Culture method of lactic acid bacteria

Abstract

The invention belongs to the field of microbial fermentation, and particularly relates to a culture method of lactic acid bacteria, which comprises culture of D-lactic acid bacteria and L-lactic acid bacteria, and is characterized in that the culture method of the D-lactic acid bacteria comprises five stages of strain screening, microbial inoculum preparation, shake-flask culture, seed culture and fermentation culture, according to the method, the five stages of D-lactic acid bacteria culture and L-lactic acid bacteria culture are divided, the process is standard, and operation is convenient. And by virtue of MRS culture medium enrichment and liquid chromatography detection, high-yield and high-purity strains are selected. Bacterial strain activity can be preserved for a long time through microbial inoculum preparation, and flask shaking and seed culture build a firm foundation for fermentation. According to the fermentation culture, parameters such as temperature, pH, dissolved oxygen and the like are accurately regulated and controlled, and batch feeding and an advanced monitoring system are adopted, so that the growth requirements of strains are met, high yield is maintained, and the yield and purity of lactic acid are improved. Components of the optimized MRS culture medium can be adjusted according to needs, the content and purity of lactic acid can be accurately measured through a detailed detection method, reliable data support is provided for culture, and it is guaranteed that the culture effect is stable and reliable.

Description

Technical Field

[0001] The invention belongs to the field of microbial fermentation, in particular to a method for culturing lactic acid bacteria. Background Art

[0002] Lactic acid, as an organic acid with wide application value, plays an important role in many fields such as food, medicine, and chemical industry. With the continuous growth of market demand, the efficient and high-purity production of lactic acid has become the focus of industry attention. At present, the production methods of lactic acid are mainly divided into two categories: chemical synthesis and fermentation. Although chemical synthesis has advantages in some aspects, its environmental problems, lack of sustainability, and high production costs have made fermentation gradually become the mainstream method of lactic acid production.

[0003] However, in the actual application of traditional lactic acid fermentation methods, these methods often face multiple challenges such as low yield, low optical purity, and difficult to control production costs. These problems not only limit the widespread application of lactic acid, but also have a negative impact on the economic benefits and market competitiveness of lactic acid production companies.

[0004] Specifically, traditional lactic acid fermentation methods lack specificity in strain screening, and it is often difficult to accurately select target strains with high yield and high optical purity. At the same time, in key links such as inoculant preparation, shake flask culture, seed culture and fermentation culture, traditional methods also have problems such as complex operations, non-standard processes, and inaccurate parameter control. These problems make it difficult to improve the efficiency of the lactic acid fermentation process, and it is difficult to stably increase the yield and optical purity.

[0005] To this end, the present invention provides a method for culturing lactic acid bacteria. Summary of the invention

[0006] In order to make up for the deficiencies of the prior art, at least one technical problem raised in the background technology is solved.

[0007] The technical solution adopted by the present invention to solve the technical problem is: a lactic acid bacteria culture method of the present invention includes the culture of D-lactic acid bacteria and L-lactic acid bacteria, and is characterized in that the culture method of D-lactic acid bacteria and the culture method of L-lactic acid bacteria both include five stages of strain screening, bacterial agent preparation, shake flask culture, seed culture and fermentation culture.

[0008] Preferably, the method for culturing D-lactic acid bacteria comprises the following steps:

[0009] S1. strain screening: collect microbial samples from natural environments, including but not limited to soil, plant surfaces, and animal intestines; place the collected samples in MRS liquid medium for enrichment culture, use high performance liquid chromatography to detect the lactic acid production and optical purity of each strain, and screen out high-yield strains with a D-lactic acid / lactic acid ratio of more than 95% for subsequent culture;

[0010] S2, preparation of bacterial agent: the selected high-yield D-lactic acid strain is expanded and cultured in MRS liquid culture medium, and the culture conditions are temperature 35-39°C, shaker speed 150-200r / min, and culture time 18-24h; after the culture, the bacterial bodies are collected by centrifugation at a speed of 6000-7000r / min for 10-15min, and the bacterial bodies are mixed with a freeze-drying protective agent containing 10-15% skim milk powder, 5-8% trehalose, and 2-4% mannitol at a ratio of 1:1-1.2, and freeze-dried at -50°C to -60°C and a vacuum degree of 10-15Pa for 24-30h to prepare bacterial powder, which is stored in an environment below -20°C for standby use;

[0011] S3, shake flask culture: take an appropriate amount of the above bacterial powder and inoculate it into MRS liquid culture medium, the inoculation amount is 0.5-1% (w / v), and shake flask culture is carried out for 12-18 hours at 30-40°C and a shaker speed of 100-140r / min to activate and proliferate the strain;

[0012] S4, seed culture: inoculate the activated bacterial solution into the seed culture medium at an inoculum rate of 3-8%, the seed culture medium contains 10-15g / L peptone, 8-12g / L beef extract, 5-8g / L yeast powder, 20-30g / L glucose, 2-3g / L potassium dihydrogen phosphate, 0.5-0.8g / L magnesium sulfate, and 0.2-0.3g / L manganese sulfate; culture at 35-39°C for 16-20h in an anaerobic environment to obtain a high-concentration seed culture solution;

[0013] S5. Fermentation culture: Inoculate the seed culture solution into the fermentation medium at an inoculation rate of 8-15%, control the temperature at 36-38°C through a temperature control system, add a buffer or an acid-base regulator using an automatic pH adjustment device to maintain the pH value of the fermentation solution at 5.5-6.5; control the dissolved oxygen concentration at 0-1 mg / L with the help of a dissolved oxygen electrode and a ventilation control system according to the aerobic characteristics of the strain; adopt a batch feed fermentation method, when the glucose concentration in the fermentation solution is lower than 5 g / L, supplement 50% (w / v) glucose solution, and when the ammonia nitrogen concentration is lower than 0.5 g / L, supplement 20% (w / v) peptone solution; and simultaneously use a fermentation control system to monitor and adjust the fermentation parameters of temperature, pH value, dissolved oxygen, and stirring speed in real time to increase the yield and optical purity of D-lactic acid.

[0014] Preferably, the method for culturing L-lactic acid bacteria comprises the following steps:

[0015] S1. Strain screening: Collect microbial samples from the natural environment, place them in MRS liquid medium for enrichment culture, and screen out strains with high L-lactic acid production capacity by high performance liquid chromatography;

[0016] S2, preparation of bacterial agent: the selected high-yield L-lactic acid strain is expanded and cultured in an optimized MRS liquid medium, wherein the carbon source is 20-30 g / L maltose, the nitrogen source is 10-15 g / L soy peptone, the culture temperature is 35-39°C, the shaker speed is 160-220 r / min, and the culture time is 20-26 h; after the culture, the bacterial bodies are collected by centrifugation at a speed of 6000-7000 r / min for 12-16 min, and mixed with a freeze-drying protective agent containing 12-18% lactose, 6-10% glycerol, and 3-5% polyvinyl pyrrolidone at a ratio of 1:1-1.3, and freeze-dried at -55°C to -65°C and a vacuum degree of 8-12 Pa for 26-32 h to prepare bacterial powder, which is stored in an environment below -25°C for standby use;

[0017] S3, shake flask culture: take an appropriate amount of the above bacterial powder and inoculate it into MRS liquid culture medium, the inoculation amount is 0.6-1.2% (w / v), and shake flask culture is carried out for 13-19 hours at 31-39°C and a shaker speed of 100-140 r / min to activate and proliferate the strain;

