阅读说明：本技术 一种高密度发酵生产l-赖氨酸的培养基及其方法 (Culture medium for producing L-lysine by high-density fermentation and method thereof ) 是由 徐庆阳 孙际宾 张玉富 郑平 李燕军 张成林 陈宁 于 2020-12-28 设计创作，主要内容包括：本发明提供了一种高密度发酵生产L-赖氨酸的培养基,属于生物工程技术领域,本发明通过对培养基中的有机氮源进行优化筛选、酶解处理,得到含有氨基酸、小分子肽、磷酸盐等的有机氮源小分子多肽水溶液。本发明还提供了将上述培养基用于高密度发酵生产L-赖氨酸的方法,包括在发酵过程中同步流加上述多肽水溶液和微量溶菌酶。本发明提供的培养基和高密度发酵方法可减少菌体生长耗能,加快菌体生长繁殖,提高生物量,达到提高L-赖氨酸糖酸转化率与产量、缩短发酵周期、降低发酵染菌几率等目的。(The invention provides a culture medium for producing L-lysine by high-density fermentation, which belongs to the technical field of bioengineering and obtains an organic nitrogen source micromolecule polypeptide aqueous solution containing amino acid, micromolecule peptide, phosphate and the like by carrying out optimized screening and enzymolysis treatment on an organic nitrogen source in the culture medium. The invention also provides a method for producing L-lysine by using the culture medium in high-density fermentation, which comprises the step of synchronously feeding the polypeptide aqueous solution and trace lysozyme in the fermentation process. The culture medium and the high-density fermentation method provided by the invention can reduce the energy consumption for the growth of the thalli, accelerate the growth and the reproduction of the thalli, improve the biomass, and achieve the purposes of improving the conversion rate and the yield of L-lysine saccharic acid, shortening the fermentation period, reducing the probability of fermentation and bacterial contamination and the like.)

1. A culture medium for producing L-lysine by high-density fermentation is characterized in that: the culture medium nitrogen source comprises micromolecular polypeptide aqueous solution obtained by enzymolysis of an organic nitrogen source.

2. The culture medium according to claim 1, wherein: the organic nitrogen source comprises corn steep liquor dry powder and soybean meal hydrolysate.

3. The culture medium according to claim 2, wherein: after the corn steep liquor dry powder is prepared into a corn steep liquor solution, pepsin, acidic phytase and alkaline protease are sequentially added for enzymolysis.

4. The culture medium of claim 3, wherein the enzymolysis conditions of the corn steep liquor solution are as follows: the enzymolysis temperature of the pepsin is 50-70 ℃, the reaction time is 0.5-3.0 h, and the reaction pH is 1.5-5.0; the enzymolysis temperature of the acidic phytase is 40-70 ℃, the reaction time is 0.5-3.0 h, and the reaction pH is 3.0-6.0; the enzymolysis temperature of the alkaline protease is 45-70 ℃, the reaction time is 0.5-3.0 h, and the reaction pH is 7.0-10.0.

5. The culture medium according to claim 2, wherein: the soybean meal hydrolysate is prepared by alkaline protease: the mass ratio of the neutral protease is 1: and (3) carrying out enzymolysis on 0.5-2 of compound enzyme.

6. The culture medium according to claim 5, wherein the enzymolysis conditions of the soybean meal hydrolysate are as follows: the enzymolysis temperature is 40-60 ℃, the enzymolysis time is 10-200 min, and the enzymolysis pH is 7.0-9.0.

7. The culture medium according to any one of claims 4 or 6, wherein: and filtering the enzymolysis liquid after enzymolysis to obtain a clear peptide solution.

8. The culture medium according to claim 7, wherein the culture medium is used for seed culture, and the raw materials comprise: 5-50 g/L of corn steep liquor dry powder, 5-50 mL/L of soybean meal hydrolysate, 2-8g/L of yeast powder, 20-60 g/L of glucose, 1-6 g/L of ammonium sulfate, 0.5-4 g/L of magnesium sulfate, 2-8g/L of monopotassium phosphate, 5-30 mg/L of ferrous sulfate, 5-30 mg/L of manganese sulfate, 1-3 mL/L of VB mixed solution, 2-10 mg/L of zinc sulfate, 1-7 mg/L of copper sulfate pentahydrate and 0.1-3.0 mg/L of biotin.

9. The culture medium according to claim 7, wherein the culture medium is used for fermentation culture, and the raw materials comprise: 5-50 g/L of corn steep liquor dry powder, 10-30 mL/L of soybean meal hydrolysate, 10-80 g/L of glucose, 1-10 g/L of ammonium sulfate, 0.3-2.0 g/L of magnesium sulfate, 1-5 g/L of potassium dihydrogen phosphate, 0.3-5.0 g/L of potassium chloride, 5-30 mg/L of ferrous sulfate, 5-30 mg/L of manganese sulfate, 1-5 mL/L of VB mixed solution, 0.2-2.0 mg/L of zinc sulfate, 0.1-5.0 mg/L of copper sulfate pentahydrate, and 0.02-1.0 mg/L of biotin.

10. A method for producing L-lysine by high-density fermentation of the culture medium of any one of claims 1 to 9, characterized in that a mixed solution of a small molecule polypeptide aqueous solution and lysozyme is fed in synchronously during the fermentation process.

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a culture medium for producing L-lysine by high-density fermentation and a method thereof.

Background

Lysine (2, 6-diaminohexanoic acid or alpha, epsilon-diaminohexanoic acid, Lysine), which has both L-and D-optical isomers, is an essential amino acid that cannot be synthesized by the organism itself, must be supplemented from food, and is the only amino acid among 8 essential amino acids that can be effectively utilized only by the L-form component. Lysine is abundant in animal food and beans and very low in cereal food, so that L-lysine is widely used in feed additives, dietary supplements, deodorants, pharmaceutical products and the like, wherein more than 90% of lysine products are used as feed additives.

In order to consider production economy and save fermentation cost as much as possible, most of organic nitrogen sources used for producing lysine by microbial fermentation at present are corn steep liquor dry powder, soybean meal hydrolysate and the like, but the fermentation is often negatively influenced due to the complex components, large molecular proteins, bacillus carrying, inhibition of thallus growth caused by the high content of phytic acid in individual components such as corn steep liquor and the like. In order to reduce the contamination probability, the corn steep liquor dry powder is usually required to be sterilized in multiple steps before use, so that the cost is increased, the workload is increased, and the loss of organic nitrogen source nutrients can be caused by repeated sterilization; insoluble substances in the corn steep liquor are attached to the tank body and the stirring shaft in the fermentation process, so that the sight is influenced, the stirring resistance is increased, and the equipment is damaged while excessive mechanical heat is generated; excessive time and energy are consumed for absorption and utilization of macromolecular protein in the fermentation process, the acid production efficiency of the thalli is greatly influenced, and the fermentation period is prolonged. Meanwhile, excessive protein causes a large amount of foam to be generated in the fermentation process, the use of a defoaming agent is increased, the cost is increased, the toxic action of the defoaming agent on microorganisms is amplified, the filling coefficient of a fermentation tank is reduced by excessive foam, and the utilization rate of equipment is reduced. The existing fermentation method directly utilizing small molecular nutrients such as finished small molecular peptides, amino acids and the like is not suitable for industrial development due to high price.

