentation of the aureomycin comprises primary fermentation, secondary fermentation and tertiary fermentation, wherein the compound protease is added in the tertiary fermentation stage and is prepared by mixing papain, neutral protease, alkaline protease and trypsin according to the ratio of 4:2:2:1, the addition amount of the compound protease is 0.1-0.5 percent of the weight of a nitrogen source in a culture medium in the tertiary fermentation stage, and the activity of the compound protease is 20 ten thousand U/g.

2. The method for improving the utilization rate of the raw materials of the aureomycin fermentation tank as recited in claim 1, characterized in that: the seed culture medium for the first-stage fermentation comprises 2% of soybean cake powder, 2.5% of corn starch, 0.2% of sodium chloride, 0.2% of ammonium sulfate, 0.25% of calcium carbonate, 1% of yeast powder, 0.02% of magnesium sulfate and 0.05% of dipotassium hydrogen phosphate, and the culture temperature is 32 ℃ and the culture period is 20-36 hours.

3. The method for improving the utilization rate of the raw materials of the aureomycin fermentation tank as recited in claim 1, characterized in that: the components of the seed culture medium for the secondary fermentation comprise 3 percent of soybean cake powder, 4 percent of corn starch, 0.2 percent of sodium chloride, 0.2 percent of ammonium sulfate, 0.25 percent of calcium carbonate, 1 percent of yeast powder, 0.02 percent of magnesium sulfate, 0.05 percent of dipotassium hydrogen phosphate and 0.08 percent of silicone oil, the culture temperature is 32 ℃, and the culture period is 10-20 hours.

4. The method for improving the utilization rate of the raw materials of the aureomycin fermentation tank as recited in claim 1, characterized in that: the components of the fermentation medium for the three-stage fermentation comprise 3.0% of peanut cake powder, 1% of soybean cake powder, 6% of corn starch, 0.01% of amylase, 0.5% of ammonium sulfate, 0.5% of calcium carbonate, 0.5% of yeast powder, 0.2% of mycoprotein, 1.5% of corn steep liquor, 0.02% of magnesium sulfate, 0.21% of ammonium chloride, 0.05% of foam killer, the culture temperature of 30 ℃, the culture period of 120 hours, the dissolved oxygen of 30%, the rotation speed of 100-150rpm, and the aeration ratio of 0.5-1.0 vvm.

Technical Field

The invention belongs to the technical field of aureomycin fermentation, and particularly relates to a method for improving the utilization rate of raw materials of an aureomycin fermentation tank.

Background

Aureomycin is a widely used antibiotic for animals, and a plurality of nitrogen sources are needed in the fermentation production of the aureomycin, wherein peanut cake powder, soybean cake powder and yeast powder are three main slow-acting nitrogen sources in the fermentation process, but the slow-acting nitrogen sources cannot be completely utilized in the fermentation production process, so that a certain burden is added to the environment and sewage treatment after each production, the utilization rate of the slow-acting nitrogen sources is increased, and the difficulty in waste treatment is reduced, thus the problem to be solved urgently is solved.

Disclosure of Invention

The invention aims to provide a method for improving the utilization rate of raw materials of a chlortetracycline fermentation tank, which reduces a delayed nitrogen source by 10% by adding compound protease and improves the fermentation titer by 6% -8%.

In order to achieve the purpose, the invention adopts the technical scheme that:

a method for improving the utilization rate of a raw material of a chlortetracycline fermentation tank comprises primary fermentation, secondary fermentation and tertiary fermentation, wherein compound protease is added in a tertiary fermentation stage, the compound protease is prepared by mixing papain, neutral protease, alkaline protease and trypsin according to a ratio of 4:2:2:1, the addition amount of the compound protease is 0.1-0.5% of the weight of a nitrogen source in a culture medium in the tertiary fermentation stage, and the activity of the compound protease is 20 ten thousand U/g.

Further, the components of the seed culture medium for the primary fermentation comprise 2% of soybean cake powder, 2.5% of corn starch, 0.2% of sodium chloride, 0.2% of ammonium sulfate, 0.25% of calcium carbonate, 1% of yeast powder, 0.02% of magnesium sulfate and 0.05% of dipotassium hydrogen phosphate, the culture temperature is 32 ℃, and the culture period is 20-36 hours.

