A method for increasing L-lysine fermentation yield

Abstract

The present invention provides a method for increasing the fermentation yield of L-lysine, and belongs to the field of L-amino acid fermentation. The method for increasing the fermentation yield of L-lysine is composed of the following steps: preparing a shake flask culture solution, preparing a seed solution, and fermenting and culturing. The method for increasing the fermentation yield of L-lysine of the present invention can effectively improve the tolerance of the bacterial body to high osmotic pressure, improve the acid production performance in the late fermentation period, and improve the single-tank yield and sugar-acid conversion rate of L-lysine; at the same time, further reduce the adverse effects of osmotic pressure regulators on the fermentation stability of L-lysine, and effectively avoid the problem of excessive addition of osmotic pressure regulators inhibiting bacterial metabolism, extending the fermentation production cycle, and reducing fermentation efficiency.

Description

Technical Field

[0001] The invention relates to the field of L-amino acid fermentation, and in particular to a method for increasing the fermentation yield of L-lysine. Background Art

[0002] In the existing amino acid fermentation process, with the generation of products and the accumulation of other ions, the ion concentration in the fermentation liquid will increase in the later stage of fermentation. According to the Van't Hoff formula, this will lead to an increase in osmotic pressure, which will in turn affect bacterial metabolism and ultimately reduce the sugar-acid conversion rate and yield in this fermentation stage.

[0003] Taking the fermentation production of L-lysine as an example, the main sources of osmotic pressure in the L-lysine fermentation process are: L-lysine and sulfate. Every 1 mol of L-lysine will consume 0.5 mol of ammonium sulfate. When the concentration of L-lysine is 200g / L (hydrochloride molecular weight 182.65), the osmotic pressure of L-lysine fermentation broth is: (200 / 182.65) × 1.5 = 1642mOsm / L. Considering other solutes in the fermentation broth, the osmotic pressure of L-lysine fermentation broth in the later stage of fermentation may be as high as 2350mOsm / L. According to analysis, during the fermentation of L-lysine, the osmotic pressure of the fermentation broth decreases continuously due to the consumption of substrate sugar in the early stage of fermentation; in the later stage of fermentation, as the concentration of L-lysine increases, the osmotic pressure increases continuously; in the later stage of fermentation, the bacteria cannot tolerate excessive osmotic pressure, resulting in a slower and slower synthesis of L-lysine. Therefore, in the later stage of L-lysine fermentation, the high osmotic pressure of the fermentation broth will greatly affect the fermentation index and fermentation effect of amino acids.

[0004] In order to solve the above problems, in the prior art, a certain concentration of osmotic pressure regulator (such as betaine) is usually added at the beginning of fermentation to adjust the osmotic pressure of the fermentation liquid during the fermentation process. Betaine is a compound widely present in nature (such as animals, plants, and microorganisms), and is mainly found in the molasses of beet sugar. Betaine, also known as trimethylamine betaine or betaine, is a quaternary amine type water-soluble alkaloid. It is white scaly or prismatic crystals or white powder at room temperature. It has high biocompatibility, is highly soluble in water, and is hygroscopic. It usually exists in the form of crystalline hydrates. Based on the cell compatibility of betaine and its good water absorption, betaine is an important buffer substance in the body and can be used as an osmotic pressure regulator to prevent sudden changes in osmotic pressure in cells, prevent water loss and salt invasion. When the body's cells are under high salt, low water, and extreme temperature conditions, betaine can alleviate and eliminate a series of adverse effects such as decreased DNA replication, protein synthesis, and cell proliferation rate, and help enhance the expression of transporter genes under hypertonic conditions; and the increase in carriers can promote the transport of betaine into cells while transferring out inorganic salts without damaging the function and structure of the cells.

[0005] However, in the late stage of L-lysine fermentation, the tolerance of the bacteria to high osmotic pressure is still not ideal, and the acid production performance in the late stage of fermentation needs to be further improved, which cannot effectively improve the single-tank yield and sugar-acid conversion rate of L-lysine. At the same time, the addition of osmotic pressure regulators will not only affect the fermentation stability of L-lysine, but also the addition process is difficult to control. Excessive osmotic pressure regulators will inhibit bacterial metabolism, prolong the fermentation production cycle, and reduce fermentation efficiency. Summary of the invention

[0006] In order to solve the technical problems existing in the prior art, the present invention provides a method for increasing the fermentation yield of L-lysine, which can effectively improve the tolerance of bacteria to high osmotic pressure, improve the acid production performance in the late fermentation period, and improve the single-tank yield and sugar-acid conversion rate of L-lysine; at the same time, the adverse effect of an osmotic pressure regulator on the fermentation stability of L-lysine is further reduced, and the problem of excessive addition of an osmotic pressure regulator inhibiting bacteria metabolism, prolonging the fermentation production cycle, and reducing the fermentation efficiency is effectively avoided.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0008] A method for improving the fermentation yield of L-lysine comprises the following steps: preparing a shaking flask culture solution, preparing a seed solution, and fermenting and culturing.