Therefore, how to obtain a culture medium suitable for high-density fermentation production of L-lysine, and the technical problems of improving the saccharic acid conversion rate and yield of the L-lysine and reducing the probability of fermentation contamination are urgently needed to be solved in the field.

Disclosure of Invention

In view of this, the present invention aims to provide a culture medium for producing L-lysine by high-density fermentation and a method thereof, which are beneficial to improving the saccharic acid conversion rate and yield in the fermentation process of L-lysine and reducing the contamination probability in the fermentation process.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides a culture medium for producing L-lysine by high-density fermentation, wherein a nitrogen source of the culture medium comprises a micromolecular polypeptide aqueous solution obtained by enzymolysis of an organic nitrogen source.

Preferably, the organic nitrogen source comprises corn steep liquor dry powder and soybean meal hydrolysate.

Preferably, after the corn steep liquor dry powder is prepared into a corn steep liquor solution, pepsin, acidic phytase and alkaline protease are sequentially added for enzymolysis.

Preferably, the enzymolysis conditions of the corn steep liquor solution are as follows: the enzymolysis temperature of the pepsin is 50-70 ℃, the reaction time is 0.5-3.0 h, and the reaction pH is 1.5-5.0; the enzymolysis temperature of the acidic phytase is 40-70 ℃, the reaction time is 0.5-3.0 h, and the reaction pH is 3.0-6.0; the enzymolysis temperature of the alkaline protease is 45-70 ℃, the reaction time is 0.5-3.0 h, and the reaction pH is 7.0-10.0.

Preferably, the soybean meal hydrolysate is prepared by alkaline protease: the mass ratio of the neutral protease is 1: and (3) carrying out enzymolysis on 0.5-2 of compound enzyme.

Preferably, the enzymolysis conditions of the soybean meal hydrolysate are as follows: the enzymolysis temperature is 40-60 ℃, the enzymolysis time is 10-200 min, and the enzymolysis pH is 7.0-9.0.

Preferably, the enzymatic hydrolysate is filtered after the enzymatic hydrolysis to obtain a clear peptide solution.

Preferably, the culture medium is used for seed culture, and the raw materials comprise: 5-50 g/L of corn steep liquor dry powder, 5-50 mL/L of soybean meal hydrolysate, 2-8g/L of yeast powder, 20-60 g/L of glucose, 1-6 g/L of ammonium sulfate, 0.5-4 g/L of magnesium sulfate, 2-8g/L of monopotassium phosphate, 5-30 mg/L of ferrous sulfate, 5-30 mg/L of manganese sulfate, 1-3 mL/L of VB mixed solution, 2-10 mg/L of zinc sulfate, 1-7 mg/L of copper sulfate pentahydrate and 0.1-3.0 mg/L of biotin.

Preferably, the culture medium is used for fermentation culture, and the raw materials comprise: 5-50 g/L of corn steep liquor dry powder, 10-30 mL/L of soybean meal hydrolysate, 10-80 g/L of glucose, 1-10 g/L of ammonium sulfate, 0.3-2.0 g/L of magnesium sulfate, 1-5 g/L of potassium dihydrogen phosphate, 0.3-5.0 g/L of potassium chloride, 5-30 mg/L of ferrous sulfate, 5-30 mg/L of manganese sulfate, 1-5 mL/L of VB mixed solution, 0.2-2.0 mg/L of zinc sulfate, 0.1-5.0 mg/L of copper sulfate pentahydrate, and 0.02-1.0 mg/L of biotin.

The invention also provides a method for producing L-lysine by high-density fermentation by using the culture medium, which comprises the following steps: synchronously adding the mixed solution of the small molecular polypeptide aqueous solution and the lysozyme in a flowing manner in the fermentation process.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, the complex organic nitrogen sources such as corn steep liquor, soybean meal hydrolysate and the like in the fermentation medium are subjected to enzymolysis by protease and acidic phytase to obtain polypeptides, amino acids, phosphates and the like with small molecular weights, the aqueous solution containing the small molecular polypeptides is obtained through operations such as filtration and the like, and is used for subsequent fermentation production, and the fed-batch addition of the small molecular polypeptide aqueous solution ensures that the thalli are in the optimum nutrient concentration at each growth stage, so that the nutrient waste is avoided, and the nutrient utilization rate is improved.

The invention uses the peptide solution after enzymolysis as a nitrogen source, can reduce the energy consumption of thallus growth, accelerate thallus growth and reproduction and improve biomass. Meanwhile, in the high-density fermentation process, the small molecular polypeptide aqueous solution and the lysozyme are added in a mixed feeding manner, the lysozyme is added to increase the permeability of cell membranes, and the product discharge is accelerated; and a certain amount of n-dodecane serving as an oxygen carrying agent is added in a biomass stabilization period, and the addition of the n-dodecane serving as the oxygen carrying agent ensures the requirement of dissolved oxygen under high thallus density, so that the aims of improving the conversion rate and yield of L-lysine saccharic acid, reducing the probability of fermentation and bacterial contamination and the like are fulfilled.

Detailed Description

The invention provides a culture medium for producing L-lysine by high-density fermentation, wherein a nitrogen source of the culture medium comprises a micromolecular polypeptide aqueous solution obtained by enzymolysis of an organic nitrogen source.

The organic nitrogen source comprises corn steep liquor dry powder, the internal quality of the corn steep liquor dry powder is the same as that of the corn steep liquor, the content of protein and trace elements is high, and the organic nitrogen source is generally applied to growth and cultivation of microorganisms. The particular source of the dry corn steep liquor powder is not limited in the present invention.

The organic nitrogen source also comprises bean pulp hydrolysate which is also called microorganism DN preparation and soybean concentrate, can replace high-price raw and auxiliary materials used in the fermentation industry such as yeast powder, peptone and the like, is an organic nitrogen source commonly applied in the microbial fermentation process, and is mainly used for amino acid fermentation. The specific source of the soybean meal hydrolysate is not limited in the invention.

The invention carries out enzymolysis on the corn steep liquor dry powder. The method comprises the step of dissolving corn steep liquor dry powder with deionized water to prepare a corn steep liquor solution, wherein the corn steep liquor solution preferably accounts for 5-10% in mass fraction, and more preferably accounts for 7% in mass fraction.

