阅读说明：本技术 一种缩短胞苷发酵迟滞期的方法 (Method for shortening cytidine fermentation lag phase ) 是由 曹华杰 张兆坤 李静 于 2020-12-31 设计创作，主要内容包括：本发明涉及一种缩短胞苷发酵迟滞期的方法,属于发酵工程技术领域,所述方法主要是在培养基中添加谷氨酸和异亮氨酸营养物且将玉米浆在发酵开始3-4h后进行缓慢流加以缩短发酵过程的停滞情况。与原有工艺相比,达到相同产量的发酵周期缩短到36h以内,节约时间约6-14h,提高批次间接降低生产成本。(The invention relates to a method for shortening cytidine fermentation lag phase, which belongs to the technical field of fermentation engineering, and is mainly characterized in that glutamic acid and isoleucine nutrients are added into a culture medium, and corn steep liquor is slowly flowed 3-4h after fermentation is started to shorten the stagnation condition of the fermentation process. Compared with the prior art, the fermentation period for achieving the same yield is shortened to be within 36h, the time is saved by about 6-14h, the batch is improved, and the production cost is indirectly reduced.)

1. A method for shortening the lag phase of cytidine fermentation is characterized in that: the method comprises the steps of adding glutamic acid and isoleucine into a fermentation medium as nutrients, adding 20-2000mg/L IPTG 3-4h after fermentation is started, and then feeding corn steep liquor at the speed of 80mL/h for 2-3 h.

2. The method for shortening cytidine fermentation lag phase according to claim 1, wherein: the method comprises the following steps:

step one, activating strains; performing slant activation on an original strain to obtain a strain for fermentation;

step two, seed culture: transferring the fermentation strain obtained in the step one to a seed culture medium for culture to obtain a seed solution; the formula of the seed culture medium is as follows: 10-30g/L of glucose, 4-10g/L of yeast powder, 1-6g/L of citric acid, 1-6g/L of peptone, 0.1-0.5g/L of glutamic acid, 0.1-0.5g/L of isoleucine, 2-9g/L of monopotassium phosphate, 0.5-2.0g/L of magnesium sulfate, 5-20ml/L of corn steep liquor, 10-40mg/L of ferrous sulfate, 1-10mg/L of manganese sulfate, 1-4mg/L of cobalt chloride, 1-4mg/L of biotin and VB₁1-4 mg/L; the culture conditions were: 36 +/-1 ℃, pH 6.5-7.5 and dissolved oxygen controlled at 25-40%;

step three, fermentation culture: inoculating the seed liquid obtained in the second step into a fermentation culture medium, wherein the formula of the fermentation culture medium is as follows: 10-30g/L of glucose, 4-10g/L of yeast powder, 1-6g/L of citric acid, 0.1-0.5g/L of glutamic acid, 0.1-0.5g/L of isoleucine, 1-6g/L of peptone, 2-9g/L of monopotassium phosphate, 0.5-2.0g/L of magnesium sulfate, 10-40mg/L of ferrous sulfate, 1-10mg/L of manganese sulfate, 1-4mg/L of cobalt chloride, 1-4mg/L of biotin and VB₁1-4 mg/L; the culture conditions were: 36 +/-1 ℃, pH 6.5-7.0, dissolved oxygen controlled at 25-40%, residual sugar controlled at 0.1-0.3%; adding 20-2000mg/L inducer 3-4h after fermentation, and feeding corn steep liquor at slow speed for 2-3 h; the fermentation period is 36 h.

Technical Field

The invention belongs to the technical field of fermentation engineering, and particularly relates to a method for shortening a cytidine fermentation lag phase.

Background

Cytidine is an important medical synthesis intermediate and can be used for synthesizing antiviral and antitumor drugs such as cytarabine, azacitidine, cytarabine and the like. At present, microorganisms used for producing cytidine by a fermentation method are basically escherichia coli modified by genetic engineering, but after the induction of genetic engineering bacteria, a period of slow growth occurs, so that the fermentation period is too long, and the industrial production is not facilitated. Therefore, it is very important to find a method for shortening the growth slowness.

Disclosure of Invention

In order to overcome the phenomenon that the growth of cytidine genetic engineering bacteria is slow after induction, the invention aims to provide a method for shortening the lag phase of cytidine fermentation. According to the method, isoleucine and glutamic acid are added into the culture medium, and the corn steep liquor is fed at a speed of 80mL/h after fermentation is started for 3-4h, so that the stagnation condition of the fermentation process is effectively shortened.

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

a method for shortening cytidine fermentation lag phase comprises adding glutamic acid and isoleucine as nutrients into a fermentation medium, adding 20-2000mg/L IPTG 3-4h after fermentation is started, and feeding corn steep liquor at 80mL/h for 2-3 h.