[0009] The method for preparing the shaking flask culture solution is to use Corynebacterium glutamicum Corynebacterium glutamicum Prepare shake flask culture medium;

[0010] The method for preparing the seed solution comprises inoculating the shake flask culture solution into a primary seed culture medium, performing primary seed culture until the absorbance OD value at a wavelength of 562 nm is 11.8-12.3, transplanting the culture solution into a secondary seed culture medium, performing secondary seed culture until the absorbance OD value at a wavelength of 562 nm is 14.8-15.3, and preparing the seed solution;

[0011] The fermentation culture method comprises the following steps: inoculating the seed liquid into the fermentation medium, performing fermentation culture, and starting to flow-add the glucose solution when the residual sugar content in the fermentation liquid reaches 1-3 g / L, and maintaining the residual sugar content in the fermentation liquid within the range of 3-5 g / L; and simultaneously starting to flow-add the osmotic pressure protective agent and the osmotic pressure adaptor, and controlling the volume ratio of the glucose solution, the osmotic pressure protective agent, and the osmotic pressure adaptor added per unit time to be 10:1-1.2:0.2-0.25;

[0012] The osmotic pressure protective agent is a deionized water solution of ectoine and betaine;

[0013] The osmotic pressure adaptor is a deionized aqueous solution of melatonin.

[0014] Furthermore, the method for preparing the shake flask culture fluid is to take the Corynebacterium glutamicum cells on the plate and inoculate them into the shake flask culture medium, and culture them in an incubator with a culture temperature of 32-35° C. and a shaking frequency of 180-200 rpm for 12-15 hours to obtain the shake flask culture fluid.

[0015] Preferably, in the preparation of the shake flask culture medium, the specific components and contents of the shake flask culture medium are: 10 g / L tryptone, 5 g / L yeast powder, 10 g / L NaCl, the solvent is deionized water, and the pH is 7.0.

[0016] Preferably, in the preparation of the seed solution, the inoculation amount of the shake flask culture solution inoculated into the primary seed culture medium is 0.8-1%;

[0017] The inoculation rate of the primary seed culture medium after transplanting to the secondary seed culture medium is 1.8-2%;

[0018] The culture temperature for primary seed culture is 32-35°C and the shaking frequency is 180-200 rpm;

[0019] The culture temperature of the secondary seed culture is 32-35°C, and the shaking frequency is 180-200 rpm.

[0020] Preferably, in the preparation of the seed solution, the specific components and contents of the primary seed culture medium are: glucose 40 g / L, KH2PO4 1.0 g / L, MgSO4 1.2 g / L, corn steep liquor 15 g / L, ammonium sulfate 10 g / L, MnSO4 0.0015 g / L, FeSO4 0.002 g / L, and the solvent is deionized water;

[0021] The specific components and contents of the secondary seed culture medium are: glucose 40g / L, KH2PO41.0g / L, MgSO41.2g / L, corn steep liquor 15g / L, ammonium sulfate 10g / L, MnSO40.0015g / L, FeSO40.002g / L, and the solvent is deionized water.

[0022] Preferably, in the fermentation culture, the inoculation amount of the seed liquid into the fermentation medium is 15.8-16%;

[0023] During the fermentation culture process, the fermentation temperature is controlled at 37.5-38.5°C, the rotation speed is 400-700rpm, and the fermentation pressure is 0.10-0.12Mpa; and the ventilation volume is controlled at 0.3-0.5vvm to maintain the dissolved oxygen volume concentration in the fermentation liquid at 15-20%; liquid ammonia is used to control the pH in the range of 6.7-6.9.

[0024] Preferably, in the fermentation culture, the concentration of the glucose solution is 650-700 g / L;

[0025] The concentration of the osmotic pressure protectant is 3-6 g / L, and the weight ratio of ectoine to betaine in the osmotic pressure protectant is 1.5-1.7:4;

[0026] The concentration of the osmotic pressure adaptor is 14-14.5 mg / L.

