Process for producing lactic acid through continuous fermentation

Abstract

The invention discloses a process for producing lactic acid through continuous fermentation. The process comprises the following steps: N fermentation tanks are connected in series to form a multi-tank continuous fermentation system; a sterile culture medium A is introduced into the first fermentation tank; when the first fermentation tank enters a stable growth period, the continuous fermentation system is started, a sterile culture medium B is added into the first fermentation tank at a stable speed, meanwhile, when the fermentation liquor in the first fermentation tank reaches 80%-85% of the volume of the first fermentation tank, the sterile culture medium B overflows into the second fermentation tank, and similarly, when the fermentation liquor in the second fermentation tank reaches 80%-85% of the volume of the second fermentation tank, the sterile culture medium B overflows into the second fermentation tank. And overflowing into the third fermentation tank, and so on until the fermentation liquid overflowing from the Nth fermentation tank is completely fermented, thereby realizing continuous overflowing discharge. According to the method disclosed by the invention, continuous culture is carried out by feeding a high-sugar-concentration culture medium with the concentration of 30%-32%, and the content of lactic acid in fermentation liquor after fermentation is finished reaches 231.58 g / L to the maximum, which is 24.79%-35.84% higher than that of single-tank fermentation.

Description

Technical Field

[0001] This invention relates to the field of lactic acid fermentation technology, specifically to a process for continuous fermentation to produce lactic acid. Background Technology

[0002] Lactic acid is an important organic acid that is widely used in the food, pharmaceutical and chemical industries. In particular, it has attracted much attention as a monomer for the synthesis of polylactic acid, a biodegradable material. Currently, in industry, lactic acid is mainly produced through microbial fermentation, with calcium hydroxide used for pH adjustment. However, in single-tank fermentation, the amount of culture medium added to the fermentation tank is limited to only about 50%-60% of the total tank volume due to the limitation on the amount of calcium hydroxide added, resulting in low tank utilization. Furthermore, due to the limitation on the maximum sugar concentration tolerated by the strain during growth, the unit acid content in the fermentation broth remains between 16%-18%. For example, patent number CN104178438A, entitled "A German Lactobacillus suitable for molasses fermentation to produce high-purity L-lactic acid and its fermentation method and application," uses the German Lactobacillus strain Lactobacillus delbrueckii SZ01, CCTCC NO: M2014268. The highest initial sugar concentration reached 23%, and with the use of calcium hydroxide to adjust the pH, the lactic acid content after single-tank fermentation was still 180.70 g / L, and could not be increased further.

[0003] Patent CN121109221A, entitled "A Strains for the Production of High Optical Purity L-Lactic Acid and Their Applications," screened out a strain for producing L-lactic acid. While the pH was adjusted using ammonia, the final lactic acid yield reached 225.5 g / L, the maximum sugar concentration was limited to 23%. Furthermore, this method discloses single-tank fermentation, which requires multiple breeding and transfer processes, as well as repeated cleaning and sterilization of the fermentation tank, consuming significant manpower, resources, and time. Patent CN115627278A, entitled "A Method for Continuous Fermentation to Prepare Lactic Acid," also utilizes continuous fermentation with strains, but it involves single-tank fermentation followed by strain collection and transfer. While this increases the inoculum size to some extent, it is still essentially single-tank fermentation. Moreover, due to limitations in alkali addition and strain sugar tolerance, the utilization rate of the fermentation tank is low, and the unit acid content in the fermentation broth cannot be increased, thus failing to truly reduce production costs for enterprises.