In the invention, pepsin, acidic phytase and alkaline protease are sequentially added into a corn steep liquor solution for enzymolysis. The invention uses pepsin to decompose the protein in the corn steep liquor into small peptide fragments, uses acid phytase to hydrolyze phytic acid and salts thereof in the corn steep liquor into inositol and phosphate, and uses alkaline protease to further hydrolyze the peptide chain of protein molecules to generate smaller peptide fragments or amino acids. Wherein the enzymolysis temperature of the pepsin is 50-70 ℃, the optimized enzymolysis temperature is 60-65 ℃, the reaction time is 0.5-3.0 h, the optimized enzymolysis time is 1.5-2.0 h, the reaction pH is 1.5-5.0, the optimized enzymolysis time is 2.0-3.5, and the enzyme adding amount is 10-100U/g, and the optimized enzymolysis time is 30-50U/g; the enzymolysis temperature of the acidic phytase is 40-70 ℃, the optimized temperature is 55-60 ℃, the reaction time is 0.5-3.0 h, the optimized time is 1.5-2.0 h, the reaction pH is 3.0-6.0, the optimized time is 4.0-5.0, and the enzyme adding amount is 10-500U/g, and the optimized time is 100-260U/g; the enzymolysis temperature of the alkaline protease is 45-70 ℃, preferably 55-60 ℃, the reaction time is 0.5-3.0 h, preferably 1.5-2.0 h, the reaction pH is 7.0-10.0, preferably 8.5-9.0, and the enzyme dosage is 5000-50000U/g, preferably 20000-35000U/g.

The invention carries out enzymolysis on the soybean meal hydrolysate. The method comprises the steps of diluting bean pulp hydrolysate with deionized water to prepare a bean pulp protein solution, wherein the mass fraction of the bean pulp protein solution is preferably 5-20%, and the mass fraction of the bean pulp protein solution is more preferably 15%; adding compound enzyme into the soybean meal protein solution for enzymolysis, wherein the compound enzyme contains alkaline protease: the mass ratio of the neutral protease is 1: 0.5 to 2.0, preferably 1: 1.5. the invention utilizes the complex enzyme to fully hydrolyze protein macromolecules in the soybean meal hydrolysate into micromolecular peptide fragments or amino acids. Wherein the enzymolysis temperature is 40-60 ℃, preferably 50-55 ℃, the enzymolysis time is 10-200 min, preferably 85-105 min, the enzymolysis pH is 7.0-9.0, preferably 7.5-8.0, the enzyme addition amount is 5000-20000U/g, preferably 10000-15000U/g.

The invention respectively filters the enzymolysis liquid obtained by enzymolysis to obtain clear peptide solution. As an alternative embodiment, the enzymatic hydrolysate can be filtered by using a ceramic membrane or a metal microfiltration membrane, and the molecular weight cut-off is 1000-5000MW, preferably 3000 MW. The specific filtering mode of the enzymolysis liquid is not limited, and the clear peptide solution is obtained after filtering.

The invention can firstly carry out large-scale enzymolysis treatment on the organic nitrogen source, and then convert the use amount of the organic nitrogen source into the use volume (V) of the treated peptide solution according to the principle that the content of available nutrients of microorganisms is not changed before and after the treatment₃)：

The amount of peptide solution added (V) obtained by treating dry corn steep liquor powder (solid organic nitrogen source)₃) The conversion method of (3):

wherein m represents the mass of the organic nitrogen source to be added to the medium, rho₂Denotes the nitrogen source density after enzymolysis, m₁Denotes the mass of the nitrogen source to be enzymolyzed, V₂Represents the volume of peptide solution after enzymolysis, p₁Denotes the substrate concentration, V₁The substrate volume is indicated.

The addition amount (V) of the peptide solution obtained by treating the soybean meal hydrolysate (liquid organic nitrogen source)₃) The conversion method of (3):

wherein v represents the volume of the organic nitrogen source to be added to the culture medium, p₂Denotes the nitrogen source density after enzymolysis, v₁Denotes the volume of the solution to be enzymolyzed, V₂Represents the volume of peptide solution after enzymolysis, p₁Denotes the substrate concentration, V₁The substrate volume is indicated.

The culture medium comprises a seed culture medium and a fermentation culture medium, and the two culture media have the same treatment modes for the corn steep liquor dry powder and the soybean meal hydrolysate.

The invention provides a detailed explanation of the process of enzymolysis of corn steep liquor dry powder:

dissolving a certain amount of corn protein powder with deionized water, preparing the corn protein powder into a corn steep liquor solution with a certain mass fraction, roughly measuring the total amount of protein in the solution by adopting a Kjeldahl method, and determining the enzyme consumption; adjusting pH value with HCl solution to optimum pH value for pepsin action, and heating to pepsin action temperature. Dissolving and activating a certain amount of enzyme by using a potassium dihydrogen phosphate buffer solution, adding the enzyme into the protein solution, and starting the reaction. Measuring and adjusting the pH value to an initial value at regular intervals, stopping heating and stirring after the preset time is reached, and finishing enzymolysis;

detecting the phytic acid content by using high performance liquid chromatography, adjusting the pH value to the optimum pH value of the action of the acidic phytase by using HCl solution again, heating to the optimum reaction temperature of the acidic phytase, adding a proper amount of magnesium sulfate to accelerate the reaction, and finishing enzymolysis;

adjusting the pH value of the enzymolysis liquid to the optimum pH value of the alkaline protease by using NaOH solution, heating to the optimum reaction temperature of the alkaline protease, and hydrolyzing for a certain time until the enzymolysis is finished.

And (3) placing the enzymolysis liquid in a filter, circularly filtering to obtain a clear peptide solution, measuring the volume, and storing at a low temperature (4 ℃) for later use.

The invention makes a detailed explanation on the process of hydrolyzing the soybean meal hydrolysate by enzymolysis:

diluting a certain volume of soybean meal hydrolysate with deionized water to prepare a soybean meal protein solution with a certain mass fraction, roughly measuring the total amount of protein in the solution by adopting a Kjeldahl method, and determining the enzyme dosage; adjusting the pH value to the optimum value of the complex enzyme action by using NaOH solution, heating to the optimum reaction temperature of the complex enzyme, dissolving and activating a proper amount of the complex enzyme by using potassium dihydrogen phosphate buffer solution, adding the solution into protein solution, and starting the reaction. Measuring and adjusting the pH value to an initial value at regular intervals, heating to an enzyme inactivation temperature after the predetermined time is reached, naturally cooling, and finishing enzymolysis.

And (3) placing the enzymolysis liquid in a filter, circularly filtering to obtain a clear peptide solution, measuring the volume, and storing at a low temperature (4 ℃) for later use.

The above description of the enzymolysis process of the corn steep liquor dry powder and the enzymolysis process of the soybean meal hydrolysate is only used as an explanation of the enzymolysis process, and is not a limitation of the present invention on the enzymolysis process.

The culture medium for seed culture of the present invention comprises: 5-50 g/L of corn steep liquor dry powder, 5-50 mL/L of soybean meal hydrolysate, 2-8g/L of yeast powder, 20-60 g/L of glucose, 1-6 g/L of ammonium sulfate, 0.5-4 g/L of magnesium sulfate, 2-8g/L of monopotassium phosphate, 5-30 mg/L of ferrous sulfate, 5-30 mg/L of manganese sulfate, 1-3 mL/L of VB mixed solution, 2-10 mg/L of zinc sulfate, 1-7 mg/L of copper sulfate pentahydrate and 0.1-3.0 mg/L of biotin.

Preferably, the culture medium for seed culture according to the present invention comprises: 15-35 g/L of corn steep liquor dry powder, 15-35 mL/L of soybean meal hydrolysate, 4-6g/L of yeast powder, 35-50 g/L of glucose, 2-4 g/L of ammonium sulfate, 2-3 g/L of magnesium sulfate, 4-6g/L of potassium dihydrogen phosphate, 18-25 mg/L of ferrous sulfate, 18-25 mg/L of manganese sulfate, 1.5-2 mL/L of VB mixed solution, 4-8 mg/L of zinc sulfate, 3-5 mg/L of copper sulfate pentahydrate and 1-1.5 mg/L of biotin.