[0027] Furthermore, during the fermentation culture, when the ammonia nitrogen content in the fermentation broth reaches 1.0-1.5 g / L, an ammonium sulfate solution with a concentration of 45-50 g / L is added to maintain the ammonia nitrogen content in the fermentation broth within the range of 1.0-1.5 g / L.

[0028] Furthermore, during the fermentation culture, when the volume of the fermented product reaches 75% of the volume of the fermenter, the material is discharged, and the discharge volume is 5% of the volume of the fermentation liquid in the fermenter, and then the fermentation is continued; the total fermentation culture time is 35-36 hours.

[0029] Preferably, in the fermentation culture, the specific components and contents of the fermentation medium are: glucose 45 g / L, H3PO4 1.5 g / L, KCl 0.8 g / L, MgSO4 0.3 g / L, corn steep liquor 100 g / L, MnSO4 0.0015 g / L, FeSO4 0.003 g / L, VB1 60 μg / L, biotin 200 μg / L, and the solvent is deionized water.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The method for increasing the fermentation yield of L-lysine of the present invention comprises the following steps: in the step of preparing the seed solution, the first-stage seed culture is carried out until the absorbance OD value at a wavelength of 562 nm is 11.8-12.3, and then the second-stage seed culture is carried out until the absorbance OD value at a wavelength of 562 nm is 14.8-15.3, so as to obtain the seed solution; then the seed solution is used for fermentation culture, and when the residual sugar content in the fermentation liquid reaches 1-3 g / L, a glucose solution and an osmotic pressure protectant and an osmotic pressure adaptant of a specific concentration are simultaneously added, the activity of the bacteria in the middle and late stages is maintained by tetrahydropyrimidine and betaine in the osmotic pressure protectant, and the osmotic pressure adaptant melatonin is used to further improve the resistance and adaptability of the bacteria under the stress of a high osmotic pressure environment, and reduce the adverse effects of the osmotic pressure protectant on the bacteria, and simultaneously improve the adaptability of the bacteria to the osmotic pressure protectant, thereby improving the fermentation. The invention can improve the stability and further improve the activity of the bacteria in the middle and late stages, so that the sugar consumption in the late stage of fermentation is significantly increased, which is beneficial to the accumulation of the fermentation product (L-lysine) of Corynebacterium glutamicum, improve the fermentation intensity in the middle and late stages, thereby increasing the fermentation yield and sugar-acid conversion rate of L-lysine, effectively improving the single-tank acid production and sugar-acid conversion rate of L-lysine fermentation production, improving production stability and production efficiency, shortening the fermentation cycle, and reducing production costs; the aforementioned technical means cooperate and work synergistically with each other, which can effectively improve the tolerance of the bacteria to high osmotic pressure, improve the acid production performance in the late stage of fermentation, and increase the single-tank output and sugar-acid conversion rate of L-lysine; at the same time, further reduce the adverse effects of existing osmotic pressure regulators on the fermentation stability of L-lysine, and effectively avoid the problems of inhibiting bacterial metabolism, extending the fermentation production cycle, and reducing fermentation efficiency caused by excessive addition of osmotic pressure regulators.

[0032] (2) The method for increasing the L-lysine fermentation yield of the present invention can achieve a fermentation acid concentration of 215 g / L, a sugar-acid conversion rate of 71.1%, and an L-lysine fermentation yield of 1965 g. It can reduce the adverse effects of the high osmotic pressure environment on the bacteria in the middle and late stages of fermentation, improve the acid production performance in the late stage of fermentation, and increase the single-tank yield and sugar-acid conversion rate of L-lysine.

[0033] (3) The method for increasing the yield of L-lysine by fermentation of the present invention has a simple operation method and is conducive to large-scale industrial production. DETAILED DESCRIPTION

[0034] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, the specific implementation methods of the present invention are now described.

[0035] The fermentation strain used in each embodiment of the present invention is the applicant's own strain M7, which is Corynebacterium glutamicum Corynebacterium glutamicum, deposit number: CGMCC No.8184, deposit time: September 13, 2013, deposit unit: General Microbiology Center of China Microbiological Culture Collection Administration; this strain has previously applied for a Chinese patent (CN103992964B).

[0036] Example 1

[0037] This embodiment provides a method for increasing the fermentation yield of L-lysine, specifically:

[0038] 1. Preparation of shake flask culture medium

[0039] The Corynebacterium glutamicum cells on the plate were inoculated into 100 ml of a shaking flask medium, and cultured in an incubator at a culture temperature of 35° C. and a shaking frequency of 200 rpm for 14 hours to obtain a shaking flask culture solution.