[0004] The patent, CN119913217A, entitled "A Method for Continuous Fermentation to Produce Lactic Acid," discloses a continuous fermentation process. However, the disclosed process involves continuous cultivation of the seed culture, in which the continuously cultivated seed culture is transferred to multiple separate fermentation tanks. After each fermentation tank completes its fermentation, it still needs to be cleaned and sterilized again before receiving the seed culture for further fermentation. This ensures the continuity of the seed culture but eliminates the need for multiple cleaning and sterilization processes in the seed tanks. However, it does not eliminate the need for multiple cleaning and sterilization processes in the fermentation tanks. Summary of the Invention

[0005] The first objective of this invention is to provide a method for continuous fermentation of lactic acid, which utilizes multiple tanks connected in series to form a continuous fermentation system. Adjacent fermenters are connected by connecting pipes, and the connecting pipes are positioned at 80%-85% of the total volume of the fermenters. This allows for transfer between fermenters via overflow, enabling continuous fermentation from a single inoculation. This increases the utilization rate of the fermenters to 80%-85%, while the overflow method saves energy consumption and significantly reduces energy consumption. The second objective of this invention is to provide a method for high-sugar fermentation of lactic acid, wherein a high-sugar culture medium with a sugar concentration of 30%-32% is stably added to the fermentation tank during a multi-tank series continuous fermentation process, thereby increasing the acid production rate per unit volume of the fermentation broth. The third objective of this invention is to provide a method for fermenting lactic acid from starch, wherein undiluted starch-based saccharification medium is stably added to the fermentation tanks during a multi-tank continuous fermentation process, thereby increasing the acid production rate per unit volume of the fermentation broth and reducing the number of fermentation steps.

[0006] According to the technical solution provided in the embodiments of this application, a process for continuous fermentation to produce lactic acid includes the following steps: 1) The first fermenter, the second fermenter, ... the Nth fermenter are connected in series by connecting pipes to form a multi-tank continuous fermentation system; 2) Sterile culture medium A and seed liquid of Lactobacillus de Germanis strain were introduced into the first fermenter for the initial fermentation culture of the strain. 3) Once the strain in the first fermenter enters the stable growth phase, the continuous fermentation system is started. Sterile culture medium B is added to the first fermenter at a stable rate. At the same time, when the fermentation broth in the first fermenter reaches 80%-85% of the total volume of the fermenter, it overflows through the connecting pipe into the second fermenter. Similarly, when the fermentation broth in the second fermenter reaches 80%-85% of the total volume of the fermenter, it overflows into the third fermenter, and so on, until the fermentation broth overflowing from the Nth fermenter is completely fermented, achieving uninterrupted overflow. The sugar concentration of sterile culture medium B is 30%-32%.

[0007] Preferably, the sterile culture medium B is one of glucose culture medium or starch saccharification culture medium. The starch saccharification in the starch saccharification culture medium can be directly added to the first fermenter, reducing the dilution process of starch saccharification.

[0008] Preferably, the sugar concentration of sterile culture medium A is 8%-12%, which is lower than that of sterile culture medium B, and is used for the proliferation and growth of strains in the initial fermentation system.

[0009] Preferably, in the continuous fermentation system, the addition rate of sterile culture medium B is 1:9 to 1:11, which is the ratio of the volume of sterile culture medium B to the volume of the first fermenter, so as to maximize the utilization rate of the fermenter.

[0010] In summary, the beneficial effects of this application are as follows: 1. This invention utilizes a multi-tank series continuous fermentation system. When the volume inside the tank reaches a fixed 80%-85%, the tank is transferred by overflow. This ensures the optimal utilization rate of the fermentation tank and improves the equipment utilization rate. Furthermore, it eliminates the need for the transfer equipment to operate, significantly reducing the use of transfer equipment and electricity, thus saving energy consumption. 2. This invention utilizes multiple tanks in series for continuous fermentation, using a low-sugar-concentration culture medium to proliferate the strain, and then continuously culturing it by adding a 30%-32% high-sugar-concentration culture medium. After fermentation, the highest lactic acid content in the fermentation broth reached 229.57 g / L, which is 46.71% higher than the lactic acid yield of 156.48 g / L obtained by single-tank fermentation using the same strain. 3. This invention utilizes undiluted starch-based saccharification medium directly added to a multi-tank continuous fermentation system, eliminating the need for dilution of the starch-based saccharification solution with water and storage equipment. Furthermore, the lactic acid content in the fermentation broth reaches 231.58 g / L after fermentation, increasing lactic acid yield while reducing raw material costs and significantly reducing the number of steps in lactic acid production. It also reduces the water content of the starch-based saccharification solution, alleviating the evaporation pressure in subsequent extraction stages. This results in a reduction of over 20% in lactic acid production costs due to lower water and steam consumption. 4. This invention utilizes multiple tanks connected in series for continuous fermentation, enabling simultaneous fermentation in multiple tanks with a single inoculation. This improves the utilization rate of fermentation equipment and saves non-fermentation time spent on repeated tank washing and sterilization, as well as the energy consumption of water and gas used for tank washing and sterilization, thus significantly reducing the cost of lactic acid production for enterprises. Attached Figure Description

