Method for producing L-lactic acid through efficient fermentation
By using core-shell structured neutralizing particles to control the pH value during fermentation, the problems of bacterial growth inhibition and enzyme activity reduction caused by sodium salt neutralization were solved, achieving efficient production and high-purity output of L-lactic acid.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HENAN JINDAN LACTIC ACID TECH CO LTD
- Filing Date
- 2025-12-05
- Publication Date
- 2026-04-14
AI Technical Summary
In the existing sodium salt neutralization method for the efficient fermentation production of L-lactic acid, excessively high local sodium salt concentrations can lead to osmotic pressure affecting microbial growth, inhibiting enzyme activity, resulting in uneven mixing of fermentation liquid, limited mass transfer, and microbial death.
The neutralizing agent is prepared by using core-shell structured neutralizing particles, with carboxymethyl chitosan as the shell and betaine and sodium hydroxide as the core. The pH value of the fermentation liquid is controlled at 5.1±0.1. The neutralizing liquid is fed with core-shell structured neutralizing particles to achieve uniform dispersion and slow-release neutralization. Betaine provides osmotic protection.
This effectively improved the yield and purity of L-lactic acid, ensured the stability of the strain and the activity of the enzyme, and achieved efficient and green production of L-lactic acid.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of lactic acid production technology. Specifically, it relates to a method for the efficient fermentation production of L-lactic acid. Background Technology
[0002] L-lactic acid is an important organic acid that is widely used in food, medicine, chemical industry, new materials and other fields.
[0003] Biological fermentation is the most commonly used method for L-lactic acid production. Microorganisms require different optimal pH values at different growth stages (such as cell growth and product synthesis), so it is necessary to dynamically control the pH value of the fermentation broth.
[0004] Traditional pH neutralizing agents are calcium carbonate and calcium hydroxide, which can convert lactic acid into calcium lactate, facilitating subsequent separation. However, this generates a large amount of calcium sulfate waste, polluting the environment and incurring high treatment costs. Sodium salt neutralization (NaOH) is an emerging method that does not produce solid waste and is environmentally friendly. However, its rapid reaction rate means that during the feeding process, the sodium salt neutralizing agent often has not had time to diffuse before the neutralization reaction occurs, leading to the large-scale generation and accumulation of sodium lactate at the feeding site. This is especially problematic in large-volume single-tank fermentations, where uneven mixing of the fermentation liquid and limited mass transfer result in excessively high local osmotic pressure, inhibiting microbial growth and reproduction, and even causing rupture and death. Furthermore, the high local concentration of sodium ions during neutralization can inhibit enzyme activity, disrupt the balance of microbial metabolic pathways, and ultimately affect the overall growth and acid production capacity of the microbial strain. Summary of the Invention
[0005] Therefore, this invention provides a method for efficient fermentation production of L-lactic acid, aiming to solve the technical problems in the existing sodium salt neutralization method for efficient fermentation production of L-lactic acid, such as the osmotic pressure affecting the growth of strains and inhibiting enzyme activity due to excessively high local sodium salt concentration, so as to improve the fermentation quality and product content of L-lactic acid.
[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution:
[0007] A method for efficiently producing L-lactic acid by fermentation includes the following steps:
[0008] S1. The biomass starch is liquefied and pre-saccharified to obtain a pre-saccharified solution;
[0009] S2. Inoculate the fermentation strain into the pre-saccharified solution for fermentation. When the pH of the fermentation solution drops to 5.1±0.1, add a neutralizing solution containing core-shell structure neutralizing particles to maintain the pH of the fermentation solution at 5.1±0.1 and continue fermentation.
[0010] S3. After fermentation, the lactic acid fermentation broth is subjected to ultrafiltration, nanofiltration and electrodialysis for dissociation, and then concentrated to obtain the finished lactic acid product.
[0011] Among them, the core-shell structured neutralizing particles are prepared using carboxymethyl chitosan as the shell and betaine and sodium hydroxide as the core.