[0040] The specific composition and content of the shake flask culture medium are: tryptone 10g / L, yeast powder 5g / L, NaCl 10g / L, the solvent is deionized water, the pH is 7.0, and it is sterilized at 121°C for 20min.

[0041] 2. Prepare seed solution

[0042] The shake flask culture fluid was inoculated into the primary seed culture medium at an inoculation rate of 1%, and the primary seed culture was carried out in an incubator at a temperature of 35°C and a shaking frequency of 200 rpm until the absorbance OD value at a wavelength of 562 nm reached 12. Then, the culture fluid was transferred into the secondary seed culture medium at an inoculation rate of 2%, and the secondary seed culture was carried out in an incubator at a temperature of 35°C and a shaking frequency of 200 rpm until the absorbance OD value at a wavelength of 562 nm reached 15, thereby obtaining the seed solution.

[0043] Among them, the specific components and contents of the primary seed culture medium are: glucose 40g / L, KH2PO41.0g / L, MgSO41.2g / L, corn steep liquor 15g / L, ammonium sulfate 10g / L, MnSO40.0015g / L, FeSO40.002g / L, the solvent is deionized water, sterilized at 121°C for 20min.

[0044] The secondary seed culture medium is the same as the primary seed culture medium.

[0045] 3. Fermentation culture

[0046] The volume of the fermentation medium in the fermentation tank with a volume of 10L is 5L. The seed liquid is introduced into the fermentation tank at an inoculation rate of 16%. The fermentation temperature is controlled to be 38°C, the rotation speed is 500rpm, and the fermentation pressure is 0.11Mpa. The ventilation volume is controlled to be 0.4vvm to maintain the volume concentration of dissolved oxygen in the fermentation liquid at 19%. Liquid ammonia is used to control the pH in the range of 6.7-6.9, and the fermentation is carried out, and the following treatments are performed:

[0047] (1) When the residual sugar content in the fermentation broth reaches 2 g / L, start adding a glucose solution with a concentration of 670 g / L to keep the residual sugar content in the fermentation broth within the range of 3-5 g / L; at the same time as starting to add the glucose solution, start adding an osmotic pressure protectant with a concentration of 4 g / L and an osmotic pressure adaptor with a concentration of 14.2 mg / L, and control the volume ratio of the glucose solution, osmotic pressure protectant, and osmotic pressure adaptor added per unit time to be 10:1.1:0.22.

[0048] The osmotic pressure protective agent is a deionized water solution of ectoine and betaine; in the osmotic pressure protective agent, the weight ratio of ectoine to betaine is 1.7:4.

[0049] The osmotic pressure adaptor is a deionized water solution of melatonin.

[0050] (2) When the ammonia nitrogen content in the fermentation liquid reaches 1.2 g / L, start adding 48 g / L ammonium sulfate solution to maintain the ammonia nitrogen content in the fermentation liquid within the range of 1.0-1.5 g / L;

[0051] During the fermentation process, when the volume of the fermented material reached 75% of the volume of the fermenter, the material was discharged, and the discharge volume was 5% of the volume of the fermentation liquid in the fermenter, and then the fermentation was continued; the total fermentation time was 36 hours. After the fermentation was completed, the fermentation acid concentration and L-lysine production were detected, and the sugar-acid conversion rate was calculated.

[0052] In the fermentation culture, the specific components and contents of the fermentation medium used are: glucose 45g / L, H3PO41.5g / L, KCl 0.8g / L, MgSO40.3g / L, corn steep liquor 100g / L, MnSO40.0015g / L, FeSO40.003g / L, VB160μg / L, biotin 200μg / L, the solvent is deionized water, and sterilized at 121°C for 20min.

[0053] In the method for increasing the fermentation yield of L-lysine in this embodiment, the fermentation acid concentration is 215 g / L, the sugar-acid conversion rate is 71.1%, and the L-lysine fermentation yield is 1965 g.

[0054] Example 2

[0055] This embodiment provides a method for increasing the fermentation yield of L-lysine, specifically:

[0056] 1. Preparation of shake flask culture medium

[0057] The Corynebacterium glutamicum cells on the plate were inoculated into 100 ml of a shaking flask culture medium, and cultured in an incubator at a culture temperature of 32° C. and a shaking frequency of 180 rpm for 12 hours to obtain a shaking flask culture solution.

[0058] The specific composition and content of the shake flask culture medium are: tryptone 10g / L, yeast powder 5g / L, NaCl 10g / L, the solvent is deionized water, the pH is 7.0, and it is sterilized at 121°C for 20min.