[0018] S4, seed culture: inoculate the activated bacterial solution into the seed culture medium at an inoculum volume of 4-9%, the seed culture medium contains 11-16g / L peptone, 9-13g / L beef extract, 6-9g / L yeast powder, 22-32g / L glucose, 2.2-3.2g / L potassium dihydrogen phosphate, 0.55-0.85g / L magnesium sulfate, and 0.22-0.32g / L manganese sulfate; culture at 36-38°C for 17-21h in an anaerobic environment to obtain a high-concentration seed culture solution;

[0019] S5. Fermentation culture: Inoculate the seed culture medium at an inoculation rate of 9-16% into the fermentation medium. According to the characteristics of L-lactic acid producing bacteria, control the temperature at 36-38°C. Add buffer or acid-base regulator through the pH automatic adjustment system to maintain the pH value of the fermentation liquid in a range that is conducive to the accumulation of L-lactic acid. For anaerobic L-lactic acid producing bacteria, use anaerobic fermentation tanks to ensure that the dissolved oxygen concentration in the fermentation liquid is lower than 0.1 mg / L. Use batch fed fermentation to replenish carbon and nitrogen sources in a timely manner according to the consumption of nutrients during the fermentation process. Use advanced fermentation control systems to monitor and accurately adjust various parameters in the fermentation process in real time. Fermentation tanks of different volumes and stirring methods can also be selected according to the characteristics of the strain and the requirements of the fermentation process to increase the yield and optical purity of L-lactic acid.

[0020] Preferably, the culture temperature is controlled at 36-38°C during the entire culture process. This temperature range is close to the optimal growth temperature of lactic acid bacteria and can ensure that the growth rate and metabolic activity of the strain are in a good state.

[0021] Preferably, during the fermentation process, the pH value of the fermentation broth is maintained within a suitable range by adding a buffer or an acid-base regulator. The suitable range for D-lactic acid bacteria is 5.5-6.5, and for L-lactic acid bacteria, the pH range is determined to be favorable for the accumulation of L-lactic acid according to the characteristics of the production bacteria.

[0022] Preferably, during the seed culture stage, anaerobic fermentation is performed, using an anaerobic incubator or adding an anaerobic gas production bag to ensure that the lactic acid bacteria grow and reproduce normally in an anaerobic or micro-aerobic environment.

[0023] Preferably, during the fermentation and cultivation stage, a batch fed fermentation method is adopted. According to the consumption of nutrients during the fermentation process, when the carbon source concentration in the fermentation broth is lower than the set value, a carbon source such as glucose or sucrose is supplemented, and when the nitrogen source concentration is lower than the set value, a nitrogen source such as peptone or yeast extract is supplemented.

[0024] The beneficial effects of the present invention are as follows:

[0025] 1. A method for cultivating lactic acid bacteria of the present invention, wherein the method constructs an independent cultivation system including strain screening, bacterial preparation, shake flask cultivation, seed cultivation and fermentation cultivation for D-lactic acid bacteria and L-lactic acid bacteria, respectively. Through this design, the conditions of each cultivation stage can be accurately regulated according to the different physiological characteristics and metabolic requirements of the two lactic acid bacteria, so as to create an optimal environment for their growth and acid production.

[0026] 2. A method for cultivating lactic acid bacteria of the present invention, in the strain screening stage, adopts a method of collecting samples extensively from the natural environment, combining with MRS liquid culture medium enrichment culture, and using a high performance liquid chromatograph to accurately measure the lactic acid production and optical purity. This design ensures that the D-lactic acid bacteria and L-lactic acid bacteria screened out have the characteristics of high yield of target lactic acid and high optical purity, respectively, laying the foundation for subsequent high-quality cultivation.

[0027] 3. A method for culturing lactic acid bacteria of the present invention performs meticulous and targeted optimization design on key parameters such as culture medium composition, culture temperature, pH value, dissolved oxygen concentration, etc. from shake flask culture to fermentation culture. For example, according to the growth and metabolic characteristics of bacteria at different stages, the ratio of carbon source, nitrogen source and inorganic salt in MRS culture medium is adjusted; according to the aerobic requirement of the strain, the dissolved oxygen concentration is strictly controlled, etc., to meet the nutritional and environmental requirements of lactic acid bacteria at different growth stages.

[0028] 4. A method for cultivating lactic acid bacteria of the present invention is designed to specifically detect the lactic acid content and optical purity in the fermentation broth. Through specific sample processing steps, such as centrifugation, extraction, vacuum distillation of the fermentation broth, etc., combined with high performance liquid chromatography technology, and different chromatographic columns, mobile phases and detection conditions are selected for the lactic acid content and optical purity, accurate analysis of the fermentation product is achieved, providing reliable data support for monitoring and optimization of the fermentation process.

[0029] 5. A lactic acid bacteria culture method of the present invention has a more refined control over the culture conditions than the traditional lactic acid bacteria culture method. The traditional method may only perform broad control over the temperature, pH value, etc., while the present method not only accurately controls the temperature at around 37°C, but also dynamically adjusts the pH value and dissolved oxygen concentration according to different stages and strain characteristics. For example, during the fermentation culture stage, these parameters are monitored and adjusted in real time through an advanced control system to ensure that the strain is always in the best growth and metabolic environment.

[0030] 6. A method for cultivating lactic acid bacteria of the present invention no longer uses a general MRS medium, but adopts an optimized MRS medium, and adjusts the ratio of carbon source, nitrogen source and inorganic salt according to the characteristics of D-lactic acid bacteria and L-lactic acid bacteria. This customized medium design can better meet the nutritional requirements of different lactic acid bacteria and improve the bacterial growth rate and lactic acid production.

[0031] 7. A method for cultivating lactic acid bacteria of the present invention introduces a batch feeding fermentation method, and uses an advanced fermentation control system to monitor and adjust the fermentation parameters in real time. The traditional cultivation method may add all nutrients at once, which is easy to cause malnutrition in the later stage or accumulation of metabolites in the early stage to inhibit bacterial growth. The batch feeding fermentation method can timely supplement carbon and nitrogen sources according to the consumption of nutrients in the fermentation process, maintain the high-yield metabolic state of the strain, and the advanced control system ensures the stability and efficiency of the fermentation process.

[0032] 8. A method for cultivating lactic acid bacteria of the present invention. Traditional lactic acid detection methods may not be accurate enough or cannot distinguish the optical isomers of lactic acid. The detection process designed by the method, from sample processing to the selection of high-performance liquid chromatography conditions, is professionally designed for lactic acid content and optical purity. For example, in the detection of lactic acid optical purity, a special chiral separation column and an optimized mobile phase are used to accurately determine the ratio of D-lactic acid and L-lactic acid, providing more comprehensive and accurate data for evaluating the quality of fermentation products.

[0033] 9. The method for cultivating lactic acid bacteria of the present invention significantly improves the yield of D-lactic acid and L-lactic acid by accurately screening high-yield strains, optimizing culture conditions, innovating culture methods and customizing culture media. During the fermentation and cultivation stage, stable temperature, suitable pH value, accurate dissolved oxygen control and timely feeding provide sufficient nutrition and a good growth environment for the strain, so that the bacteria can synthesize lactic acid in large quantities, meeting the demand for lactic acid production in industrial production.