[0012] Furthermore, the preparation process of core-shell structured neutralizing particles is as follows:
[0013] A1. Add carboxymethyl chitosan to deionized water, heat and stir to obtain a chitosan solution;
[0014] A2. Add betaine to the chitosan solution, mix well, heat and concentrate, then cool to room temperature to obtain a mixed slurry;
[0015] A3. Under stirring conditions, sodium hydroxide is added to the mixed slurry, dried, and then ground into powder to obtain core-shell structured neutral particles.
[0016] Furthermore, in the carboxymethyl chitosan solution, the mass fraction of carboxymethyl chitosan is 1.8-2.2%.
[0017] Furthermore, the amount of betaine added is 4-6% of the mass of carboxymethyl chitosan, and the mass fraction of sodium hydroxide in the core-shell structure neutralizing particles is 15-25%.
[0018] Furthermore, in the neutralization solution, the mass fraction of core-shell structured neutralized particles is 25-35%. Furthermore, the raw material for biomass starch is one or a mixture of two or more of rice, corn, wheat, potatoes, and sweet potatoes.
[0019] Furthermore, the fermentation strain is Bacillus coagulans.
[0020] Furthermore, during the liquefaction process, the biomass starch is prepared into starch milk, amylase is added, and spray liquefaction is performed. The spray temperature is controlled at 108-110℃ and maintained for 4-5 minutes. Then, the temperature is reduced to 90-100℃, the pH value is adjusted to 5.0-5.3, and maintained for 20-50 minutes to obtain liquefied sugar solution.
[0021] Furthermore, during the pre-saccharification process, the temperature is 60-62℃, the pH value is 4.2-4.5, the amount of saccharifying enzyme added is 0.35-0.40 mg / g of the total sugar mass in the liquefied sugar solution, and the saccharification time is 3-5 h.
[0022] Furthermore, after inoculation in the fermenter, the fermentation temperature is controlled at 50-52℃ and the aeration rate is 0.1-0.12 vvm.
[0023] The technical solution of the present invention achieves the following beneficial technical effects:
[0024] 1. This invention utilizes a slightly acidic fermentation environment to ensure the activity of saccharifying enzymes, enabling simultaneous lactic acid fermentation and saccharification. Simultaneously, it inhibits the growth of unwanted microorganisms and the production of unwanted acids, effectively guaranteeing the optical purity of lactic acid. Furthermore, by using a sodium salt neutralizing agent with a specific structure and controlling fermentation process parameters, it solves problems such as excessively high local sodium salt concentrations during neutralization, which lead to inhibited microbial growth and reduced enzyme activity. This effectively improves the yield and purity of L-lactic acid, achieving efficient and green production of L-lactic acid.
[0025] 2. Chitosan itself is a membrane-like material that acts as a slow-release neutralizer. Its carboxymethyl group has excellent hydrophilicity, enabling it to carry sodium salt neutralizer (NaOH) and betaine, achieving uniform dispersion and effective slow release in the acidic fermentation broth. This effectively prevents the accumulation of sodium salt neutralizer at the feeding site. Furthermore, betaine has strong osmotic protection capabilities, accumulating around the microbial cells while the neutralizer is active, forming an "invisible protective shield." This avoids and reduces the impact of the high sodium salt osmotic pressure after the neutralization reaction on the microbial strain, effectively maintaining the stability of the cell structure and function. Betaine also has good compatibility and stability, maintaining good activity in the acidic fermentation broth. Ultimately, carboxymethyl chitosan, betaine, and sodium salt neutralizer work synergistically to maintain stable pH control of the fermentation broth and efficient fermentation, significantly improving the fermentation quality and product content of L-lactic acid. Detailed Implementation
[0026] Example 1
[0027] A method for efficiently producing L-lactic acid by fermentation includes the following steps:
[0028] S1. Corn starch was adjusted to a corn starch slurry with a dry matter content of 28% by mass. 0.14 mg / g of heat-resistant α-amylase was added to the corn starch, and the mixture was spray-liquefied. The spray temperature was controlled at 109℃ and maintained at this high temperature for 4 minutes. Then, the temperature was lowered to 95℃, and the pH was adjusted to 5.2 and maintained for 30 minutes to obtain a liquefied sugar solution. Pre-saccharification was then performed at 61℃, with a pH of 4.3. The amount of saccharifying enzyme added was 0.38 mg / g (based on the total sugar content in the liquefied sugar solution), and the saccharification time was 4 hours to obtain a pre-saccharified solution.