[0059] 2. Prepare seed solution

[0060] The shake flask culture fluid was inoculated into the primary seed culture medium at an inoculation rate of 0.8%, and the primary seed culture was carried out in an incubator at a temperature of 32° C. and a shaking frequency of 180 rpm until the absorbance OD value at a wavelength of 562 nm reached 11.8. Then, the culture fluid was transferred into the secondary seed culture medium at an inoculation rate of 1.8%, and the secondary seed culture was carried out in an incubator at a temperature of 32° C. and a shaking frequency of 180 rpm until the absorbance OD value at a wavelength of 562 nm reached 14.8, thereby obtaining the seed solution.

[0061] Among them, the specific components and contents of the primary seed culture medium are: glucose 40g / L, KH2PO41.0g / L, MgSO41.2g / L, corn steep liquor 15g / L, ammonium sulfate 10g / L, MnSO40.0015g / L, FeSO40.002g / L, the solvent is deionized water, sterilized at 121°C for 20min.

[0062] The secondary seed culture medium is the same as the primary seed culture medium.

[0063] 3. Fermentation culture

[0064] The volume of the fermentation medium in the fermentation tank with a volume of 10L is 5L. The seed liquid is introduced into the fermentation tank at an inoculation rate of 15.8%. The fermentation temperature is controlled to be 37.5°C, the rotation speed is 400rpm, and the fermentation pressure is 0.10Mpa. The ventilation volume is controlled to be 0.3vvm to maintain the volume concentration of dissolved oxygen in the fermentation liquid at 15%. Liquid ammonia is used to control the pH in the range of 6.7-6.9, and the fermentation is carried out, and the following treatments are performed:

[0065] (1) When the residual sugar content in the fermentation broth reaches 1 g / L, start adding a glucose solution with a concentration of 650 g / L to keep the residual sugar content in the fermentation broth within the range of 3-5 g / L; at the same time as starting to add the glucose solution, start adding an osmotic pressure protectant with a concentration of 3.5 g / L and an osmotic pressure adaptor with a concentration of 14 mg / L, and control the volume ratio of the glucose solution, osmotic pressure protectant, and osmotic pressure adaptor added per unit time to be 10:1:0.2.

[0066] The osmotic pressure protective agent is a deionized water solution of ectoine and betaine; in the osmotic pressure protective agent, the weight ratio of ectoine to betaine is 1.5:4.

[0067] The osmotic pressure adaptor is a deionized water solution of melatonin.

[0068] (2) When the ammonia nitrogen content in the fermentation liquid reaches 1.0 g / L, start adding 45 g / L ammonium sulfate solution to maintain the ammonia nitrogen content in the fermentation liquid within the range of 1.0-1.5 g / L;

[0069] During the fermentation process, when the volume of the fermented material reached 75% of the volume of the fermenter, the material was discharged, and the discharge volume was 5% of the volume of the fermentation liquid in the fermenter, and then the fermentation was continued; the total fermentation time was 36 hours. After the fermentation was completed, the fermentation acid concentration and L-lysine production were detected, and the sugar-acid conversion rate was calculated.

[0070] In the fermentation culture, the specific components and contents of the fermentation medium used are: glucose 45g / L, H3PO41.5g / L, KCl 0.8g / L, MgSO40.3g / L, corn steep liquor 100g / L, MnSO40.0015g / L, FeSO40.003g / L, VB160μg / L, biotin 200μg / L, the solvent is deionized water, and sterilized at 121°C for 20min.

[0071] In the method for increasing the fermentation yield of L-lysine in this embodiment, the fermentation acid concentration is 212 g / L, the sugar-acid conversion rate is 70.8%, and the L-lysine fermentation yield is 1925 g.

[0072] Example 3

[0073] This embodiment provides a method for increasing the fermentation yield of L-lysine, specifically:

[0074] 1. Preparation of shake flask culture medium

[0075] The Corynebacterium glutamicum cells on the plate were inoculated into 100 ml of a shaking flask culture medium, and cultured in an incubator at a culture temperature of 34° C. and a shaking frequency of 190 rpm for 15 hours to obtain a shaking flask culture solution.

[0076] The specific composition and content of the shake flask culture medium are: tryptone 10g / L, yeast powder 5g / L, NaCl 10g / L, the solvent is deionized water, the pH is 7.0, and it is sterilized at 121°C for 20min.