More preferably, the culture medium for seed culture according to the present invention is: 15-35 g/L of corn steep liquor dry powder, 15-35 mL/L of soybean meal hydrolysate, 4-6g/L of yeast powder, 35-50 g/L of glucose, 2-4 g/L of ammonium sulfate, 2-3 g/L of magnesium sulfate, 4-6g/L of potassium dihydrogen phosphate, 18-25 mg/L of ferrous sulfate, 18-25 mg/L of manganese sulfate, 1.5-2 mL/L of VB mixed solution, 4-8 mg/L of zinc sulfate, 3-5 mg/L of copper sulfate pentahydrate and 1-1.5 mg/L of biotin.

The culture medium for fermentation culture comprises: 5-50 g/L of corn steep liquor dry powder, 10-30 ml/L of soybean meal hydrolysate, 10-80 g/L of glucose, 1-10 g/L of ammonium sulfate, 0.3-2.0 g/L of magnesium sulfate, 1-5 g/L of potassium dihydrogen phosphate, 0.3-5.0 g/L of potassium chloride, 5-30 mg/L of ferrous sulfate, 5-30 mg/L of manganese sulfate, V_B1-5 mL/L of mixed solution, 0.2-2.0 mg/L of zinc sulfate, 0.1-5.0 mg/L of blue vitriol, and 0.02-1.0 mg/L of biotin.

Preferably, the culture medium for fermentation culture of the present invention comprises: 15-25 g/L of corn steep liquor dry powder, 15-25 ml/L of soybean meal hydrolysate, 40-60 g/L of glucose, 4-6g/L of ammonium sulfate, 1-1.5 g/L of magnesium sulfate, 2-3 g/L of potassium dihydrogen phosphate, 2-5 g/L of potassium chloride, 15-20 mg/L of ferrous sulfate, 15-20 mg/L of manganese sulfate, V_B2-3 mL/L of mixed solution, 1-1.5 mg/L of zinc sulfate, 2-3 mg/L of blue vitriol and 0.5-0.8 mg/L of biotin.

More preferably, the culture medium for fermentation culture of the present invention is: 15-25 g/L of corn steep liquor dry powder, 15-25 ml/L of soybean meal hydrolysate, 40-60 g/L of glucose, 4-6g/L of ammonium sulfate, 1-1.5 g/L of magnesium sulfate, 2-3 g/L of potassium dihydrogen phosphate, 2-5 g/L of potassium chloride, 15-20 mg/L of ferrous sulfate, 15-20 mg/L of manganese sulfate, V_B2-3 mL/L of mixed solution, 1-1.5 mg/L of zinc sulfate, 2-3 mg/L of blue vitriol and 0.5-0.8 mg/L of biotin.

The invention utilizes the culture medium to carry out high-density fermentation to produce the L-lysine. In-fermentor biomass (OD) for high-density fermentation production of L-lysine in the invention₅₆₂X 50) is more than or equal to 80, and the dissolved oxygen is 30-50 percent, preferably 45 percent.

The process for producing the L-lysine by high-density fermentation by using the culture medium comprises strain activation, seed culture and fermentation culture.

The strain activation comprises the following steps: the production bacteria are connected to a first-generation slant culture medium from a glycerol bacteria-protecting tube, the first-generation slant culture medium is cultured for 22-24 hours at a constant temperature of 30-35 ℃, a small amount of bacteria on the first-generation slant are connected to a second-generation slant and cultured for 22-24 hours at a constant temperature of 30-35 ℃, and all bacteria on the second-generation slant are used for seed tank culture.

The seed culture conditions include: selecting the seed culture medium, and controlling the pH to be 7.0-7.2, the temperature to be 32-34 ℃ and the dissolved oxygen to be 30-50% by feeding ammonia water; when the dissolved oxygen is lower than 30%, the rotating speed and the air quantity are alternately increased, and the seed maturity index is OD₅₆₂×50≥30。

The fermentation culture conditions comprise: selecting the fermentation medium, and controlling the pH to be 7.0-7.2, the temperature to be 35-37 ℃ and the dissolved oxygen to be 30-50% by feeding ammonia water; when the dissolved oxygen is lower than 30%, the rotating speed and the air quantity are alternately increased, when the residual sugar in the fermentation tank is reduced to 0.5-1.0 g/L, the glucose is fed, the sugar in the whole fermentation process is controlled to be 3-10 g/L, and the glucose concentration is 750-850 g/L.

Preferably, the invention can also flow and add small molecule polypeptide aqueous solution and lysozyme in the above-mentioned fermentation process. The invention makes a detailed explanation on the feeding mode of the micromolecule polypeptide aqueous solution and the lysozyme in the high-density fermentation process:

in the fermentation process, the micromolecule polypeptide aqueous solution and the lysozyme are uniformly mixed and then synchronously added: taking corn steep liquor dry powder enzymolysis micromolecule polypeptide aqueous solution and soybean meal hydrolysate micromolecule polypeptide aqueous solution according to a formula proportion of a fermentation medium, uniformly mixing, equally dividing into 2 parts, sterilizing one part of the mixture and other components in the fermentation medium, pouring the sterilized mixture into a fermentation tank at one time, adjusting the pH of the other part of the mixture to 6.4-6.6, sterilizing, cooling, mixing with lysozyme under aseptic operation, and feeding the mixture for use in the fermentation process. The using amount of the lysozyme in the mixed solution is 0.08-0.12 mg/L, and the preferable using amount is 0.1 mg/L. The feeding mode is non-feedback quantitative feeding, and continuous pulse feeding is preferred; the feeding amount is 0.4-0.6% of the volume of the fed-batch fermentation liquor per hour, and the preferred feeding amount is 0.5%. The invention discovers that feedback inhibition of high nutrient concentration at the initial stage of fermentation on thallus growth can be effectively removed through the fed-batch mode, and through continuous fed-batch after nutrient division, the activity of strains and the utilization rate of nutrient substances at the later stage of fermentation are improved under the condition that the total nutrient is not increased, the problem that the activity of the thallus is reduced due to lack of nutrient at the middle and later stages of fermentation is solved, so that the saccharic acid conversion rate and the yield in the L-lysine fermentation process are improved, and the contamination probability in the fermentation process is reduced.

The oxygen carrying agent is added when the fermentation process just enters a biomass stabilization period, and the oxygen carrying agent is added into the fermentation tank once after sterilization and sterile operation. As an optional implementation mode, the oxygen carrier can be n-dodecane, and the addition amount of the oxygen carrier is 30-70 mg/L, and more preferably 50-55 mg/L. The biomass stationary phase is the strain growth stationary phase. The present invention is not limited to the manner of determining the biological stationary phase. The invention adds the oxygen carrying agent in the biomass stabilization period, thereby ensuring the demand of dissolved oxygen under high thallus density, and further achieving the purposes of improving the L-lysine saccharic acid conversion rate and yield and reducing the probability of fermentation contamination.