[0029] S2. Using Bacillus coagulans as the fermentation strain, the culture medium comprises the following substances: sugar water with a total sugar content of 220 g / L, yeast powder (as a nitrogen source, with a carbon-to-nitrogen ratio of sugar water to yeast powder of 19:1), 2.0 g / L diammonium citrate, 2.0 g / L K₂HPO₄, 0.2 g / L MgSO₄·7H₂O, 0.05 g / L MnSO₄·H₂O, 0.03 g / L NaCl, 0.01 g / L FeSO₄·7H₂O, 5.0 g / L sodium acetate trihydrate, and 1.0 m L / L Tween; The inoculation was carried out in the fermenter using a gradual expansion method, with the inoculation amount being 10% of the pre-fermentation liquid volume in the fermenter. After inoculation, the fermentation temperature was controlled at 51℃, the pressure at 0.02MPa, and the aeration rate at 0.11vvm. Fermentation was carried out at natural pH. When the pH of the fermentation liquid decreased to 5.1±0.1, a neutralization solution containing core-shell structure neutralizing particles was added, with a mass fraction of 30% of the core-shell structure neutralizing particles in the neutralization solution. The pH of the fermentation liquid was maintained at 5.1±0.1, and fermentation continued. When the OD of the fermentation liquid... 620 When the concentration reaches 14 or higher, the liquid in the fermenter can be used as a starter culture and inoculated into a larger fermenter.
[0030] S3. Ferment until the glucose concentration in the fermentation liquid drops to 0, stop aeration, continue fermentation for 2 hours, and the fermentation ends, yielding lactic acid fermentation broth. This broth is then subjected to ultrafiltration, nanofiltration, and electrodialysis for dissociation, and concentrated to obtain the final lactic acid product.
[0031] The preparation process of the core-shell structured neutralizing particles is as follows:
[0032] A1. Add carboxymethyl chitosan to deionized water, heat to 55°C, stir for 5 minutes to obtain a chitosan solution with a mass fraction of 2.0%;
[0033] A2. Add betaine to the chitosan solution, wherein the amount of betaine added is 5% of the mass of carboxymethyl chitosan. After mixing evenly, heat and concentrate, then cool to room temperature to obtain a mixed slurry.
[0034] A3. Under stirring conditions, sodium hydroxide is added to the mixed slurry, dried and ground into powder, and passed through a 60-mesh sieve to obtain core-shell structured neutral particles, wherein the mass fraction of sodium hydroxide in the core-shell structured neutral particles is 20%.
[0035] Example 2
[0036] A method for efficiently producing L-lactic acid by fermentation includes the following steps:
[0037] S1. Corn starch was adjusted to a corn starch slurry with a dry matter content of 28% by mass. 0.14 mg / g of heat-resistant α-amylase was added to the corn starch, and the mixture was spray-liquefied. The spray temperature was controlled at 108℃, and the high temperature was maintained for 5 minutes. Then, the temperature was lowered to 90℃, and the mixture was kept at pH 5.0 for 50 minutes to obtain a liquefied sugar solution. Pre-saccharification was then performed at 60℃, pH 4.2, with 0.35 mg / g of saccharifying enzyme added (based on the total sugar content in the liquefied sugar solution), and the saccharification time was 5 hours to obtain a pre-saccharified solution.
[0038] S2. The fermentation strain is Bacillus coagulans. The initial culture medium includes the following substances: sugar water with a total sugar content of 220 g / L, yeast powder (as a nitrogen source, with a carbon-to-nitrogen ratio of sugar water to yeast powder of 19:1), 2.0 g / L diammonium citrate, 2.0 g / L K₂HPO₄, 0.2 g / L MgSO₄·7H₂O, 0.05 g / L MnSO₄·H₂O, 0.03 g / L NaCl, 0.01 g / L FeSO₄·7H₂O, 5.0 g / L sodium acetate trihydrate, and 1 0.0 mL / L Tween was used; the inoculation was carried out in the fermenter in a stepwise manner, with the inoculation amount being 10% of the pre-fermentation liquid volume in the fermenter. After inoculation, the fermentation temperature was controlled at 50℃, the pressure at 0.02 MPa, and the aeration rate at 0.1 vvm. Fermentation was carried out, and when the pH of the fermentation liquid decreased to 5.1±0.1, a neutralization solution containing core-shell structure neutralizing particles was added, with a mass fraction of 35% of the core-shell structure neutralizing particles in the neutralization solution. The pH of the fermentation liquid was maintained at 5.1±0.1, and fermentation continued. When the OD of the fermentation liquid... 620 When the concentration reaches 14 or higher, the liquid in the fermenter can be used as a starter culture and inoculated into a larger fermenter.