[0034] 10. The lactic acid bacteria culture method of the present invention, the specially designed strain screening standard and the high performance liquid chromatography detection method ensure that the D-lactic acid and L-lactic acid obtained by culture have high optical purity. During strain screening, strains with high D-lactic acid / lactic acid or L-lactic acid / lactic acid ratios are selected, and the metabolic characteristics of the strains are maintained through fine condition control during the subsequent culture process, so that the optical purity of lactic acid in the final product meets the high requirements of application scenarios, such as the fields of medicine, food and other industries with strict standards for optical purity.

[0035] 11. The method for cultivating lactic acid bacteria of the present invention, the advanced fermentation control system and the innovative cultivation method make the whole cultivation process more stable. Real-time monitoring and adjustment of fermentation parameters can timely discover and solve problems arising in the cultivation process, and avoid abnormal bacterial growth or unstable acid production caused by environmental fluctuations. The batch feeding fermentation method ensures the continuous supply of nutrients, reduces the inhibitory effect of metabolic product accumulation on bacterial growth, and thus improves the stability and repeatability of the cultivation process.

[0036] 12. The lactic acid bacteria culture method of the present invention provides a high-yield and high-optical-purity lactic acid product, which provides high-quality raw materials for its application in the fields of food, medicine, and chemical industry. In the food industry, it can be used to produce acidulants, preservatives, etc.; in the medical field, it can be used to synthesize drug carriers, biodegradable materials, etc. High-quality lactic acid raw materials help improve the quality and performance of related products and expand the application range of lactic acid bacteria fermentation products.

[0037] 13. A method for culturing lactic acid bacteria of the present invention designs independent systems covering strain screening, bacterial agent preparation, shake flask culture, seed culture and fermentation culture for D-lactic acid bacteria and L-lactic acid bacteria, respectively. In the strain screening stage, samples are widely collected from the natural environment, enriched and cultured using MRS liquid culture medium, and then the lactic acid production and optical purity are accurately measured by high-performance liquid chromatography to screen out target strains with high yield and high optical purity. In each culture stage, parameters such as culture medium composition, temperature, pH value, and dissolved oxygen concentration are optimized in a targeted manner. For example, according to different stages and strain characteristics, the ratio of carbon source, nitrogen source and inorganic salt in the MRS culture medium is adjusted, the temperature is accurately controlled at about 37°C, and the dissolved oxygen concentration is regulated according to the aerobic requirement of the strain. At the same time, a unique detection process is designed to accurately analyze the lactic acid content and optical purity in the fermentation broth;

[0038] Compared with traditional methods, this culture method has achieved improvements in many aspects. The culture conditions are more precisely controlled, not only the temperature is precisely controlled, but also the pH value and dissolved oxygen concentration are dynamically adjusted; the culture medium uses the optimized MRS culture medium, and the proportion of ingredients is customized according to the characteristics of different lactic acid bacteria; the innovative batch feeding fermentation method is used, and the advanced fermentation control system is used to monitor and adjust the fermentation parameters in real time; the detection method is more professional and can accurately determine the lactic acid content and optical purity;

[0039] These improvements have produced significant results, greatly increasing the production of D-lactic acid and L-lactic acid to meet the needs of industrial production. At the same time, the optical purity of lactic acid has been improved to meet the standards of industries with high purity requirements such as medicine and food. Advanced control systems and culture methods have enhanced the stability and repeatability of the culture process, providing guarantees for continuous production. High-yield, high-purity lactic acid products provide high-quality raw materials for the fields of food, medicine, and chemicals, expanding the application range of lactic acid fermentation products and helping to improve the quality and performance of related products. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The present invention will be further described below in conjunction with the accompanying drawings.

[0041] Figure 1 This is a step diagram of the D-lactic acid bacteria culture method of the present invention;

[0042] Figure 2 It is a step diagram of the L-lactic acid bacteria culture method in the present invention. DETAILED DESCRIPTION

[0043] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0044] A method for culturing lactic acid bacteria according to an embodiment of the present invention includes culturing D-lactic acid bacteria and L-lactic acid bacteria, and is characterized in that the culturing method of D-lactic acid bacteria and the culturing method of L-lactic acid bacteria both sequentially include five stages: strain screening, bacterial agent preparation, shake flask culture, seed culture and fermentation culture.

[0045] The method for culturing D-lactic acid bacteria comprises the following steps:

[0046] S1. strain screening: collect microbial samples from natural environments, including but not limited to soil, plant surfaces, and animal intestines; place the collected samples in MRS liquid medium for enrichment culture, use high performance liquid chromatography to detect the lactic acid production and optical purity of each strain, and screen out high-yield strains with a D-lactic acid / lactic acid ratio of more than 95% for subsequent culture;

[0047] S2, preparation of bacterial agent: the selected high-yield D-lactic acid strain is expanded and cultured in MRS liquid culture medium, and the culture conditions are temperature 35-39°C, shaker speed 150-200r / min, and culture time 18-24h; after the culture, the bacterial bodies are collected by centrifugation at a speed of 6000-7000r / min for 10-15min, and the bacterial bodies are mixed with a freeze-drying protective agent containing 10-15% skim milk powder, 5-8% trehalose, and 2-4% mannitol at a ratio of 1:1-1.2, and freeze-dried at -50°C to -60°C and a vacuum degree of 10-15Pa for 24-30h to prepare bacterial powder, which is stored in an environment below -20°C for standby use;

[0048] S3, shake flask culture: take an appropriate amount of the above bacterial powder and inoculate it into MRS liquid culture medium, the inoculation amount is 0.5-1% (w / v), and shake flask culture is carried out for 12-18 hours at 30-40°C and a shaker speed of 100-140r / min to activate and proliferate the strain;

[0049] S4, seed culture: inoculate the activated bacterial solution into the seed culture medium at an inoculum rate of 3-8%, the seed culture medium contains 10-15g / L peptone, 8-12g / L beef extract, 5-8g / L yeast powder, 20-30g / L glucose, 2-3g / L potassium dihydrogen phosphate, 0.5-0.8g / L magnesium sulfate, and 0.2-0.3g / L manganese sulfate; culture at 35-39°C for 16-20h in an anaerobic environment to obtain a high-concentration seed culture solution;

[0050] S5. Fermentation culture: Inoculate the seed culture solution into the fermentation medium at an inoculation rate of 8-15%, control the temperature at 36-38°C through a temperature control system, add a buffer or an acid-base regulator using an automatic pH adjustment device to maintain the pH value of the fermentation solution at 5.5-6.5; control the dissolved oxygen concentration at 0-1 mg / L with the help of a dissolved oxygen electrode and a ventilation control system according to the aerobic characteristics of the strain; adopt a batch feed fermentation method, when the glucose concentration in the fermentation solution is lower than 5 g / L, supplement 50% (w / v) glucose solution, and when the ammonia nitrogen concentration is lower than 0.5 g / L, supplement 20% (w / v) peptone solution; and simultaneously use a fermentation control system to monitor and adjust the fermentation parameters of temperature, pH value, dissolved oxygen, and stirring speed in real time to increase the yield and optical purity of D-lactic acid.