In the specific embodiment of the invention, the selected strain is Corynebacterium glutamicum (Corynebacterium glutamicum) which is purchased from the laboratory of metabolic engineering of Tianjin university of science and technology. The present invention is not limited to a specific source of Corynebacterium glutamicum.

The corn steep liquor dry powder selected in the specific embodiment of the invention is purchased from Kyoto Dakai Biotech Co., Ltd; the soybean meal hydrolysate was purchased from Shandong Yubao Biotech Co., Ltd. The specific sources of the corn steep liquor dry powder and the soybean meal hydrolysate are not limited.

The enzymes selected in the specific examples of the present invention are: alkaline protease (50000-100000U/g) from mountain Biotech, Inc., Sichuan province; neutral proteases (100000-500000U/g) from Nanning Pont bioengineering, Inc.; pepsin (1000-; the acid phytase (5000-; lysozyme (50000-100000U/g) purchased from Shandong Zun Macro Biotech Co., Ltd; n-dodecane, available from Shanghai Allantin Biotech Co., Ltd. The present invention is not limited to the particular sources of the various enzymes and reagents.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

100g of corn steep liquor dry powder is taken and dissolved by deionized water to prepare a corn steep liquor solution with the mass fraction of 7 percent, and the total amount of protein in the solution is roughly measured to be 47.5g by adopting a Kjeldahl method. The corn steep liquor solution is adjusted to pH 2.5 with HCl solution and heated to 60 ℃ in a constant temperature water bath. After 0.23g of pepsin was dissolved and activated in 10mL of potassium dihydrogen phosphate buffer (0.1M), it was added to the corn steep liquor solution to start the reaction. Measuring and adjusting the pH value to the initial value every 20min after the reaction, stopping heating and stirring after reacting for 2.5h, and finishing enzymolysis;

detecting the phytic acid content to be 0.3g by utilizing a high performance liquid chromatography, adjusting the pH value to be 4.0 by using an HCl solution again, heating the mixture to 55 ℃ in a constant-temperature water bath kettle, adding 0.016g of acidic phytase, and adding 0.05% (weight to volume) of magnesium sulfate to accelerate the reaction for 1h, and finishing the enzymolysis;

adjusting the pH value of the enzymolysis liquid to 8.0 by using NaOH solution, heating the enzymolysis liquid to 55 ℃ in a constant-temperature water bath kettle, adding 9.5g of alkaline protease for hydrolysis for 2h, and finishing enzymolysis.

And (3) placing the enzymolysis liquid in a ceramic membrane filter with the molecular weight cutoff of 3000MW, circularly filtering to obtain 1.02L of clear peptide solution, and storing at low temperature (4 ℃) for later use.

Example 2

This example differs from example 1 in that:

adjusting the pH value of the corn steep liquor solution to 2 by using HCl solution, heating the corn steep liquor solution to 65 ℃ in a constant-temperature water bath kettle, adding pepsin, reacting for 2 hours, stopping heating and stirring, and finishing enzymolysis;

adjusting the pH value to 5.0 again by using HCl solution, heating to 60 ℃ in a constant-temperature water bath kettle, adding the acidic phytase and magnesium sulfate to react for 0.5h, and finishing enzymolysis;

adjusting the pH value of the enzymolysis liquid to 9.0 by using NaOH solution, heating the enzymolysis liquid to 60 ℃ in a constant-temperature water bath kettle, adding alkaline protease for hydrolysis for 1.5h, and finishing enzymolysis.

And (3) placing the enzymolysis liquid in a ceramic membrane filter with the molecular weight cutoff of 4000MW, circularly filtering to obtain 970mL of clear peptide solution, and storing at low temperature (4 ℃) for later use.

Example 3

Diluting 100mL of soybean meal hydrolysate with deionized water to prepare a soybean meal protein solution with the mass fraction of 15%, roughly measuring the total amount of protein in the solution by adopting a Kjeldahl method to be 16.6g, and carrying out alkaline protease in complex enzyme: the mass ratio of the neutral protease is 1: 1.5, the dosage of the alkaline protease is 0.474g, and the dosage of the neutral protease is 0.711 g; adjusting the pH value to 8.0 by using NaOH solution, heating to 50 ℃ in a constant-temperature water bath kettle, dissolving and activating the complex enzyme by using 10mL of potassium dihydrogen phosphate buffer solution (0.1M), adding the solution into the protein solution, and starting the reaction. Measuring and adjusting the pH value to the initial value every 10min after the reaction, heating to 90 ℃ after the reaction for 1h to inactivate the enzyme, naturally cooling, and finishing the enzymolysis.

And (3) placing the enzymolysis liquid in a ceramic membrane filter with the molecular weight cutoff of 3000MW, circularly filtering to obtain 530mL of clear peptide solution, and storing at low temperature (4 ℃) for later use.

Example 4

This example differs from example 3 in that:

regulating the pH value of the soybean meal hydrolysate to 7.5 by using NaOH solution, heating the soybean meal hydrolysate to 55 ℃ in a constant-temperature water bath kettle, dissolving and activating the complex enzyme by using potassium dihydrogen phosphate buffer solution, adding the complex enzyme into the protein solution, and starting reaction. Measuring the reaction every 10min, adjusting the pH value to the initial value, reacting for 150min, heating until the enzyme is inactivated, naturally cooling, and finishing enzymolysis.

And (3) placing the enzymolysis liquid in a ceramic membrane filter with the molecular weight cutoff of 4000MW, circularly filtering to obtain 510mL of clear peptide solution, and storing at low temperature (4 ℃) for later use.

Example 5

The seed culture medium comprises: 20g/L of corn steep liquor dry powder, 20mL/L of soybean meal hydrolysate, 4g/L of yeast powder, 40g/L of glucose, 4g/L of ammonium sulfate, 2g/L of magnesium sulfate, 4g/L of monopotassium phosphate, 20mg/L of ferrous sulfate, 20mg/L of manganese sulfate, 2mL/L of VB mixed solution, 6mg/L of zinc sulfate, 4mg/L of copper sulfate pentahydrate and 1.5mg/L of biotin.

The corn steep liquor dry powder and the soybean meal hydrolysate are subjected to enzymolysis by the methods in example 1 and example 3 respectively.

Seed medium preparation (1L dose):

(1) according to the formula (1L) of the culture medium, respectively weighing the culture medium raw materials except the corn steep liquor dry powder, the soybean meal hydrolysate and the glucose, mixing and dissolving with deionized water;

(2) measuring 0.204L of corn steep liquor dry powder enzymatic hydrolysate and 0.106L of bean pulp enzymatic hydrolysate according to the formula, adding into the mixed solution in the step (1), fixing the volume to 0.8L by using deionized water, and sterilizing at 121 ℃ for 15min for later use;

(3) weighing 40g of glucose, fixing the volume to 0.1L by using deionized water, and sterilizing for 15min at 121 ℃ for later use;

(4) and (4) mixing the solutions in the steps (2) to (3) to obtain the seed culture medium.