[0039] S3. Ferment until the glucose concentration in the fermentation liquid drops to 0, stop aeration, continue fermentation for 2 hours, and the fermentation ends, yielding lactic acid fermentation broth. This broth is then subjected to ultrafiltration, nanofiltration, and electrodialysis for dissociation, and concentrated to obtain the final lactic acid product.
[0040] The preparation process of the core-shell structured neutralizing particles is as follows:
[0041] A1. Add carboxymethyl chitosan to deionized water, heat to 50°C, stir for 5 minutes to obtain a chitosan solution with a mass fraction of 1.8%;
[0042] A2. Add betaine to the chitosan solution, wherein the amount of betaine added is 4% of the mass of carboxymethyl chitosan. After mixing evenly, heat and concentrate, then cool to room temperature to obtain a mixed slurry.
[0043] A3. Under stirring conditions, sodium hydroxide is added to the mixed slurry, dried and ground into powder, and passed through a 60-mesh sieve to obtain core-shell structured neutral particles, wherein the mass fraction of sodium hydroxide in the core-shell structured neutral particles is 15%.
[0044] Example 3
[0045] A method for efficiently producing L-lactic acid by fermentation includes the following steps:
[0046] S1. Corn starch was adjusted to a corn starch slurry with a dry matter content of 28% by mass. 0.14 mg / g of heat-resistant α-amylase was added to the corn starch, and spray liquefaction was performed. The spray temperature was controlled at 110℃, and the high temperature was maintained for 4 minutes. Then, the temperature was lowered to 100℃, and the solution was maintained at pH 5.3 for 20 minutes to obtain a liquefied sugar solution. Pre-saccharification was then performed at a temperature of 62℃, a pH of 4.5, and an enzyme dosage of 0.40 mg / g (based on the total sugar content in the liquefied sugar solution). The saccharification time was 5 hours to obtain a pre-saccharified solution.
[0047] S2. The fermentation strain is Bacillus coagulans. The initial culture medium includes the following substances: sugar water with a total sugar content of 220 g / L, yeast powder (as a nitrogen source, with a carbon-to-nitrogen ratio of sugar water to yeast powder of 19:1), 2.0 g / L diammonium citrate, 2.0 g / L K2HPO4, 0.2 g / L MgSO4·7H2O, 0.05 g / L MnSO4·H2O, 0.03 g / L NaCl, 0.01 g / L FeSO4·7H2O, 5.0 g / L sodium acetate trihydrate, and 1. 0 mL / L Tween was used; the inoculation was carried out in the fermenter in a stepwise manner, with the inoculation amount being 10% of the pre-fermentation liquid volume in the fermenter. After inoculation, the fermentation temperature was controlled at 52℃, the pressure at 0.02 MPa, and the aeration rate at 0.12 vvm. Fermentation was carried out, and when the pH of the fermentation liquid decreased to 5.1±0.1, a neutralization solution containing core-shell structure neutralizing particles was added, with a mass fraction of 35% of the core-shell structure neutralizing particles in the neutralization solution. The pH of the fermentation liquid was maintained at 5.1±0.1, and fermentation continued. When the OD of the fermentation liquid... 620 When the concentration reaches 14 or higher, the liquid in the fermenter can be used as a starter culture and inoculated into a larger fermenter.
[0048] S3. Ferment until the glucose concentration in the fermentation liquid drops to 0, stop aeration, continue fermentation for 2 hours, and the fermentation ends, yielding lactic acid fermentation broth. This broth is then subjected to ultrafiltration, nanofiltration, and electrodialysis for dissociation, and concentrated to obtain the final lactic acid product.