[0077] 2. Prepare seed solution

[0078] The shake flask culture fluid was inoculated into the primary seed culture medium at an inoculation rate of 0.9%, and the primary seed culture was carried out in an incubator at a culture temperature of 34°C and a shaking frequency of 190 rpm until the absorbance OD value at a wavelength of 562 nm reached 12.3. Then, the culture fluid was transferred into the secondary seed culture medium at an inoculation rate of 1.9%, and the secondary seed culture was carried out in an incubator at a culture temperature of 34°C and a shaking frequency of 190 rpm until the absorbance OD value at a wavelength of 562 nm reached 15.3, thereby obtaining the seed solution.

[0079] Among them, the specific components and contents of the primary seed culture medium are: glucose 40g / L, KH2PO41.0g / L, MgSO41.2g / L, corn steep liquor 15g / L, ammonium sulfate 10g / L, MnSO40.0015g / L, FeSO40.002g / L, the solvent is deionized water, sterilized at 121°C for 20min.

[0080] The secondary seed culture medium is the same as the primary seed culture medium.

[0081] 3. Fermentation culture

[0082] The volume of the fermentation medium in the fermentation tank with a volume of 10L is 5L. The seed liquid is introduced into the fermentation tank at an inoculation rate of 15.9%. The fermentation temperature is controlled to be 38.5°C, the rotation speed is 700rpm, and the fermentation pressure is 0.12Mpa. The ventilation volume is controlled to be 0.5vvm to maintain the volume concentration of dissolved oxygen in the fermentation liquid at 20%. Liquid ammonia is used to control the pH in the range of 6.7-6.9, and the fermentation is carried out, and the following treatments are performed:

[0083] (1) When the residual sugar content in the fermentation broth reaches 3 g / L, start adding a glucose solution with a concentration of 700 g / L to keep the residual sugar content in the fermentation broth within the range of 3-5 g / L; at the same time as starting to add the glucose solution, start adding an osmotic pressure protectant with a concentration of 5 g / L and an osmotic pressure adaptor with a concentration of 14.5 mg / L, and control the volume ratio of the glucose solution, osmotic pressure protectant, and osmotic pressure adaptor added per unit time to be 10:1.2:0.25.

[0084] The osmotic pressure protective agent is a deionized water solution of ectoine and betaine; in the osmotic pressure protective agent, the weight ratio of ectoine to betaine is 1.6:4.

[0085] The osmotic pressure adaptor is a deionized water solution of melatonin.

[0086] (2) When the ammonia nitrogen content in the fermentation liquid reaches 1.5 g / L, start adding 50 g / L ammonium sulfate solution to maintain the ammonia nitrogen content in the fermentation liquid within the range of 1.0-1.5 g / L;

[0087] During the fermentation process, when the volume of the fermented material reached 75% of the volume of the fermenter, the material was discharged, and the discharge volume was 5% of the volume of the fermentation liquid in the fermenter, and then the fermentation was continued; the total fermentation time was 36 hours. After the fermentation was completed, the fermentation acid concentration and L-lysine production were detected, and the sugar-acid conversion rate was calculated.

[0088] In the fermentation culture, the specific components and contents of the fermentation medium used are: glucose 45g / L, H3PO41.5g / L, KCl 0.8g / L, MgSO40.3g / L, corn steep liquor 100g / L, MnSO40.0015g / L, FeSO40.003g / L, VB160μg / L, biotin 200μg / L, the solvent is deionized water, and sterilized at 121°C for 20min.

[0089] In the method for increasing the fermentation yield of L-lysine in this embodiment, the fermentation acid concentration is 214 g / L, the sugar-acid conversion rate is 71.0%, and the L-lysine fermentation yield is 1952 g.

[0090] Comparative Example 1

[0091] The technical solution of Example 1 is adopted, but the differences are as follows: 1) in the fermentation culture step, ectoine is omitted from the osmotic pressure protectant, and only betaine is used; 2) in the fermentation culture step, no osmotic pressure adaptor is added.

[0092] In the method for increasing the fermentation yield of L-lysine in Comparative Example 1, the fermentation acid concentration was 203 g / L, the sugar-acid conversion rate was 68.0%, and the L-lysine fermentation yield was 1805 g.

[0093] Comparative Example 2

[0094] The technical solution of Example 1 is adopted, but the difference is that in the fermentation culture step, no osmotic pressure protectant and osmotic pressure adaptor are added.

[0095] In the method for increasing the fermentation yield of L-lysine of Comparative Example 2, the fermentation acid concentration was 189 g / L, the sugar-acid conversion rate was 65.4%, and the L-lysine fermentation yield was 1618 g.