[0051] The method for culturing L-lactic acid bacteria comprises the following steps:

[0052] S1. Strain screening: Collect microbial samples from the natural environment, place them in MRS liquid medium for enrichment culture, and screen out strains with high L-lactic acid production capacity by high performance liquid chromatography;

[0053] S2, preparation of bacterial agent: the selected high-yield L-lactic acid strain is expanded and cultured in an optimized MRS liquid medium, wherein the carbon source is 20-30 g / L maltose, the nitrogen source is 10-15 g / L soy peptone, the culture temperature is 35-39°C, the shaker speed is 160-220 r / min, and the culture time is 20-26 h; after the culture, the bacterial bodies are collected by centrifugation at a speed of 6000-7000 r / min for 12-16 min, and mixed with a freeze-drying protective agent containing 12-18% lactose, 6-10% glycerol, and 3-5% polyvinyl pyrrolidone at a ratio of 1:1-1.3, and freeze-dried at -55°C to -65°C and a vacuum degree of 8-12 Pa for 26-32 h to prepare bacterial powder, which is stored in an environment below -25°C for standby use;

[0054] S3, shake flask culture: take an appropriate amount of the above bacterial powder and inoculate it into MRS liquid culture medium, the inoculation amount is 0.6-1.2% (w / v), and shake flask culture is carried out for 13-19 hours at 31-39°C and a shaker speed of 100-140 r / min to activate and proliferate the strain;

[0055] S4, seed culture: inoculate the activated bacterial solution into the seed culture medium at an inoculum volume of 4-9%, the seed culture medium contains 11-16g / L peptone, 9-13g / L beef extract, 6-9g / L yeast powder, 22-32g / L glucose, 2.2-3.2g / L potassium dihydrogen phosphate, 0.55-0.85g / L magnesium sulfate, and 0.22-0.32g / L manganese sulfate; culture at 36-38°C for 17-21h in an anaerobic environment to obtain a high-concentration seed culture solution;

[0056] S5. Fermentation culture: Inoculate the seed culture medium at an inoculation rate of 9-16% into the fermentation medium. According to the characteristics of L-lactic acid producing bacteria, control the temperature at 36-38°C. Add buffer or acid-base regulator through the pH automatic adjustment system to maintain the pH value of the fermentation liquid in a range that is conducive to the accumulation of L-lactic acid. For anaerobic L-lactic acid producing bacteria, use anaerobic fermentation tanks to ensure that the dissolved oxygen concentration in the fermentation liquid is lower than 0.1 mg / L. Use batch fed fermentation to replenish carbon and nitrogen sources in a timely manner according to the consumption of nutrients during the fermentation process. Use advanced fermentation control systems to monitor and accurately adjust various parameters in the fermentation process in real time. Fermentation tanks of different volumes and stirring methods can also be selected according to the characteristics of the strain and the requirements of the fermentation process to increase the yield and optical purity of L-lactic acid.

[0057] The culture temperature is controlled at 36-38°C during the entire culture process. This temperature range is close to the optimal growth temperature of lactic acid bacteria, which can ensure that the growth rate and metabolic activity of the strain are in a good state.

[0058] During the fermentation process, the pH value of the fermentation broth is maintained within an appropriate range by adding a buffer or an acid-base regulator. The appropriate range for D-lactic acid bacteria is 5.5-6.5, and for L-lactic acid bacteria, the pH range is determined to be favorable for the accumulation of L-lactic acid according to the characteristics of the production bacteria.

[0059] During the seed culture stage, anaerobic fermentation is carried out, using an anaerobic incubator or adding anaerobic gas production bags to ensure that lactic acid bacteria grow and reproduce normally in an anaerobic or micro-aerobic environment.

[0060] During the fermentation and cultivation stage, a batch fed fermentation method is adopted. According to the consumption of nutrients in the fermentation process, when the carbon source concentration in the fermentation liquid is lower than the set value, a carbon source such as glucose or sucrose is supplemented. When the nitrogen source concentration is lower than the set value, a nitrogen source such as peptone or yeast extract is supplemented.

[0061] Specifically:

[0062] Cultivation method of D-lactic acid bacteria

[0063] Strain screening: Microbial samples are widely collected from a variety of natural environments such as soil, plant surfaces, and animal intestines. The collected samples are inoculated into MRS liquid culture medium for enrichment culture to create a suitable growth environment for the target strains and promote their rapid increase in number. The lactic acid production and optical purity of each strain are accurately determined using high-performance liquid chromatography, and high-yield strains with a D-lactic acid / lactic acid ratio of more than 90% are strictly selected as the starting material for subsequent culture.

[0064] Preparation of bacterial agents: The selected high-yield D-lactic acid strains are inoculated into the nutrient-rich MRS liquid culture medium, and the culture is expanded under suitable temperature, ventilation and other conditions to make the number of strains grow exponentially. After the culture reaches a certain bacterial concentration, the centrifugal technology is used to achieve efficient separation of the bacteria and the culture solution under specific speed and time parameters. The collected bacteria are fully mixed with the carefully formulated freeze-dried protective agent, and then freeze-dried to make bacterial powder. Finally, the bacterial powder is placed in a refrigerator at -20°C or lower for low temperature storage and standby use to ensure that the bacterial powder maintains a good active state before use.

[0065] Shake flask culture: Take out an appropriate amount of the low-temperature preserved bacterial powder and inoculate it into fresh MRS liquid culture medium. In a constant temperature shaker, control the temperature at 30-40°C and oscillate at an appropriate speed to allow the bacteria to fully contact the culture medium, promote oxygen transfer and nutrient absorption, achieve rapid activation and proliferation of the strain, and provide a sufficient number of bacteria with good activity for subsequent seed culture.

[0066] Seed culture: The activated bacterial solution from the shake flask culture is accurately inoculated into the seed culture medium in a certain proportion. The formula of the seed culture medium has been optimized and is rich in various nutrients required for bacterial growth. Anaerobic fermentation culture is used in this stage. By using anaerobic incubators or adding anaerobic gas production bags, an anaerobic or micro-aerobic growth environment is created for the bacteria, which promotes the rapid growth and reproduction of the bacteria at this stage, accumulates sufficient biomass, and obtains a high-concentration seed culture solution.

[0067] Fermentation culture: The seed culture solution is inoculated into the fermentation medium at an appropriate inoculation amount. The temperature is stably controlled at around 37°C through a precise temperature control system; a buffer or acid-base regulator is added using an automatic pH adjustment device to maintain the pH value of the fermentation solution in the appropriate range of 5.5-6.5; the dissolved oxygen concentration is accurately adjusted based on the aerobic characteristics of the strain with the help of a dissolved oxygen electrode and aeration control system. At the same time, a batch-fed fermentation method is adopted. Based on the real-time consumption of nutrients during the fermentation process, carbon sources (such as glucose, sucrose, etc.) and nitrogen sources (such as peptone, yeast extract, etc.) are supplemented in a timely manner through feedback from an online monitoring system. Advanced fermentation control systems are used to conduct all-round real-time monitoring and precise adjustment of fermentation parameters such as temperature, pH value, dissolved oxygen, and stirring speed to ensure the stability and efficiency of the fermentation process and significantly increase the yield and optical purity of D-lactic acid.

[0068] Cultivation method of L-lactic acid bacteria

[0069] Strain screening: Microbial samples are also collected extensively from the natural environment, and enriched and cultured using MRS liquid culture medium, focusing on the diversity of different sampling locations and sample types to increase the probability of screening high-yield L-lactic acid strains. Through specific screening methods such as plate streaking separation and gradient dilution coating, combined with high-performance liquid chromatography analysis, strains with high L-lactic acid production capacity are accurately screened.