[0011] Other features, objects, and advantages of this application will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This is a schematic diagram illustrating the effect of sugar concentration on lactic acid content during single-tank fermentation of the strain of this invention. Detailed Implementation

[0012] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings.

[0013] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0014] Seed culture: Lactic acid bacteria (laboratory-prepared strains) were activated using solid LB medium slant, then inoculated into liquid medium using an inoculation loop and cultured. The culture was then further expanded to a seed tank. Once the strains had proliferated to the stable phase, the seed culture was obtained.

[0015] The lactic acid bacteria strain used is patent number CN104178438A, entitled "A strain of Lactobacillus de Germanis suitable for the production of high-purity L-lactic acid by molasses fermentation, and the fermentation method and application described therein". Lactobacillus delbrueckii SZ01, CCTCC NO: M2014268.

[0016] Example 1: The highest sugar concentration yielded by acid production in a single-tank fermentation The seed culture was inoculated into a 10L fermenter at an inoculation rate of 8%-10%. Sterile culture medium was added to the fermenter and the volume was adjusted to 6L before incubation. The incubation conditions were: rotation speed of 150r / min, temperature of 55℃, pH of 6.8, and calcium hydroxide solution was used as a neutralizing agent with a concentration of 20%-23%. After fermentation, the acid production rate was measured.

[0017] The initial sugar concentrations of the sterile culture medium were set at 16%, 18%, 20%, 22%, and 24% (w / v). The fermentation medium formulation with an initial sugar concentration of 16% is as follows: glucose 16%, ammonium sulfate 3%, Tween 80 0.1%, sodium acetate 0.5%, triammonium citrate 0.2%, dipotassium hydrogen phosphate 0.2%, magnesium sulfate heptahydrate 0.02%, and manganese sulfate monohydrate 0.005%. The fermentation medium formula with an initial sugar concentration of 18% is as follows: glucose 18%, ammonium sulfate 3%, Tween 80 0.1%, sodium acetate 0.5%, triammonium citrate 0.2%, dipotassium hydrogen phosphate 0.2%, magnesium sulfate heptahydrate 0.02%, and manganese sulfate monohydrate 0.005%. The fermentation medium formula with an initial sugar concentration of 20% is as follows: glucose 20%, ammonium sulfate 3%, Tween 80 0.1%, sodium acetate 0.5%, triammonium citrate 0.2%, dipotassium hydrogen phosphate 0.2%, magnesium sulfate heptahydrate 0.02%, and manganese sulfate monohydrate 0.005%. The fermentation medium formulation with an initial sugar concentration of 22% is as follows: glucose 22%, ammonium sulfate 3%, Tween 80 0.1%, sodium acetate 0.5%, triammonium citrate 0.2%, dipotassium hydrogen phosphate 0.2%, magnesium sulfate heptahydrate 0.02%, and manganese sulfate monohydrate 0.005%. The fermentation medium formulation with an initial sugar concentration of 24% is as follows: glucose 24%, ammonium sulfate 3%, Tween 80 0.1%, sodium acetate 0.5%, triammonium citrate 0.2%, dipotassium hydrogen phosphate 0.2%, magnesium sulfate heptahydrate 0.02%, and manganese sulfate monohydrate 0.005%. The fermentation process ends when the residual sugar in the fermentation broth is less than 0.5 g / L.