[0096] It can be seen from Examples 1-3 that the method for increasing the fermentation yield of L-lysine of the present invention comprises the following steps: in the step of preparing the seed solution, the first-level seed culture is carried out until the absorbance OD value at a wavelength of 562 nm is 11.8-12.3, and then the second-level seed culture is carried out until the absorbance OD value at a wavelength of 562 nm is 14.8-15.3, so as to obtain the seed solution; then the seed solution is used for fermentation culture, and when the residual sugar content in the fermentation liquid reaches 1-3 g / L, the glucose solution and the osmotic pressure protective agent and the osmotic pressure adaptor of a specific concentration are simultaneously added, the activity of the bacteria in the middle and late stages is maintained by the tetrahydropyrimidine and betaine in the osmotic pressure protective agent, and the osmotic pressure adaptor melatonin is used to further improve the resistance and adaptability of the bacteria under the stress of the high osmotic pressure environment, and reduce the adverse effects of the osmotic pressure protective agent on the bacteria, and simultaneously improve the adaptability of the bacteria to the osmotic pressure protective agent. , improve fermentation stability, and further improve the activity of the bacteria in the middle and late stages, so that the sugar consumption in the late fermentation stage is significantly increased, which is beneficial to the accumulation of Corynebacterium glutamicum fermentation products (L-lysine), and improve the fermentation intensity in the middle and late stages, thereby increasing the L-lysine fermentation yield and sugar-acid conversion rate, effectively increasing the single-tank acid production and sugar-acid conversion rate of L-lysine fermentation production, improving production stability and production efficiency, shortening the fermentation cycle, and reducing production costs; the aforementioned technical means cooperate and work synergistically with each other, which can effectively improve the tolerance of the bacteria to high osmotic pressure, improve the acid production performance in the late fermentation stage, and increase the single-tank output and sugar-acid conversion rate of L-lysine; at the same time, further reduce the adverse effects of existing osmotic pressure regulators on the fermentation stability of L-lysine, and effectively avoid the problems of inhibiting bacterial metabolism, extending the fermentation production cycle, and reducing fermentation efficiency caused by excessive addition of osmotic pressure regulators.

[0097] It can be seen from Comparative Example 1 that in the fermentation culture step, ectoine in the osmotic pressure protective agent and the osmotic pressure adaptor are omitted, and the activity of the bacteria in the middle and late stages cannot be maintained synergistically by ectoine and betaine, and the resistance and adaptability of the bacteria under the stress of the high osmotic pressure environment cannot be further improved by the osmotic pressure protective agent melatonin. In the high osmotic pressure environment, the activity of the bacteria in the middle and late stages of fermentation decreases to a certain extent, and the fermentation intensity in the middle and late stages decreases, which is specifically manifested in that the fermentation acid concentration, the sugar-acid conversion rate and the L-lysine fermentation yield all decrease to a certain extent.

[0098] It can be seen from Comparative Example 2 that in the fermentation culture step, no osmotic pressure protective agent and osmotic pressure adaptor are added. In the later stage of fermentation, with the increase of L-lysine concentration, the osmotic pressure continues to increase, and the bacteria cannot tolerate the excessively high osmotic pressure, resulting in a significant decrease in the activity of the bacteria in the middle and late stages, and a decrease in the fermentation intensity and efficiency in the middle and late stages, which is specifically manifested in a significant decrease in the fermentation acid concentration, sugar-acid conversion rate and L-lysine fermentation yield.

[0099] Unless otherwise specified, all percentages used in the present invention are by mass.