[0070] Preparation of bacterial agents: The selected high-yield L-lactic acid strains are expanded and cultured in the optimized MRS liquid culture medium, and the culture conditions, such as temperature, pH value, ventilation volume, etc., are strictly controlled to meet the growth requirements of the strains. When the bacterial concentration reaches a certain level, the bacteria are collected by centrifugation, mixed evenly with a freeze-drying protective agent specially developed for the characteristics of L-lactic acid bacteria, and then freeze-dried to make bacterial powder, which is stored at low temperature for future use to ensure that the bacterial powder can still maintain a high activity during long-term storage.

[0071] Shake flask culture: Take an appropriate amount of cryopreserved bacterial powder and inoculate it into the freshly prepared MRS liquid culture medium, place it in a constant temperature shaker, and culture it under appropriate temperature, oscillation frequency and other conditions. The strain is activated quickly, the growth and metabolic capacity is restored, and the number of bacteria is increased through continuous proliferation, providing sufficient high-quality bacteria for subsequent seed culture.

[0072] Seed culture: The activated bacterial solution from the shake flask culture is inoculated into the seed culture medium in proportion, and the anaerobic fermentation culture method is adopted. In an anaerobic environment, by controlling the temperature, nutrient concentration and other conditions, the bacteria are promoted to grow and reproduce rapidly, accumulating abundant biomass, thereby obtaining a high-concentration seed culture solution.

[0073] Fermentation culture: After the seed culture is inoculated into the fermentation medium, the fermentation conditions are finely adjusted according to the unique characteristics of the L-lactic acid producing bacteria. The temperature is usually controlled at around 37°C, but it needs to be fine-tuned according to the specific strain; through the pH automatic adjustment system, add appropriate buffers or acid-base regulators to maintain the pH value of the fermentation broth in a range that is conducive to the accumulation of L-lactic acid; for anaerobic L-lactic acid producing bacteria, strict anaerobic culture technology is adopted, such as using anaerobic fermentation tanks to ensure that the dissolved oxygen concentration in the fermentation broth is extremely low. The same batch feeding fermentation method is adopted. According to the consumption of nutrients during the fermentation process, carbon and nitrogen sources are supplemented in time, and advanced fermentation control systems are used to monitor and accurately adjust various parameters in the fermentation process in real time. In addition, according to the characteristics of the strain and the requirements of the fermentation process, suitable fermentation equipment can be flexibly selected, such as fermentation tanks of different volumes, stirring methods, etc., and the operation mode can be optimized, such as the timing of feeding and the adjustment of stirring speed, so as to maximize the L-lactic acid yield and optical purity.

[0074] Culture conditions:

[0075] Culture medium composition: Using optimized MRS culture medium, according to the characteristics of different strains of D-lactic acid bacteria and L-lactic acid bacteria, the proportion of carbon source, nitrogen source and inorganic salt is precisely adjusted to meet their specific nutritional needs and improve the utilization efficiency of nutrients.

[0076] Culture temperature: The culture temperature is controlled at close to 37°C, which is close to the optimal growth temperature of lactic acid bacteria. This can ensure that the growth rate and metabolic activity of the strain are in a good state and promote its efficient acid production.

[0077] pH value: By adding buffers or acid-base regulators, the pH value of the fermentation broth is maintained within an appropriate range, providing a stable acid-base environment for the growth and metabolism of lactic acid bacteria, which is conducive to the accumulation of lactic acid.

[0078] Dissolved oxygen concentration: Precisely adjust according to the aerobic requirements of the strain. For anaerobic lactic acid bacteria, strictly control the dissolved oxygen concentration in the fermentation broth to avoid adverse effects on the growth and metabolism of the strain; for facultative anaerobic or microaerobic lactic acid bacteria, provide a suitable dissolved oxygen environment to ensure the normal growth and metabolism of the strain.

[0079] Cultivation method: Batch feeding fermentation is adopted. During the fermentation and cultivation stage, carbon and nitrogen sources are supplemented in time according to the consumption of nutrients in the fermentation process to maintain the high-yield metabolic state of the strain. At the same time, advanced fermentation control systems are used to monitor and adjust fermentation parameters in real time to ensure the stability and efficiency of the fermentation process. The system can automatically adjust key parameters such as temperature, pH value, dissolved oxygen concentration, etc. according to the preset parameter range, respond to changes in the fermentation process in a timely manner, and avoid fermentation abnormalities caused by parameter fluctuations.

[0080] Fermentation broth detection method:

[0081] Lactic acid content detection:

[0082] Sample processing: Accurately pipette 1mL of fermentation liquid into a 1.5mL EP tube, centrifuge at 12000r / min for 2min in a high-speed centrifuge to fully separate the bacteria from the fermentation supernatant. Set a series of fermentation supernatant addition gradients (50μL, 100μL, 200μL, 300μL, 400μL, 500μL), use H2O2 with a mass ratio of 30% as a clarifier, and set the corresponding addition gradient (10μL, 20μL, 25μL, 30μL, 50μL, 100μL, 200μL), add water to 1000μL, and finally filter through a 0.22μm water-based polyethersulfone filter head into a sample injection bottle to obtain the sample to be tested for liquid phase detection.

[0083] HPLC conditions: A Shim-pack GISTC18 (4.6 ID × 250 mm, 5 μm) column was selected, mobile phase A was 0.01 mol / L dipotassium hydrogen phosphate solution, mobile phase B was 100% methanol, the ratio of the two was 0.95:0.05, the mobile phase flow rate was controlled at 1 mL / min, the column box temperature was maintained at 30 ° C, and the detection wavelength was set to 210 nm.

[0084] Lactic acid optical purity content detection:

[0085] Sample treatment: Accurately pipette 5 mL of fermentation liquid into a 15 mL centrifuge tube (liquid volume 5 mL / 15 mL), and use 6 mol / L HCl to adjust the pH of the fermentation liquid to about 2.0. Add the fermentation supernatant and an equal volume of anhydrous ether into a separatory funnel, shake vigorously for 1 min, and take the organic phase on the upper layer of the separatory funnel after standing and stratification.

[0086] Collect lactic acid by vacuum distillation: Add the organic phase liquid on the upper layer of the separatory funnel and 2 mL of ultrapure water into a round-bottom flask and install it in the distillation area of ​​the rotary evaporator. Use a vacuum pump to accurately control the negative pressure of the rotary evaporator at about 0.9 MPa, stabilize the temperature of the rotary evaporator at 35°C through a water bath heater, and set the temperature of the cooling water circulation pump to 5°C to collect lactic acid by vacuum distillation.

[0087] HPLC conditions: chiral separation column Chirex3126 (D) -penicillam (i4.6mmi.d × 250mmL, 5μm), mobile phase A was 2mmol / LCuSO4·5H2O salt solution, mobile phase B was 100% pure isopropanol, the ratio of the two (A:B) was 95:5, the mobile phase flow rate was 1mL / min, the column box temperature was 26°C, and the detection wavelength was 254nm.

[0088] Effect:

[0089] Improve acid production and optical purity: Through strict strain screening, we have obtained D-lactic acid bacteria and L-lactic acid bacteria strains with high production capacity and high optical purity. In the subsequent cultivation process, we accurately optimize the culture conditions and fermentation methods according to the characteristics of different strains, which significantly improves the production and optical purity of D-lactic acid and L-lactic acid, meeting the needs of various industries for high-quality lactic acid bacteria products.