Further, a method for shortening the cytidine fermentation lag phase comprises the following steps:

step one, activating strains; performing slant activation on an original strain to obtain a strain for fermentation;

step two, seed culture: transferring the fermentation strain obtained in the step one to a seed culture medium for culture to obtain a seed solution; the formula of the seed culture medium is as follows: 10-30g/L glucose, 4-10g/L yeast powder, 1-6g/L citric acid, 1-6g/L peptone, 0.1-0.5g/L glutamic acid, 0.1-0.5g/L isoleucine, 2-9g/L potassium dihydrogen phosphate, 0.5-2.0g/L magnesium sulfate, 5-20ml/L corn steep liquor, 10-40mg/L ferrous sulfate, 1-10mg/L manganese sulfate, 1-4mg/L cobalt chloride, 1-4mg/L biotin and VB₁1-4 mg/L; the culture conditions were: 36 +/-1 ℃, pH 6.5-7.5 and dissolved oxygen controlled at 25-40%;

step three, fermentation culture: inoculating the seed liquid obtained in the second step into a fermentation culture medium, wherein the formula of the fermentation culture medium is as follows: 10-30g/L of glucose, 4-10g/L of yeast powder, 1-6g/L of citric acid, 0.1-0.5g/L of glutamic acid, 0.1-0.5g/L of isoleucine, 1-6g/L of peptone, 2-9g/L of monopotassium phosphate, 0.5-2.0g/L of magnesium sulfate, 10-40mg/L of ferrous sulfate, 1-10mg/L of manganese sulfate, 1-4mg/L of cobalt chloride, 1-4mg/L of biotin and VB₁1-4 mg/L; the culture conditions were: 36 +/-1 ℃, pH 6.5-7.0, dissolved oxygen controlled at 25-40%, residual sugar controlled at 0.1-0.3%; adding 20-2000mg/L IPTG 3-4h after fermentation, and then feeding the corn steep liquor at the speed of 80mL/h for 2-3 h; the fermentation period is 36 h.

Has the advantages that:

compared with the prior art, the fermentation period for achieving the same yield is shortened to be within 36h, the time is saved by about 6-14h, the batch is improved, the production cost is indirectly reduced, and the fermentation stability is improved.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

Example 1

A method for shortening cytidine fermentation lag phase comprises the following specific steps:

1. activating strains; inoculating the original strain into a slant solid culture medium for activation to obtain a strain for fermentation;

2. seed culture: transferring the fermentation strain obtained in the step one to a seed culture medium for culture to obtain a seed solution; the formula of the seed culture medium is as follows: 10g/L glucose, 4g/L yeast powder, 6g/L citric acid, 6g/L peptone, 0.1g/L isoleucine, 0.5g/L glutamic acid, 2g/L potassium dihydrogen phosphate, 2.0g/L magnesium sulfate, 5ml/L corn steep liquor, 10mg/L ferrous sulfate, 5mg/L manganese sulfate, 4mg/L cobalt chloride, 4mg/L biotin and VB₁4 mg/L; the culture conditions were: the pH value is 7.0 at 37 ℃, and the dissolved oxygen is controlled at 25%;

3. fermentation culture: inoculating the seed liquid obtained in the second step into a fermentation culture medium, wherein the fermentation culture formula is as follows: 30g/L glucose, 6g/L yeast powder, 3g/L citric acid, 4g/L peptone, 0.3g/L isoleucine, 0.5g/L glutamic acid, 6g/L potassium dihydrogen phosphate, 2g/L magnesium sulfate, 10mg/L ferrous sulfate, 3 mg/L manganese sulfate, 2 mg/L cobalt chloride, 2 mg/L biotin and VB₁2 mg/L. The culture conditions were: at 37 deg.C, pH 7.0, dissolved oxygen at 25%, and residual sugar at 0.1%. After fermenting for 3h, 200mg/L of inducer IPTG was added, and then corn steep liquor was fed at a rate of 80mL/h for 2.5 h. After fermentation for 36 hours, the concentration of cytidine in the fermentation liquor can reach 80g/L, and the sugar-acid conversion rate can reach 30%.

Example 2

A method for shortening cytidine fermentation lag phase comprises the following specific steps:

1. activating strains; inoculating the original strain into a slant solid culture medium for activation to obtain a strain for fermentation;

2. seed culture: transferring the strain for fermentation obtained in the step one toCulturing in a seed culture medium to obtain a seed solution; the formula of the seed culture medium is as follows: 30g/L glucose, 10g/L yeast powder, 3g/L citric acid, 1g/L peptone, 0.1g/L isoleucine, 0.5g/L glutamic acid, 9g/L potassium dihydrogen phosphate, 0.5g/L magnesium sulfate, 20ml/L corn steep liquor, 40mg/L ferrous sulfate, 10mg/L manganese sulfate, 1mg/L cobalt chloride, 1mg/L biotin and VB₁1 mg/L; the culture conditions were: the pH value is 7.0 at 37 ℃, and the dissolved oxygen is controlled at 25%;

3. fermentation culture: inoculating the seed liquid obtained in the second step into a fermentation culture medium, wherein the fermentation culture formula is as follows: 10g/L glucose, 10g/L yeast powder, 6g/L citric acid, 6g/L peptone, 0.3g/L isoleucine, 0.5g/L glutamic acid, 9g/L potassium dihydrogen phosphate, 0.5g/L magnesium sulfate, 40mg/L ferrous sulfate, 10mg/L manganese sulfate, 4mg/L cobalt chloride, 4mg/L biotin and VB₁1 mg/L. The culture conditions were: at 37 deg.C, pH 7.0, dissolved oxygen at 25%, and residual sugar at 0.1%. After fermenting for 3h, 200mg/L of inducer IPTG was added, and then corn steep liquor was fed at a rate of 80mL/h for 2.5 h. After fermentation for 36 hours, the concentration of cytidine in the fermentation liquor can reach 85g/L, and the sugar-acid conversion rate can reach 31%.

It should be noted that the above-mentioned embodiments illustrate rather than limit the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that certain insubstantial modifications and adaptations of the present invention can be made without departing from the spirit and scope of the invention.