Furthermore, the components of the seed culture medium for the secondary fermentation comprise 3% of soybean cake powder, 4% of corn starch, 0.2% of sodium chloride, 0.2% of ammonium sulfate, 0.25% of calcium carbonate, 1% of yeast powder, 0.02% of magnesium sulfate, 0.05% of dipotassium hydrogen phosphate and 0.08% of silicone oil, the culture temperature is 32 ℃, and the culture period is 10-20 hours.

Further, the components of the fermentation medium for the three-stage fermentation comprise 3.0% of peanut cake powder, 1% of soybean cake powder, 6% of corn starch, 0.01% of amylase, 0.5% of ammonium sulfate, 0.5% of calcium carbonate, 0.5% of yeast powder, 0.2% of mycoprotein, 1.5% of corn steep liquor, 0.02% of magnesium sulfate, 0.21% of ammonium chloride, 0.05% of natural enemy, the culture temperature of 30 ℃, the culture period of 120 hours, the dissolved oxygen of 30%, the rotation speed of 100-150rpm, and the aeration ratio of 0.5-1.0 vvm.

The invention has the advantages that: because some proteins in the slow-acting nitrogen source such as peanut cake powder and soybean cake powder used for fermentation of the aureomycin are difficult to utilize, enzymes such as neutral protease in the compound protease can effectively assist in degrading macromolecular proteins in the soybean meal, the degradation effect has a time effect, TCA-NSI and DH in the peanut cake powder and the soybean cake powder rise along with the increase of the addition of the enzymes, the rise gradually becomes smaller and tends to be gentle after the addition reaches a certain value, experiments prove that the slow-acting nitrogen source is reduced by 10% by adding the compound protease, and the fermentation titer is improved by 6% -8%.

Detailed Description

Examples

A method for improving the utilization rate of a raw material of a aureomycin fermentation tank comprises primary fermentation, secondary fermentation and tertiary fermentation, wherein composite protease is added in a tertiary fermentation stage, the composite protease is prepared by mixing papain, neutral protease, alkaline protease and trypsin according to a ratio of 4:2:2:1, the composite protease is added in an amount which is 0.1-0.5% of the weight of a nitrogen source in a culture medium in the tertiary fermentation stage, and the activity of the composite protease is 20 ten thousand U/g; the components of the seed culture medium for the first-stage fermentation comprise 2% of soybean cake powder, 2.5% of corn starch, 0.2% of sodium chloride, 0.2% of ammonium sulfate, 0.25% of calcium carbonate, 1% of yeast powder, 0.02% of magnesium sulfate and 0.05% of dipotassium phosphate, the culture temperature is 32 ℃, and the culture period is 20-36 hours; the components of the seed culture medium for the secondary fermentation comprise 3 percent of soybean cake powder, 4 percent of corn starch, 0.2 percent of sodium chloride, 0.2 percent of ammonium sulfate, 0.25 percent of calcium carbonate, 1 percent of yeast powder, 0.02 percent of magnesium sulfate, 0.05 percent of dipotassium phosphate and 0.08 percent of silicone oil, the culture temperature is 32 ℃, and the culture period is 10-20 hours; the components of the fermentation medium for the three-stage fermentation comprise 3.0 percent of peanut cake powder, 1 percent of soybean cake powder, 6 percent of corn starch, 0.01 percent of amylase, 0.5 percent of ammonium sulfate, 0.5 percent of calcium carbonate, 0.5 percent of yeast powder and 0.2 percent of mycoprotein, fermented glutamic acid residue is adopted, the fermentation medium is rich in protein and a plurality of amino acid vitamins, 1.5 percent of corn steep liquor, 0.02 percent of magnesium sulfate, 0.21 percent of ammonium chloride and 0.05 percent of foam enemy, the culture temperature is 30 ℃, the culture period is 120 hours, the dissolved oxygen is controlled at 30 percent, the rotation speed is 100-150rpm, and the aeration ratio is 0.5-1.0 vvm; because some proteins in the slow-acting nitrogen source such as peanut cake powder and soybean cake powder used for fermentation of the aureomycin are difficult to utilize, enzymes such as neutral protease in the compound protease can effectively assist in degrading macromolecular proteins in the soybean meal, the degradation effect has a time effect, TCA-NSI and DH in the peanut cake powder and the soybean cake powder rise along with the increase of the addition of the enzymes, the rise gradually becomes smaller and tends to be gentle after the addition reaches a certain value, experiments prove that the slow-acting nitrogen source is reduced by 10% by adding the compound protease, and the fermentation titer is improved by 6% -8%.