[0090] Stabilize the fermentation process: Using advanced fermentation control systems, key parameters such as temperature, pH value, dissolved oxygen concentration, etc. in the fermentation process are monitored in real time and accurately adjusted, effectively avoiding fermentation abnormalities caused by parameter fluctuations, ensuring the stability and reliability of the fermentation process, and improving production efficiency and product quality consistency.

[0091] Reduce production costs: The optimized culture medium formula and batch feeding fermentation method improve the utilization efficiency of nutrients and reduce the waste of raw materials. At the same time, the stable fermentation process reduces the losses caused by fermentation failure, thereby reducing the overall production cost and improving the economic benefits of the enterprise.

[0092] Broad application prospects: The high-quality D-lactic acid bacteria and L-lactic acid bacteria cultivated by the present invention can be widely used in food fermentation, biopharmaceuticals, feed additives and other fields, providing high-quality microbial resources for the development of these industries and promoting technological progress and innovative development of related industries.

[0093] Example of D-lactic acid bacteria culture

[0094] Strain screening: A total of 50 microbial samples were collected from the soil of an orchard, the leaves of surrounding plants, and the intestines of local farmed animals. Each sample was inoculated into a triangular flask containing 100 mL of MRS liquid culture medium and cultured at 37°C and 150 r / min for 48 hours for enrichment culture. Afterwards, the enriched bacterial liquid was inoculated onto MRS solid culture medium using the plate streaking method, cultured anaerobically at 37°C for 48 hours, and a single colony was picked and inoculated into MRS liquid culture medium, and cultured at 37°C for 24 hours. The lactic acid production and optical purity of each strain were detected by high-performance liquid chromatography, and finally three high-yield strains with a D-lactic acid / lactic acid ratio of more than 95% were screened out, marked as D1, D2, and D3 respectively.

[0095] Preparation of bacterial agent: Inoculate the selected D1 strain into 500 mL of MRS liquid culture medium and expand the culture at 37°C and 180 r / min for 24 hours. After the culture is completed, transfer the bacterial solution to a centrifuge tube and centrifuge it at 4°C and 8000 r / min for 10 minutes to collect the bacteria. Mix the bacteria with a freeze-drying protective agent containing 10% skim milk powder, 5% trehalose, and 2% mannitol in a 1:1 ratio, place it in a freeze dryer, freeze-dry it at -50°C and 10 Pa for 24 hours, make it into bacterial powder, and store it in a -20°C refrigerator for later use.

[0096] Shake flask culture: Take out an appropriate amount of D1 bacterial powder from a -20℃ refrigerator and inoculate it into a conical flask containing 100mL MRS liquid culture medium. Culture it in a constant temperature shaker at 37℃ and 150r / min for 12h to activate and proliferate the strain.

[0097] Seed culture: The D1 bacterial liquid activated by shake flask culture was inoculated at a 5% inoculum volume into an anaerobic culture bottle containing 500 mL of seed culture medium (10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 20 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 0.58 g / L magnesium sulfate, and 0.25 g / L manganese sulfate), and cultured in an anaerobic incubator at 37°C for 18 h to obtain a high-concentration seed culture solution.

[0098] Fermentation culture: The seed culture solution is inoculated at 10% into a fermentation tank containing 2L fermentation medium (30g / L glucose, 15g / L peptone, 8g / L yeast powder, 12g / L beef extract, 3g / L potassium dihydrogen phosphate, 0.6g / L magnesium sulfate, 0.3g / L manganese sulfate). The temperature is controlled at 37°C by the temperature control system, and 5mol / L sodium hydroxide solution is added by the pH automatic adjustment device to maintain the pH value of the fermentation liquid at 6.0. According to the monitoring results of the dissolved oxygen electrode, the air flow is adjusted by the ventilation control system to keep the dissolved oxygen concentration at 0.5mg / L. Batch fed fermentation is adopted. When the glucose concentration in the fermentation liquid is lower than 5g / L, 50% glucose solution is added; when the ammonia nitrogen concentration is lower than 0.5g / L, 20% peptone solution is added. The fermentation control system is used to monitor and adjust the temperature, pH value, dissolved oxygen, stirring speed and other parameters in the fermentation process in real time. After 48 h of fermentation, the D-lactic acid yield reached 120 g / L with an optical purity of 98%.

[0099] Example of L-lactic acid bacteria culture

[0100] Strain screening: 80 microbial samples were collected from soil, plant rhizosphere and aquatic animal intestines in different regions. The samples were inoculated into MRS liquid culture medium and cultured at 37°C for 48 hours for enrichment culture. The enriched bacterial liquid was inoculated onto MRS solid culture medium by gradient dilution coating method, cultured anaerobically at 37°C for 48 hours, and single colonies were picked for liquid culture. Five strains with high L-lactic acid production were screened out by high performance liquid chromatography, among which the L5 strain performed the best, with an L-lactic acid / lactic acid ratio of 96%.

[0101] Preparation of inoculant: Inoculate the L5 strain into 1L optimized MRS liquid medium (carbon source adjusted to 25g / L maltose, nitrogen source adjusted to 12g / L soy peptone), and expand the culture at 37℃ and 200r / min for 30h. After the culture, centrifuge at 4℃ and 10000r / min for 15min to collect the bacteria, mix with a freeze-drying protective agent containing 15% lactose, 8% glycerol, and 3% polyvinyl pyrrolidone at a ratio of 1:1.2, freeze-dry for 30h at -55℃ and 8Pa to make bacterial powder, and store it in a -25℃ refrigerator.

[0102] Shake flask culture: Take an appropriate amount of L5 bacterial powder and inoculate it into a triangular flask containing 150 mL of MRS liquid culture medium, and culture it in a constant temperature shaker at 37°C and 180 r / min for 15 hours to activate and proliferate the strain.

[0103] Seed culture: The L5 bacterial liquid activated by shake flask culture was inoculated at an inoculum rate of 8% into an anaerobic culture bottle containing 800 mL of seed culture medium (12 g / L peptone, 8 g / L beef extract, 6 g / L yeast powder, 25 g / L glucose, 2.5 g / L dipotassium hydrogen phosphate, 0.6 g / L magnesium sulfate, and 0.3 g / L manganese sulfate), and cultured in an anaerobic incubator at 37°C for 20 h to obtain a high-concentration seed culture solution.

[0104] Fermentation culture: The seed culture solution was inoculated at a rate of 12% into 3 L of fermentation medium (glucose 35 g / L, peptone 18 g / L, yeast powder 10 g / L, beef extract 15 g / L, hydrogen phosphate).

[0105] principle:

[0106] Principle of strain screening: There are diverse microorganisms in the natural environment. By connecting the collected samples to MRS liquid culture medium for enrichment culture, a suitable growth environment is provided for the target lactic acid bacteria, which promotes their increase in number. Then, using a high-performance liquid chromatograph, based on the differences in lactic acid production and optical purity of different strains, strains with high production of a specific type of lactic acid (D-lactic acid or L-lactic acid) and high optical purity are accurately identified. For example, D-lactic acid bacteria strains with a D-lactic acid / lactic acid ratio of more than 90% are selected to provide high-quality starting strains for subsequent culture.

[0107] Principle of bacterial preparation: The selected high-yield strains are cultured in a nutrient-rich MRS liquid medium to allow the strains to multiply in large quantities. After reaching a certain bacterial concentration, the bacteria and the culture medium are separated by centrifugation technology. The bacteria are mixed with a lyophilization protectant, which can protect the cell structure and activity of the bacteria during the lyophilization process. The bacteria are then lyophilized to produce bacterial powder. Low-temperature storage can reduce the loss of bacterial powder activity and maintain its good activity state.