(the seed culture medium may be supplemented with 0.1L of a second generation seed washing solution when used)

Example 6

The seed culture medium is as follows: 24g/L of corn steep liquor dry powder; 20mL/L of soybean meal hydrolysate; 5g/L of yeast powder; glucose 40 g/L; 1.5g/L of ammonium sulfate; 3g/L of magnesium sulfate; 6g/L potassium dihydrogen phosphate; 20mg/L of ferrous sulfate; 20mg/L of manganese sulfate; 2mL/L of VB mixed solution; 6mg/L of zinc sulfate; 6mg/L of blue vitriol; biotin 1.5 mg/L.

The corn steep liquor dry powder and the soybean meal hydrolysate are subjected to enzymolysis by the methods in example 1 and example 3 respectively.

Seed medium preparation (1L dose):

(1) according to the formula (1L) of the culture medium, respectively weighing the culture medium raw materials except the corn steep liquor dry powder, the soybean meal hydrolysate and the glucose, mixing and dissolving with deionized water;

(2) measuring 0.2448L of corn steep liquor dry powder enzymatic hydrolysate and 0.106L of bean pulp enzymatic hydrolysate according to the formula, adding into the mixed solution in the step (1), fixing the volume to 0.8L by using deionized water, and sterilizing at 121 ℃ for 15min for later use;

(3) weighing 40g of glucose, fixing the volume to 0.1L by using deionized water, and sterilizing for 15min at 121 ℃ for later use;

(4) and (4) mixing the solutions in the steps (2) to (3) to obtain the seed culture medium.

(the seed culture medium may be supplemented with 0.1L of a second generation seed washing solution when used)

Example 7

The seed culture medium is as follows: 24g/L of corn steep liquor dry powder; 20mL/L of soybean meal hydrolysate; 5g/L of yeast powder; glucose 40 g/L; 1.5g/L of ammonium sulfate; 3g/L of magnesium sulfate; 6g/L potassium dihydrogen phosphate; 20mg/L of ferrous sulfate; 20mg/L of manganese sulfate; 2mL/L of VB mixed solution; 6mg/L of zinc sulfate; 6mg/L of blue vitriol; biotin 1.5 mg/L.

The corn steep liquor dry powder and the soybean meal hydrolysate are subjected to enzymolysis by the methods in example 2 and example 4 respectively.

Seed medium preparation (1L dose):

(1) according to the formula (1L) of the culture medium, respectively weighing the culture medium raw materials except the corn steep liquor dry powder, the soybean meal hydrolysate and the glucose, mixing and dissolving with deionized water;

(2) measuring 0.2328L of corn steep liquor dry powder enzymatic hydrolysate and 0.102L of bean pulp enzymatic hydrolysate according to the formula, adding into the mixed solution in the step (1), fixing the volume to 0.8L with deionized water, and sterilizing at 121 ℃ for 15min for later use;

(3) weighing 40g of glucose, fixing the volume to 0.1L by using deionized water, and sterilizing for 15min at 121 ℃ for later use;

(4) and (4) mixing the solutions in the steps (2) to (3) to obtain the seed culture medium.

(the seed culture medium may be supplemented with 0.1L of a second generation seed washing solution when used)

Example 8

The fermentation medium comprises: 20g/L of corn steep liquor dry powder, 25mL/L of soybean meal hydrolysate, 40g/L of glucose, 6g/L of ammonium sulfate, 1g/L of magnesium sulfate, 2.5g/L of monopotassium phosphate, 1.5g/L of potassium chloride, 16mg/L of ferrous sulfate, 16mg/L of manganese sulfate, 2mL/L of VB mixed solution, 1mg/L of zinc sulfate, 1.5mg/L of copper sulfate pentahydrate and 0.75mg/L of biotin.

The corn steep liquor dry powder and the soybean meal hydrolysate are subjected to enzymolysis by the methods in example 1 and example 3 respectively.

Preparation of fermentation medium (1L dose):

(1) according to the formula (1L) of the culture medium, respectively weighing the culture medium raw materials except the corn steep liquor dry powder, the soybean meal hydrolysate and the glucose, mixing and dissolving with deionized water;

(2) measuring 0.204L of corn steep liquor dry powder enzymolysis liquid and 0.1325L of soybean meal hydrolysis liquid according to the formula, adding into the mixed liquid in the step (1), fixing the volume to 0.75L with deionized water, and sterilizing at 121 ℃ for 15min for later use;

(3) weighing 40g of glucose, fixing the volume to 0.04L by using deionized water, and sterilizing for 15min at 121 ℃ for later use;

(4) and (4) mixing the solutions in the steps (2) to (3) to obtain the fermentation medium.

(the fermentation Medium may be supplemented with 0.21L of seed solution for specific use.)

Example 9

The fermentation medium comprises: 20g/L of corn steep liquor dry powder, 25mL/L of soybean meal hydrolysate, 40g/L of glucose, 6g/L of ammonium sulfate, 1g/L of magnesium sulfate, 2.5g/L of monopotassium phosphate, 1.5g/L of potassium chloride, 16mg/L of ferrous sulfate, 16mg/L of manganese sulfate, 2mL/L of VB mixed solution, 1mg/L of zinc sulfate, 1.5mg/L of copper sulfate pentahydrate and 0.75mg/L of biotin.

The corn steep liquor dry powder and the soybean meal hydrolysate are subjected to enzymolysis by the methods in example 1 and example 3 respectively.

Preparation of fermentation medium (1L dose):

(1) measuring 0.204L of corn steep liquor dry powder enzymolysis liquid and 0.1325L of soybean meal hydrolysis liquid according to the formula, uniformly mixing, dividing into two parts with equal volume, adjusting the pH value of one part to 6.5, and then filling into a supplement bottle, and using the other part for later use.

(2) And (3) respectively weighing the culture medium raw materials except the corn steep liquor dry powder, the soybean meal hydrolysate and the glucose according to the culture medium formula (1L), uniformly mixing the culture medium raw materials with the small molecular polypeptide aqueous solution prepared in the step (1), and fixing the volume to 0.537L by using deionized water.

(3) Weighing 40g of glucose, fixing the volume to 0.04L by using deionized water, and sterilizing for 15min at 121 ℃ for later use;

and (4) mixing the solutions in the steps (2) to (3) to obtain the fermentation medium.

(0.25475L seed solution is added when the fermentation medium is used specifically; 0.16825L enzymolysis mixed solution in a supplement bottle is added according to the continuous pulse type flow addition of 0.5 percent of the volume of the fermentation solution per hour)

Example 10

The fermentation medium comprises: 30g/L of corn steep liquor dry powder; 20ml/L of soybean meal hydrolysate; glucose 30 g/L; 5g/L of ammonium sulfate; magnesium sulfate 0.87 g/L; 2.5g/L of monopotassium phosphate; potassium chloride 0.53 g/L; 20mg/L of ferrous sulfate; 20mg/L of manganese sulfate; VB mixed solution is 3 mL/L; 0.6mg/L of zinc sulfate; 0.6mg/L of blue vitriol; biotin 0.85mg/L

The corn steep liquor dry powder and the soybean meal hydrolysate are subjected to enzymolysis by the methods in example 2 and example 4 respectively.