[0018] Depend on Figure 1 It can be seen that when using laboratory-prepared lactic acid bacteria for fermentation, the sugar in the fermenter can be completely consumed when the initial sugar concentration is 16%-20%, that is, the fermentation can be complete; however, when the initial sugar concentration is 24%, the sugar in the fermenter cannot be completely consumed, which also indicates that the maximum sugar concentration that this strain can tolerate is not 24%. When the sugar concentration in the culture medium is higher than 24%, the strain may not be able to grow normally due to osmotic pressure.

[0019] Meanwhile, at the end of fermentation, the fermenter volumes corresponding to initial sugar concentrations of 16%, 18%, 20%, and 22% were 7 (±0.3) L, 7.3 (±0.3) L, 7.7 (±0.3) L, and 8.0 (±0.3) L, respectively. This means the utilization rate of a 10 L fermenter was less than 8.0 L, i.e., below 80%. In actual industrial production, 350M... 3 The volumetric accuracy of fermenters can have significant errors. Therefore, in industrial production, fermenters are typically filled to around 50% capacity, resulting in lower utilization rates. Furthermore, the concentration of calcium hydroxide is not entirely constant during actual production; it is affected by various factors such as human intervention during preparation and the moisture content of raw materials. This causes variations in the volume added to the fermenter for acid adjustment. In single-tank fermentation, these factors are taken into account, ensuring the fermenter has sufficient volume to hold the calcium hydroxide solution, ultimately reducing the tank's utilization rate to below 80%.

[0020] Example 2:

[0021] Continuous fermentation using a multi-tank series system N fermenters are connected in series by connecting pipes, with the connecting pipes connecting adjacent fermenters at 80%-85% of their volume. The connecting pipes are equipped with on / off valves.

[0022] First, introduce sterile culture medium A into the first fermenter, bringing the volume to 50%-60%. Then, inoculate the seed culture into the first fermenter at an inoculum rate of 8%-10%. Fermentation is carried out under the following conditions: rotation speed 150 r / min, temperature 55℃, pH 6.8, using calcium hydroxide solution as a neutralizing agent. Once the bacterial strain in the fermenter has reached the stationary phase, introduce sterile culture medium B through the inlet of the first fermenter. Simultaneously, open the valve on the connecting pipe between the first and second fermenters, allowing the first fermenter to... The fermentation broth in the first fermenter overflows into the second fermenter through the connecting pipe. As the sterile culture medium B is continuously added to the first fermenter, the liquid in the second fermenter will continuously increase until the fermentation broth in the second fermenter overflows into the third fermenter through the connecting pipe connecting the first and second fermenters. Similarly, the third fermenter overflows into the fourth fermenter, and so on, until it overflows into the Nth fermenter. When the residual sugar in the fermentation broth at the overflow point of the Nth fermenter is less than 0.5 g / L, the fermentation can be considered complete.