[0108] Principle of shake flask culture: Take an appropriate amount of low-temperature stored bacterial powder and inoculate it into fresh MRS liquid culture medium. Under the suitable temperature (30-40℃) and oscillation conditions provided by the constant temperature shaker, the bacteria are fully in contact with the culture medium to promote oxygen transfer and nutrient absorption, activate dormant strains, and quickly restore their growth and reproduction capabilities, providing sufficient number of active bacteria for seed culture.

[0109] Seed culture principle: The activated bacterial liquid from the shake flask culture is inoculated into the seed culture medium with an optimized formula in a certain proportion, and the anaerobic fermentation culture method is adopted. An anaerobic incubator or anaerobic gas production bag is used to create an anaerobic or micro-aerobic environment, in which lactic acid bacteria can grow and reproduce rapidly, accumulate a large amount of biomass, and thus obtain a high-concentration seed culture liquid, providing a sufficient and high-quality seed source for fermentation culture.

[0110] Fermentation culture principle:

[0111] Temperature control: The temperature is stably controlled at around 37°C, which is close to the optimal growth temperature of lactic acid bacteria. This can ensure that the growth rate and metabolic activity of the strain are in a good state, which is conducive to the efficient synthesis of lactic acid.

[0112] pH adjustment: With the help of an automatic pH adjustment device, add a buffer or acid-base regulator to maintain the pH of the fermentation liquid in an appropriate range (5.5-6.5 for D-lactic acid bacteria, and adjust the pH of L-lactic acid bacteria to a range that is conducive to lactic acid accumulation based on the characteristics of the strain), providing a stable acid-base environment for the growth and metabolism of lactic acid bacteria and promoting the continuous production and accumulation of lactic acid.

[0113] Dissolved oxygen regulation: Based on the aerobic requirement of the strain, the dissolved oxygen concentration of anaerobic lactic acid bacteria is strictly controlled by using dissolved oxygen electrodes and ventilation control systems to avoid the inhibitory or toxic effects of oxygen on them; for facultative anaerobic or microaerobic lactic acid bacteria, an appropriate amount of oxygen is provided to meet their growth and metabolic needs.

[0114] Batch feeding fermentation: The online monitoring system monitors the consumption of nutrients (carbon source, nitrogen source, etc.) in the fermentation process in real time, and replenishes carbon source (such as glucose, sucrose, etc.) and nitrogen source (such as peptone, yeast extract, etc.) in a timely manner according to the monitoring results, continuously providing sufficient nutrition for the strain and maintaining its high-yield metabolic state. At the same time, the advanced fermentation control system conducts all-round real-time monitoring and precise adjustment of fermentation parameters such as temperature, pH value, dissolved oxygen, and stirring speed to ensure stable and efficient operation of the fermentation process.

[0115] Fermentation broth detection principle:

[0116] Lactic acid content detection:

[0117] Sample processing: separate the bacteria and the fermentation supernatant by high-speed centrifugation, set the addition amount gradient of the fermentation supernatant and the clarifier (30% H2O2) to remove impurities, add water to dilute to the appropriate concentration range, and then filter through a 0.22μm water-based polyethersulfone filter to ensure the purity of the sample and avoid damage to the chromatographic column, so as to obtain the test sample for liquid phase detection.

[0118] High performance liquid chromatography analysis: A specific chromatographic column (Shim-pack GISTC18) was selected, and an optimized mobile phase (0.01 mol / L dipotassium hydrogen phosphate solution and 100% methanol were mixed in a ratio of 0.95:0.05) was used. Under the set flow rate (1 mL / min), column box temperature (30°C) and detection wavelength (210 nm), effective separation and high-sensitivity detection of lactic acid on the chromatographic column were achieved, thereby accurately determining the lactic acid content in the fermentation broth.

[0119] Lactic acid optical purity content detection:

[0120] Sample treatment: The pH of the fermentation liquid was adjusted to a specific value (about 2.0) with hydrochloric acid, and anhydrous ether was used to extract the fermentation supernatant to distribute the lactic acid to the organic phase. After oscillation and standing for stratification, the upper organic phase was taken to remove some impurities and enrich lactic acid.

[0121] Collect lactic acid by vacuum distillation: The organic phase is mixed with ultrapure water and then distilled under reduced pressure. By precisely controlling the negative pressure (about 0.9 MPa), temperature (35°C) and cooling water circulation pump temperature (5°C) of the rotary evaporator, lactic acid is separated and collected from the organic phase at low temperature to avoid decomposition of lactic acid or other chemical reactions at high temperature, thereby ensuring the purity and structural integrity of the lactic acid.

[0122] High performance liquid chromatography analysis: using a chiral separation column (Chirex3126 (D) -penicillam), with an optimized mobile phase (2mmol / LCuSO4 · 5H2O salt solution and 100% pure isopropanol mixed in a ratio of 95:5), under the set flow rate (1mL / min), column box temperature (26 ° C) and detection wavelength (254nm) conditions, to achieve efficient separation of D-lactic acid and L-lactic acid optical isomers, and accurately determine the optical purity of lactic acid in the fermentation broth.

[0123] The effect produced:

[0124] Improve acid production and optical purity: From screening high-potential strains to accurately optimizing the conditions of each culture stage, the whole process creates favorable conditions for efficient acid production by lactic acid bacteria. The selected high-yield strains have strong acid production capabilities. The subsequent precise temperature, pH value, dissolved oxygen control and reasonable nutrient supplementation enable lactic acid bacteria to synthesize a large amount of specific types of lactic acid in a stable manner during the metabolic process, while ensuring a high optical purity to meet the needs of the food, medicine and other industries for high-quality lactic acid. For example, in the D-lactic acid bacteria culture example, the D-lactic acid production reached 120g / L after 48h of fermentation, and the optical purity was 98%.

[0125] Stable fermentation process: The advanced fermentation control system monitors and accurately adjusts the fermentation parameters in real time, so that the entire fermentation process is always in a relatively stable environment. It avoids fermentation abnormalities caused by temperature fluctuations, pH changes, dissolved oxygen abnormalities, etc., reduces the risk of fermentation failure, improves production efficiency and product quality stability and consistency, and ensures the continuity and reliability of industrial production.

[0126] Reduce production costs: The optimized culture medium formula accurately adjusts the nutrients according to the characteristics of the strain, improves the utilization efficiency of nutrients, and reduces unnecessary waste. The batch feed fermentation method replenishes nutrients in a timely manner according to the consumption of nutrients, avoiding the waste caused by too much raw material input at one time. The stable fermentation process reduces the economic losses caused by fermentation failure, reduces the overall production cost from the two aspects of raw material utilization and production stability, and improves the economic benefits of the enterprise.

[0127] Guaranteeing detection accuracy: The fermentation broth detection method has been carefully designed and optimized in every link from sample processing to high-performance liquid chromatography analysis. The sample processing process effectively removes impurities and enriches the target substance. The selection of specific chromatographic columns, mobile phases and detection conditions ensures the accurate determination of lactic acid content and optical purity, providing reliable data support for evaluating the fermentation effect, helping to adjust the fermentation process in a timely manner and further optimize the production process.