Preparation of fermentation medium (1L dose):

(1) according to the formula (1L) of the culture medium, respectively weighing the culture medium raw materials except the corn steep liquor dry powder, the soybean meal hydrolysate and the glucose, mixing and dissolving with deionized water;

(2) measuring 0.291L of corn steep liquor dry powder enzymatic hydrolysate and 0.102L of soybean meal hydrolysate according to the formula, adding into the mixed solution obtained in the step (1), diluting to 0.75L with deionized water, and sterilizing at 121 deg.C for 15 min;

(3) weighing 40g of glucose, fixing the volume to 0.04L by using deionized water, and sterilizing for 15min at 121 ℃ for later use;

(4) and (4) mixing the solutions in the steps (2) to (3) to obtain the fermentation medium.

(the fermentation Medium may be supplemented with 0.21L of seed solution for specific use.)

Example 11

The fermentation medium comprises: 30g/L of corn steep liquor dry powder; 20ml/L of soybean meal hydrolysate; glucose 30 g/L; 5g/L of ammonium sulfate; magnesium sulfate 0.87 g/L; 2.5g/L of monopotassium phosphate; potassium chloride 0.53 g/L; 20mg/L of ferrous sulfate; 20mg/L of manganese sulfate; VB mixed solution is 3 mL/L; 0.6mg/L of zinc sulfate; 0.6mg/L of blue vitriol; biotin 0.85mg/L

The corn steep liquor dry powder and the soybean meal hydrolysate are subjected to enzymolysis by the methods in example 2 and example 4 respectively.

Preparation of fermentation medium (1L dose):

(1) measuring 0.291L of corn steep liquor dry powder enzymolysis liquid and 0.102L of soybean meal hydrolysis liquid according to the formula, uniformly mixing, dividing into two parts with equal volume, adjusting the pH value of one part to 6.5, and then filling into a supplement bottle, and using the other part for later use.

(2) According to the formula of the culture medium, the culture medium raw materials except the corn steep liquor dry powder, the soybean meal hydrolysate and the glucose are respectively weighed and uniformly mixed with the micromolecule polypeptide aqueous solution prepared in the step (1), and the volume is fixed to 0.537L by using deionized water.

(3) Weighing 40g of glucose, fixing the volume to 0.04L by using deionized water, and sterilizing for 15min at 121 ℃ for later use;

(4) and (4) mixing the solutions in the steps (2) to (3) to obtain the fermentation medium.

(0.2265L of seed liquid is added when the fermentation medium is used specifically; 0.1965L of enzymolysis mixed liquid in a supplement bottle is added according to the volume of 0.5 percent of the volume of the fermentation liquid per hour in a continuous pulse mode.)

Example 12

This example shows a method for producing L-lysine by high density fermentation, which comprises strain activation, seed culture, and fermentation culture.

Strain activation: placing the strain in a glycerol-preserving tube on a slant culture medium, uniformly scratching the slant of a test tube by using an inoculating loop, and carrying out inversion constant-temperature culture at 32 ℃ for 24h to obtain a generation of seeds; taking a first generation of seeds by an inoculating loop, uniformly mixing the seeds in an oblique plane of an eggplant-shaped bottle, and carrying out inversion constant-temperature culture at 32 ℃ for 24h to obtain second generation seeds.

The slant culture medium is: 5g/L of glucose; 10g/L of beef extract; 10g/L of yeast powder; 2.5g/L of urea; 2g/L of sodium chloride; 1g/L potassium dihydrogen phosphate; magnesium sulfate 0.3 g/L; 25g/L of agar powder.

Seed culture: the initial fermentation temperature is 34 deg.C, pH is controlled to 7.2 by feeding ammonia water, dissolved oxygen is 40%, rotation speed and air volume are alternately increased when dissolved oxygen is less than 30%, and seed maturity index is OD₅₆₂And (5) fermenting when the multiplied by 50 is more than or equal to 30.

Fermentation culture: in the fermentation process, dissolved oxygen is controlled at 40%, the fermentation temperature is 37 ℃, when the dissolved oxygen is lower than 30%, the rotating speed and the air volume are alternately increased, the pH value is controlled to be 7.0 by feeding ammonia water, when the residual sugar in the fermentation tank is reduced to 0.8g/L, the sugar is fed in a flow supplementing manner, and the sugar in the whole fermentation process is controlled to be 6 g/L.

The L-lysine yield is determined by thin layer chromatography, wherein the chromatography carrier is chromatography silica gel (GF254) -thin plate purchased from tetramethyl biochemical plastics factory in Zhejiang province; the chromatographic solution is n-butanol: water: glacial acetic acid 4: 2: 1.

example 13

This example used the fermentation conditions in example 12 for high density fermentation to produce L-lysine: the seed medium components of example 5 and the fermentation medium components of example 8 were used.

The seed culture adopts a 5L fermentation tank, and 2L seed culture medium is prepared: the method comprises the steps of diluting the mixed solution of the components except glucose to a constant volume of 1.6L for sterilization, diluting the glucose to a constant volume of 200mL for independent sterilization, cleaning the second generation seeds with 200mL of sterile water, pouring the sterilized mixed solution and the second generation seed cleaning solution into a tank under strict aseptic operation, simultaneously pouring a glucose solution, and culturing until the seeds are mature and then inoculating into a fermentation tank.

The fermentation culture adopts a 5L fermentation tank, the inoculation amount is 20% (600mL), and 3L fermentation medium dosage is prepared: and (3) diluting the mixed solution of the components except the glucose to a constant volume of 2.2L for sterilization, diluting the glucose to a constant volume of 200mL for independent sterilization, pouring the sterilized mixed solution and the seed solution into a tank under strict aseptic operation, and simultaneously pouring the glucose solution into the tank for fermentation culture.

Two batches of repeated fermentation experiments are carried out, and the average value of the data of the two experiments is recorded and named as 5L-1. And (3) measuring the final L-lysine concentration, the concentration of the midway liquid-discharging lysine, the saccharic acid conversion rate, the fermentation period, the maximum filling coefficient of the fermentation tank and the maximum biomass.

Example 14

This example used the fermentation conditions in example 12 for high density fermentation to produce L-lysine: the seed medium components of example 5 and the fermentation medium components of example 8 were used.

The seed culture adopts a 5L fermentation tank, and 3L seed culture medium is prepared: the method comprises the steps of diluting the mixed solution of the components except glucose to a constant volume of 2.6L for sterilization, diluting the glucose to a constant volume of 200mL for independent sterilization, cleaning the second generation seeds with 200mL of sterile water, pouring the sterilized mixed solution and the second generation seed cleaning solution into a tank under strict aseptic operation, simultaneously pouring a glucose solution, and culturing until the seeds are mature and then inoculating into a fermentation tank.

The fermentation culture adopts a 30L fermentation tank, the inoculation amount is 20% (2.8L), and 14L fermentation medium dosage is prepared: and (3) diluting the mixed solution of the components except the glucose to a constant volume of 10.6L for sterilization, diluting the glucose to a constant volume of 600mL for independent sterilization, pouring the sterilized mixed solution and the seed solution into a tank under strict aseptic operation, and simultaneously pouring the glucose solution into the tank for fermentation culture.