[0100] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for increasing L-lysine fermentation yield, characterized in that: The method comprises the following steps: preparing a shake flask culture solution, preparing a seed solution, and fermenting and culturing; The method for preparing the shaking flask culture solution is to use Corynebacterium glutamicum Corynebacterium glutamicum Prepare shake flask culture medium; Corynebacterium glutamicum Corynebacterium glutamicum , the deposit number is CGMCC No.8184; The method for preparing the seed solution comprises inoculating the shake flask culture solution into a primary seed culture medium, performing primary seed culture until the absorbance OD value at a wavelength of 562 nm is 11.8-12.3, transplanting the culture solution into a secondary seed culture medium, performing secondary seed culture until the absorbance OD value at a wavelength of 562 nm is 14.8-15.3, and preparing the seed solution; The specific components and contents of the primary seed culture medium are: glucose 40 g / L, KH2PO4 1.0 g / L, MgSO4 1.2 g / L, corn steep liquor 15 g / L, ammonium sulfate 10 g / L, MnSO4 0.0015 g / L, FeSO4 0.002 g / L, and the solvent is deionized water; The specific components and contents of the secondary seed culture medium are: glucose 40 g / L, KH2PO4 1.0 g / L, MgSO4 1.2 g / L, corn steep liquor 15 g / L, ammonium sulfate 10 g / L, MnSO4 0.0015 g / L, FeSO4 0.002 g / L, and the solvent is deionized water; The fermentation culture method comprises the following steps: inoculating the seed liquid into the fermentation medium, performing fermentation culture, and starting to flow-add the glucose solution when the residual sugar content in the fermentation liquid reaches 1-3 g / L, and maintaining the residual sugar content in the fermentation liquid within the range of 3-5 g / L; and simultaneously starting to flow-add the osmotic pressure protective agent and the osmotic pressure adaptor, and controlling the volume ratio of the glucose solution, the osmotic pressure protective agent, and the osmotic pressure adaptor added per unit time to be 10:1-1.2:0.2-0.25; The specific components and contents of the fermentation medium are: glucose 45 g / L, H3PO4 1.5 g / L, KCl 0.8 g / L, MgSO4 0.3 g / L, corn steep liquor 100 g / L, MnSO4 0.0015 g / L, FeSO4 0.003 g / L, VB1 60 μg / L, biotin 200 μg / L, and the solvent is deionized water; The osmotic pressure protective agent is a deionized water solution of ectoine and betaine; the concentration of the osmotic pressure protective agent is 3-6 g / L, and the weight ratio of ectoine to betaine in the osmotic pressure protective agent is 1.5-1.7:4; The osmotic pressure adaptor is a deionized water solution of melatonin; the concentration of the osmotic pressure adaptor is 14-14.5 mg / L.

2. The method for increasing L-lysine fermentation yield according to claim 1, characterized in that: The method for preparing the shake flask culture liquid is to take the Corynebacterium glutamicum cells on the plate and inoculate them into the shake flask culture medium, and culture them for 12-15 hours in an incubator with a culture temperature of 32-35° C. and a shaking frequency of 180-200 rpm to obtain the shake flask culture liquid.

3. The method for increasing L-lysine fermentation yield according to claim 2, characterized in that: In the preparation of the shake flask culture medium, the specific components and contents of the shake flask culture medium are: 10 g / L tryptone, 5 g / L yeast powder, 10 g / L NaCl, the solvent is deionized water, and the pH is 7.

0.

4. The method for increasing L-lysine fermentation yield according to claim 1, characterized in that: In the preparation of the seed solution, the inoculation amount of the shake flask culture solution inoculated into the primary seed culture medium is 0.8-1%; The inoculation rate of the primary seed culture medium after transplanting to the secondary seed culture medium is 1.8-2%; The culture temperature for primary seed culture is 32-35°C and the shaking frequency is 180-200 rpm; The culture temperature of the secondary seed culture is 32-35°C, and the shaking frequency is 180-200 rpm.

5. The method for increasing L-lysine fermentation yield according to claim 1, characterized in that: In the fermentation culture, the inoculation amount of the seed liquid into the fermentation medium is 15.8-16%; During the fermentation culture process, the fermentation temperature is controlled at 37.5-38.5°C, the rotation speed is 400-700rpm, and the fermentation pressure is 0.10-0.12Mpa; and the ventilation volume is controlled at 0.3-0.5vvm to maintain the dissolved oxygen volume concentration in the fermentation liquid at 15-20%; liquid ammonia is used to control the pH in the range of 6.7-6.

9.

6. The method for increasing L-lysine fermentation yield according to claim 1, characterized in that: In the fermentation culture, the concentration of the glucose solution is 650-700 g / L.

7. The method for increasing L-lysine fermentation yield according to claim 1, characterized in that: During the fermentation culture, when the ammonia nitrogen content in the fermentation liquid reaches 1.0-1.5 g / L, an ammonium sulfate solution with a concentration of 45-50 g / L is added to maintain the ammonia nitrogen content in the fermentation liquid within the range of 1.0-1.5 g / L.

8. The method for increasing L-lysine fermentation yield according to claim 1, characterized in that: During the fermentation culture, when the volume of the fermented product reaches 75% of the volume of the fermenter, discharge begins, and the discharge volume is 5% of the volume of the fermentation liquid in the fermenter, and then fermentation continues; the total fermentation culture time is 35-36 hours.