Examples of the experiments

In order to determine the appropriate enzyme dosage, the enzyme dosage is 0.1%, 0.3% and 0.5% (weight percentage) of the nitrogen source, the efficiency of decomposing the nitrogen source by the protease is evaluated by adopting a method for measuring the amino nitrogen, a comparison experiment is carried out by adopting a plurality of enzymes, and the appropriate enzyme, alkaline protease, neutral protease, papain and compound protease are determined, wherein the activities are all 20 ten thousand U/g and are provided by Dongheng Huadao, a single-factor experiment is firstly carried out, and the data before and after the amino nitrogen comparison hydrolysis are determined after the soybean cake powder and the peanut cake powder are respectively hydrolyzed by using the proteases for 1 h; the experimental result shows that the larger the enzyme amount is, the higher the hydrolysis efficiency is, the 0.5% enzyme amount is, and the ratio of the amino nitrogen treatment group to the control group is measured at 0.5h, so that the hydrolysis effect of the papain and the compound protease is better; the hydrolysis advantage of the compound protease is obvious when the compound protease is analyzed by the amino nitrogen ratio value at 1h, and the specific result is shown in table 1.

TABLE 1

And then selecting compound protease to hydrolyze the slow-acting nitrogen source of the basic tank. The substrate concentration is calculated according to the content of the fermentation medium, the formula is all components of the basic tank fermentation, the used protease is added with the dosage of 0.5 percent of the used nitrogen source, the natural pH is 200r/min, the step of only adding the protease is completely carried out according to the temperature of a control group by a normal process, and the step of maintaining the optimum temperature for 60 minutes is added on the basis of the control group in the experimental group so as to facilitate the decomposition of the protease. Wherein, the production is carried out without adding strains, amino nitrogen is detected when the enzymolysis is carried out for 01h, and the enzymolysis effect is analyzed, which is shown in table 2.

TABLE 2

When the compound protease is used for hydrolyzing the whole culture medium, the increase of the amino nitrogen treatment group is determined to be higher than that of the control group; multiple experiments were performed with 0.5% enzyme treatment of the total medium with 1.3 times the amino nitrogen compared to the control. This corresponds to an increase of 30mg/100ml of amino nitrogen.

This experiment was performed at the fermentation level to examine the effect of adding protease to the basal medium. Firstly, inoculating the slant spores into a first-level seeding tank, wherein the culture temperature is 32 ℃, the culture period is 20-36 hours, and the rotation speed is 100-150 rpm; the inoculation amount is 10% in the first-stage and second-stage seed transfer, the second-stage culture temperature is 32 ℃, the rotation speed is 100-150rpm, and the culture period is 10-20 hours; the inoculation amount of the secondary inoculation fermentation is 20%, the culture temperature is 30 ℃, the rotating speed is 100-150rpm, the culture period is 120 hours, the tank is placed for detecting the titer and the amino nitrogen, and all indexes are counted.

Based on the formula used in production, the control group was the normal process formula (marked as group 1), only the protease test group (marked as group 2) was added, the nitrogen source test group (marked as group 3) was added with protease and 10% reduction, and the amino nitrogen after digestion, fermentation termination titer, and integrated fermentation titer and fermentation termination amino nitrogen were measured to determine which protocol was most suitable. Compared with the group 1, the amino nitrogen after digestion is basically kept flat, the titer is improved by 6%, the amino nitrogen after tank placing is reduced by 16%, the effect of adding protease and reducing nitrogen source addition is proved, and the amino nitrogen after digestion is increased by 2 compared with the group 1, but the titer is not greatly changed. It was thus possible to obtain a 10% reduction in the nitrogen source of the lag phase by adding the protease, the specific results of which are shown in Table 3.

TABLE 3