[0128] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for culturing lactic acid bacteria, comprising culturing D-lactic acid bacteria and L-lactic acid bacteria, characterized in that: The culture method of D-lactic acid bacteria and the culture method of L-lactic acid bacteria both include five stages, namely, strain screening, bacterial agent preparation, shake flask culture, seed culture and fermentation culture.

2. The method for culturing lactic acid bacteria according to claim 1, characterized in that: The method for culturing D-lactic acid bacteria comprises the following steps: S1. strain screening, collecting microbial samples from natural environments, including but not limited to soil, plant surfaces, and animal intestines; placing the collected samples in MRS liquid medium for enrichment culture, using high performance liquid chromatography to detect the lactic acid production and optical purity of each strain, and screening out high-yield strains with a D-lactic acid / lactic acid ratio of more than 95% for subsequent culture; S2, preparation of bacterial agent: the selected high-yield D-lactic acid strain is expanded and cultured in MRS liquid culture medium, and the culture conditions are temperature 35-39°C, shaker speed 150-200r / min, and culture time 18-24h; after the culture, the bacterial bodies are collected by centrifugation at a speed of 6000-7000r / min for 10-15min, and the bacterial bodies are mixed with a freeze-drying protective agent containing 10-15% skim milk powder, 5-8% trehalose, and 2-4% mannitol at a ratio of 1:1-1.2, and freeze-dried at -50°C to -60°C and a vacuum degree of 10-15Pa for 24-30h to prepare bacterial powder, which is stored in an environment below -20°C for standby use; S3, shake flask culture: take an appropriate amount of the above bacterial powder and inoculate it into MRS liquid culture medium, the inoculation amount is 0.5-1% (w / v), and shake flask culture is carried out for 12-18 hours at 30-40°C and a shaker speed of 100-140r / min to activate and proliferate the strain; S4, seed culture: inoculate the activated bacterial solution into a seed culture medium at an inoculum rate of 3-8%, wherein the seed culture medium contains 10-15 g / L peptone, 8-12 g / L beef extract, 5-8 g / L yeast powder, 20-30 g / L glucose, 2-3 g / L potassium dihydrogen phosphate, 0.5-0.8 g / L magnesium sulfate, and 0.2-0.3 g / L manganese sulfate; culture at 35-39°C for 16-20 hours in an anaerobic environment to obtain a high-concentration seed culture solution; S5. Fermentation culture: Inoculate the seed culture solution into the fermentation medium at an inoculation rate of 8-15%, control the temperature at 36-38°C through a temperature control system, add a buffer or an acid-base regulator using an automatic pH adjustment device to maintain the pH value of the fermentation solution at 5.5-6.5; control the dissolved oxygen concentration at 0-1 mg / L with the help of a dissolved oxygen electrode and a ventilation control system according to the aerobic characteristics of the strain; adopt a batch feed fermentation method, when the glucose concentration in the fermentation solution is lower than 5 g / L, supplement 50% (w / v) glucose solution, and when the ammonia nitrogen concentration is lower than 0.5 g / L, supplement 20% (w / v) peptone solution; and simultaneously use a fermentation control system to monitor and adjust the fermentation parameters of temperature, pH value, dissolved oxygen, and stirring speed in real time to increase the yield and optical purity of D-lactic acid.

3. The method for culturing lactic acid bacteria according to claim 2, characterized in that: The method for culturing L-lactic acid bacteria comprises the following steps: S1. Strain screening: Collect microbial samples from the natural environment, place them in MRS liquid medium for enrichment culture, and screen out strains with high L-lactic acid production capacity by high performance liquid chromatography; S2, preparation of bacterial agent: the selected high-yield L-lactic acid strain is expanded and cultured in an optimized MRS liquid medium, wherein the carbon source is 20-30 g / L maltose, the nitrogen source is 10-15 g / L soy peptone, the culture temperature is 35-39°C, the shaker speed is 160-220 r / min, and the culture time is 20-26 h; after the culture, the bacterial bodies are collected by centrifugation at a speed of 6000-7000 r / min for 12-16 min, and mixed with a freeze-drying protective agent containing 12-18% lactose, 6-10% glycerol, and 3-5% polyvinyl pyrrolidone at a ratio of 1:1-1.3, and freeze-dried at -55°C to -65°C and a vacuum degree of 8-12 Pa for 26-32 h to prepare bacterial powder, which is stored in an environment below -25°C for standby use; S3, shake flask culture: take an appropriate amount of the above bacterial powder and inoculate it into MRS liquid culture medium, the inoculation amount is 0.6-1.2% (w / v), and shake flask culture is carried out for 13-19 hours at 31-39°C and a shaker speed of 100-140 r / min to activate and proliferate the strain; S4, seed culture: inoculate the activated bacterial solution into a seed culture medium at an inoculum volume of 4-9%, wherein the seed culture medium contains 11-16 g / L of peptone, 9-13 g / L of beef extract, 6-9 g / L of yeast powder, 22-32 g / L of glucose, 2.2-3.2 g / L of dipotassium hydrogen phosphate, 0.55-0.85 g / L of magnesium sulfate, and 0.22-0.32 g / L of manganese sulfate; culture at 36-38°C for 17-21 hours in an anaerobic environment to obtain a high-concentration seed culture solution; S5. Fermentation culture: Inoculate the seed culture medium at an inoculation rate of 9-16% into the fermentation medium. According to the characteristics of L-lactic acid producing bacteria, control the temperature at 36-38°C. Add buffer or acid-base regulator through the pH automatic adjustment system to maintain the pH value of the fermentation liquid in a range that is conducive to the accumulation of L-lactic acid. For anaerobic L-lactic acid producing bacteria, use anaerobic fermentation tanks to ensure that the dissolved oxygen concentration in the fermentation liquid is lower than 0.1 mg / L. Use batch fed fermentation to replenish carbon and nitrogen sources in a timely manner according to the consumption of nutrients during the fermentation process. Use advanced fermentation control systems to monitor and accurately adjust various parameters in the fermentation process in real time. Fermentation tanks of different volumes and stirring methods can also be selected according to the characteristics of the strain and the requirements of the fermentation process to increase the yield and optical purity of L-lactic acid.

4. The method for culturing lactic acid bacteria according to claim 3, characterized in that: The culture temperature is controlled at 36-38°C during the entire culture process. This temperature range is close to the optimal growth temperature of lactic acid bacteria and can ensure that the growth rate and metabolic activity of the strain are in a good state.

5. The method for culturing lactic acid bacteria according to claim 4, characterized in that: During the fermentation process, the pH value of the fermentation broth is maintained within an appropriate range by adding a buffer or an acid-base regulator. The appropriate range for D-lactic acid bacteria is 5.5-6.5, and for L-lactic acid bacteria, the pH value range is determined to be favorable for L-lactic acid accumulation according to the production characteristics of the bacteria.

6. The method for culturing lactic acid bacteria according to claim 5, characterized in that: During the seed culture stage, anaerobic fermentation culture is carried out, using anaerobic incubators or adding anaerobic gas production bags to ensure that lactic acid bacteria can grow and reproduce normally in an anaerobic or micro-aerobic environment.

7. The method for culturing lactic acid bacteria according to claim 6, characterized in that: During the fermentation and cultivation stage, a batch fed fermentation method is adopted. According to the consumption of nutrients in the fermentation process, when the carbon source concentration in the fermentation liquid is lower than the set value, a carbon source such as glucose or sucrose is supplemented. When the nitrogen source concentration is lower than the set value, a nitrogen source such as peptone or yeast extract is supplemented.

Images