[0049] The preparation process of the core-shell structured neutralizing particles is as follows:
[0050] A1. Add carboxymethyl chitosan to deionized water, heat to 60°C, stir for 5 minutes to obtain a chitosan solution with a mass fraction of 2.2%;
[0051] A2. Add betaine to the chitosan solution, wherein the amount of betaine added is 6% of the mass of carboxymethyl chitosan. After mixing evenly, heat and concentrate, then cool to room temperature to obtain a mixed slurry.
[0052] A3. Under stirring conditions, sodium hydroxide is added to the mixed slurry, dried and ground into powder, and passed through a 60-mesh sieve to obtain core-shell structured neutral particles, wherein the mass fraction of sodium hydroxide in the core-shell structured neutral particles is 25%.
[0053] Comparative Example 1
[0054] The only difference between this comparative example and Example 1 is that, in step S2 of the fermentation process, core-shell neutralizing particles are not added; instead, sodium hydroxide solution is added directly, as detailed below:
[0055] S2. Using Bacillus coagulans as the fermentation strain, the culture medium comprises the following substances: sugar water with a total sugar content of 220 g / L, yeast powder (as a nitrogen source, with a carbon-to-nitrogen ratio of sugar water to yeast powder of 19:1), 2.0 g / L diammonium citrate, 2.0 g / L K₂HPO₄, 0.2 g / L MgSO₄·7H₂O, 0.05 g / L MnSO₄·H₂O, 0.03 g / L NaCl, 0.01 g / L FeSO₄·7H₂O, and 5.0 g / L... Sodium acetate trihydrate and 1.0 mL / L Tween were inoculated into the fermenter using a gradual expansion method, with the inoculation amount being 10% of the pre-fermentation liquid volume in the fermenter. After inoculation, the fermentation temperature was controlled at 51℃, the pressure at 0.02 MPa, and the aeration rate at 0.11 vvm. Fermentation was carried out at natural pH. When the pH of the fermentation liquid decreased to 5.1 ± 0.1, a 6% sodium hydroxide solution was added to maintain the pH of the fermentation liquid at 5.1 ± 0.1, and fermentation continued. When the OD of the fermentation liquid... 620 When the concentration reaches 14 or higher, it is inoculated into a larger fermentation tank.
[0056] Comparative Example 2
[0057] The only difference between this comparative example and Example 1 is that betaine is not added in the preparation of the core-shell structure and particles, as detailed below:
[0058] A1. Add carboxymethyl chitosan to deionized water, heat to 55°C, stir for 5 minutes to obtain a chitosan solution with a mass fraction of 2.0%;
[0059] A2. Add the chitosan solution to the solution, heat and concentrate, then cool to room temperature to obtain a mixed slurry;
[0060] A3. Under stirring conditions, sodium hydroxide is added to the mixed slurry, dried and ground into powder, and passed through a 60-mesh sieve to obtain core-shell structured neutral particles, wherein the mass fraction of sodium hydroxide in the core-shell structured neutral particles is 20%.
[0061] Comparative Example 3
[0062] The only difference between this comparative example and Example 1 is that carboxymethyl chitosan is not added in the preparation of the core-shell structure and particles, as detailed below:
[0063] A1. Add betaine to deionized water, wherein the amount of betaine added is 5% of the amount of deionized water. After mixing evenly, heat and concentrate, then cool to room temperature to obtain a mixed slurry.
[0064] A2. Add sodium hydroxide under stirring conditions, dry and grind into powder, pass through a 60-mesh sieve to obtain core-shell structured neutral particles, wherein the mass fraction of sodium hydroxide in the core-shell structured neutral particles is 20%.
[0065] The main raw materials and strains used in the above embodiments and comparative examples are as follows:
[0066] Corn starch, Henan Jindan Lactic Acid Technology Co., Ltd., moisture content ≤14%;
[0067] Thermoresistant α-amylase: Enzyme activity 20000 U / ml;
[0068] Glycoamylase: Enzyme activity 50000 U / ml;
[0069] Bacillus coagulans, JDKJ 1.1201-2312, isolated and preserved by Henan Provincial Lactic Acid Engineering Technology Research Center.