Two batches of repeated fermentation experiments are also carried out, and the average value of the data of the two experiments is recorded and named as 30L-1. And (3) measuring the final L-lysine concentration, the concentration of the midway liquid-discharging lysine, the saccharic acid conversion rate, the fermentation period, the maximum filling coefficient of the fermentation tank and the maximum biomass.

Example 15

This example used the fermentation conditions in example 12 for high density fermentation to produce L-lysine: the seed medium components of example 5 and the fermentation medium components of example 8 were used, but the seed medium and the organic nitrogen source in the fermentation medium were directly used with the non-enzymatic corn steep liquor dry powder and the soybean meal hydrolysate.

The seed and fermentation culture steps and methods were the same as in example 13.

Two batches of repeated fermentation experiments were also carried out, and the average of the data of the two experiments was recorded and named 5L-2. And (3) measuring the final L-lysine concentration, the concentration of the midway liquid-discharging lysine, the saccharic acid conversion rate, the fermentation period, the maximum filling coefficient of the fermentation tank and the maximum biomass.

Example 16

This example used the fermentation conditions in example 12 for high density fermentation to produce L-lysine: the seed medium components of example 5 and the fermentation medium components of example 8 were used, but the seed medium and the organic nitrogen source in the fermentation medium were directly used with the non-enzymatic corn steep liquor dry powder and the soybean meal hydrolysate.

The seed and fermentation culture steps and methods were the same as in example 14.

Two batches of repeated fermentation experiments are also carried out, and the average value of the data of the two experiments is recorded and named as 30L-2. And (3) measuring the final L-lysine concentration, the concentration of the midway liquid-discharging lysine, the saccharic acid conversion rate, the fermentation period, the maximum filling coefficient of the fermentation tank and the maximum biomass.

TABLE 1 comparison of fermentation results for batches

As can be seen from Table 1, the final L-lysine concentrations of 5L-1 and 30L-1 subjected to the enzymatic hydrolysis treatment by the organic nitrogen source in the culture medium are 263.5g/L and 269.7g/L respectively, which are increased by 19.0% and 18.8% respectively compared with 5L-2 and 30L-2 not subjected to the enzymatic hydrolysis treatment by the organic nitrogen source in the culture medium; the concentration of discharged liquid lysine of 5L-1 and 30L-1 of the culture medium organic nitrogen source subjected to enzymolysis treatment is 122.5g/L and 131.6g/L respectively, and is respectively increased by 4.4% and 2.2% compared with that of 5L-2 and 30L-2 of the culture medium organic nitrogen source which is not subjected to enzymolysis treatment; the saccharic acid conversion rates of 5L-1 and 30L-1 of the culture medium organic nitrogen source subjected to enzymolysis treatment are respectively 0.727g/g and 0.722g/g, and are respectively improved by 6.9 percent and 5.7 percent compared with 5L-2 and 30L-2 of the culture medium organic nitrogen source not subjected to enzymolysis treatment. And the maximum canning coefficient, the fermentation period and the maximum biomass of 5L-1 and 30L-1 of the culture medium organic nitrogen source subjected to enzymolysis are higher than those of 5L-2 and 30L-2 of the culture medium organic nitrogen source which is not subjected to enzymolysis.

Shows that: the method for producing the L-lysine by fermentation can effectively improve the biomass, the saccharic acid conversion rate and the L-lysine yield, and when the volume of the fermentation tank is increased from 5L to 30L, the maximum biomass, the saccharic acid conversion rate and the L-lysine yield of the fermentation reaction are not reduced, the bacteria contamination probability is not increased, and the equipment utilization rate is greatly improved.

Example 17

This example performed high density fermentation on different seed media and fermentation media combinations (same conditions for fermentation except for media selection, lysozyme and oxygen carrier addition).

Control group: adopting the components of the seed culture medium of the embodiment 5 and the components of the fermentation culture medium of the embodiment 8, directly adopting the corn steep liquor dry powder without enzymolysis and the soybean meal hydrolysate as the organic nitrogen source in the seed culture medium and the fermentation culture medium, and not adding lysozyme and an oxygen carrying agent in the fermentation process;

experimental groups: the seed medium formulation of example 5/6/7, the fermentation medium formulation of example 8/9/10/11 and the addition method were used with/without addition of n-dodecane, an oxygen carrier, during the biomass stabilization phase, and the corresponding experimental groups were numbered. Example (c): the numbers 5-8-1, 5-8-2 and 6-9-1 respectively represent: n-dodecane was added during the biomass stabilization phase using the seed medium formulation of example 5, the fermentation medium formulation of example 8 and the addition method; the seed medium formulation of example 5, the fermentation medium formulation of example 8 and the addition method were used, with no n-dodecane added during the biomass stabilization phase; n-dodecane was added during the biomass stabilization phase using the seed medium formulation of example 6, the fermentation medium formulation and method of example 9, and so on for all experimental group numbers.

Each group was subjected to two repeated fermentation experiments, and the average of the data of the two experiments was recorded. And (3) measuring the final L-lysine concentration, the concentration of the midway liquid-discharging lysine, the saccharic acid conversion rate, the fermentation period, the maximum filling coefficient of the fermentation tank and the maximum biomass, and recording whether the strain is infected or not.

TABLE 2 comparison of fermentation results of different batches

As can be seen from Table 2, the culture medium uses the small molecular polypeptide aqueous solution to replace the traditional corn steep liquor and bean concentration, so that the yield, the conversion rate and the biomass of the L-lysine are improved to a large extent, and the equipment utilization rate is improved by 4 percent; different optimized seed culture media do not influence the final fermentation result, so that a scheme as few as possible can be selected according to the material saving principle.

The addition of the oxygen carrying agent n-dodecane slightly improves the yield and the conversion rate of the L-lysine, shortens the fermentation period and improves the efficiency, for example, the yield of 5-8-2 is improved by 1.2 percent compared with the yield of 5-8-1L-lysine, the conversion rate is improved by 0.4 percent, and the fermentation period is shortened by 1 hour; the fed-batch addition of the lysozyme and the polypeptide aqueous solution greatly improves the yield and the conversion rate of the L-lysine, for example, the yield of 7-9-1 is improved by 1.3 percent compared with the yield of 7-8-1, and the conversion rate is improved by 2.5 percent; the conversion rate is not improved even reduced to a certain extent due to the increase of the concentration of nutrient substances in the fermentation medium, and the yield and the biomass are greatly improved. For example, the yield of 6-11-2 is improved by 1.5 percent, the biomass is improved by 1.2 percent and the conversion rate is reduced by 0.9 percent compared with the yield of 6-9-2. In addition, the optimized groups for carrying out enzymolysis treatment on the organic nitrogen source of the culture medium are all in the condition of bacterial contamination, and the control group is infected with bacteria, so that the fermentation efficiency is greatly influenced.

In conclusion, the fermentation production of the L-lysine can effectively improve the biomass, the saccharic acid conversion rate and the L-lysine yield, can well control the contamination probability, improves the equipment utilization rate to a certain extent, advances the fermentation period by 1 hour, and has positive significance for the industrial fermentation production of the L-lysine.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.