[0070] The main equipment used in the above embodiments and comparative examples was the pilot production line for strain expansion at the Henan Province Biodegradable Materials Pilot Plant.
[0071] Proof of effectiveness
[0072] The fermentation processes of Examples 1-3 and Comparative Examples 1-3 were scaled up to a single 90 cubic meter fermentation unit. The L-lactic acid content of the lactic acid fermentation broth and the optical purity of the finished lactic acid were tested. The specific results are shown in Table 1.
[0073] Table 1
[0074]
[0075] Results Analysis
[0076] As can be seen from Examples 1-3 and Comparative Examples 1-3, and in conjunction with the data in Table 1, the efficient fermentation method for producing L-lactic acid provided by the present invention (Examples 1-3) produces an L-lactic acid fermentation broth with an L-lactic acid content of over 160.7 g / L and an optical purity of over 99.5% for the finished lactic acid, exhibiting high acid production and optical purity.
[0077] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of the claims of this patent application.
Claims
1. A method for efficient fermentation production of L-lactic acid, characterized in that, Includes the following steps: S1. The biomass starch is liquefied and pre-saccharified to obtain a pre-saccharified solution; S2. Inoculate the fermentation strain into the presaccharified solution for fermentation. When the pH of the fermentation solution drops to 5.1±0.1, add a neutralizing solution containing core-shell structure neutralizing particles to maintain the pH of the fermentation solution at 5.1±0.1 and continue fermentation. S3. After fermentation, the lactic acid fermentation broth is subjected to ultrafiltration, nanofiltration and electrodialysis for dissociation, and then concentrated to obtain the finished lactic acid product; Among them, the core-shell structured neutralizing particles are prepared using carboxymethyl chitosan as the shell and betaine and sodium hydroxide as the core.
2. The method for efficient fermentation production of L-lactic acid according to claim 1, characterized in that, The preparation process of core-shell structured neutralizing particles is as follows: A1. Add carboxymethyl chitosan to deionized water, heat and stir to obtain a chitosan solution; A2. Add betaine to the chitosan solution, mix well, heat and concentrate, then cool to room temperature to obtain a mixed slurry; A3. Under stirring conditions, sodium hydroxide is added to the mixed slurry, dried, and then ground into powder to obtain core-shell structured neutral particles.
3. The method for efficient fermentation production of L-lactic acid according to claim 2, characterized in that, In the carboxymethyl chitosan solution, the mass fraction of carboxymethyl chitosan is 1.8-2.2%.
4. The method for efficient fermentation production of L-lactic acid according to claim 2, characterized in that, The amount of betaine added is 4-6% of the mass of carboxymethyl chitosan, and the mass fraction of sodium hydroxide in the core-shell structure neutralizing particles is 15-25%.
5. The method for efficient fermentation production of L-lactic acid according to claim 1, characterized in that, In the neutralization solution, the mass fraction of core-shell structured neutralized particles is 25-35%.
6. The method for efficient fermentation production of L-lactic acid according to claim 1, characterized in that, The raw materials for biomass starch are one or more of rice, corn, wheat, potatoes and sweet potatoes.
7. The method for efficient fermentation production of L-lactic acid according to claim 1, characterized in that, The fermentation strain is Bacillus coagulans.
8. The method for efficient fermentation production of L-lactic acid according to claim 1, characterized in that, During the liquefaction process, biomass starch is prepared into starch milk, amylase is added, and spray liquefaction is carried out. The spray temperature is controlled at 108-110℃ and maintained for 4-5 minutes. Then, the temperature is reduced to 90-100℃, the pH value is adjusted to 5.0-5.3, and maintained for 20-50 minutes to obtain liquefied sugar solution.
9. The method for efficient fermentation production of L-lactic acid according to claim 1, characterized in that, During the pre-saccharification process, the temperature is 60-62℃, the pH value is 4.2-4.5, the amount of saccharifying enzyme added is 0.35-0.40 mg / g of the total sugar mass in the liquefied sugar solution, and the saccharification time is 3-5 h.
10. The method for efficient fermentation production of L-lactic acid according to claim 1, characterized in that, After inoculation of the fermenter, control the fermentation temperature at 50-52℃ and the aeration rate at 0.1-0.12 vvm.