[0023] The sterile culture medium A consists of: 8-12% glucose, 3% ammonium sulfate, 0.1% Tween 80, 0.5% sodium acetate, 0.2% triammonium citrate, 0.2% dipotassium hydrogen phosphate, 0.02% magnesium sulfate heptahydrate, and 0.005% manganese sulfate monohydrate. The initial sugar concentration of sterile culture medium B was set as follows: 22%, 26%, 28%, 30%, 32%, 34%. The sterile culture medium B with an initial sugar concentration of 22% consisted of: 22% glucose, 3% ammonium sulfate, 0.1% Tween 80, 0.5% sodium acetate, 0.2% triammonium citrate, 0.2% dipotassium hydrogen phosphate, 0.02% magnesium sulfate heptahydrate, and 0.005% manganese sulfate monohydrate. The sterile culture medium B with an initial sugar concentration of 26% consisted of: 26% glucose, 3% ammonium sulfate, 0.1% Tween 80, 0.5% sodium acetate, 0.2% triammonium citrate, 0.2% dipotassium hydrogen phosphate, 0.02% magnesium sulfate heptahydrate, and 0.005% manganese sulfate monohydrate. The sterile culture medium B with an initial sugar concentration of 28% consisted of: 28% glucose, 3% ammonium sulfate, 0.1% Tween 80, 0.5% sodium acetate, 0.2% triammonium citrate, 0.2% dipotassium hydrogen phosphate, 0.02% magnesium sulfate heptahydrate, and 0.005% manganese sulfate monohydrate. The sterile culture medium B with an initial sugar concentration of 30% consists of: 30% glucose, 3% ammonium sulfate, 0.1% Tween 80, 0.5% sodium acetate, 0.2% triammonium citrate, 0.2% dipotassium hydrogen phosphate, 0.02% magnesium sulfate heptahydrate, and 0.005% manganese sulfate monohydrate. The sterile culture medium B with an initial sugar concentration of 32% consisted of: 32% glucose, 3% ammonium sulfate, 0.1% Tween 80, 0.5% sodium acetate, 0.2% triammonium citrate, 0.2% dipotassium hydrogen phosphate, 0.02% magnesium sulfate heptahydrate, and 0.005% manganese sulfate monohydrate. The sterile culture medium B with an initial sugar concentration of 34% consisted of: 34% glucose, 3% ammonium sulfate, 0.1% Tween 80, 0.5% sodium acetate, 0.2% triammonium citrate, 0.2% dipotassium hydrogen phosphate, 0.02% magnesium sulfate heptahydrate, and 0.005% manganese sulfate monohydrate. In actual fermentation, when the initial sugar concentration of the fed-batch medium was 34%, the growth of the strain showed signs of decline when the OD value was measured by sampling the first fermenter. This may be because the high concentration of sugar fed into the fermenter resulted in a sugar concentration greater than 20%, exceeding the maximum sugar tolerance concentration of the strain and thus limiting its growth.

[0024] Once fermentation is complete, take the fermentation liquid overflowing from the Nth fermentation tank and test the concentration of acid produced in the fermentation liquid.

[0025] Table 1. Effects of different fed-batch culture media—sterile medium B—on lactic acid production.

[0026] As can be seen from Table 1, when the sugar concentration of fed-batch medium-sterile medium B is 30%-32%, the lactic acid yield can reach 212.75 g / L and 229.57 g / L, which is 24.79%-34.66% higher than the lactic acid yield of 170.48 g / L obtained by single-tank fermentation in Example 1.

[0027] The data above shows that when multiple tanks are used for continuous fermentation in series, the use of a high-sugar fed-batch culture medium can increase the lactic acid content per unit volume of fermentation broth by 35.96%-46.71% after fermentation. Since the solution in each fermenter overflows at 80%-85%, it indicates that the utilization rate of each fermenter in the series system reaches 80%-85%, which is significantly higher than the utilization rate of the tanks when fermenting in a single tank.

[0028] Example 3:

[0029] Continuous fermentation of starch for sugar production Starch saccharification process: After adjusting the pH of starch milk to 5.5-5.8, liquefying enzyme is added and liquefaction is carried out by spraying. Saccharifying enzyme is added to adjust the pH to 4.2-4.5 for saccharification. After 36 hours, the reducing sugar content in starch is determined to be 90%-96% using the Fehling's method.

[0030] Since the sugar content of starch-based saccharification reaches over 90%, and considering that the strain's sugar tolerance value is 20%, the starch-based saccharification solution needs to be diluted before being mixed with the basic nutrient culture medium during single-tank fermentation. This ensures that the sugar concentration in the fermentation medium is below 20%, guaranteeing the normal fermentation and growth of the strain.

[0031] In Example 2, it was found that the sugar concentration of the fed-batch medium could reach 30%-32% during continuous fermentation. However, in real life, a fermentation medium with a sugar concentration of 30%-32% can be directly prepared using undiluted starch saccharification and a basic nutrient medium.

[0032] Preparation process of starch-to-sugar culture medium: Preparation of basic nutrient culture medium: ammonium sulfate 9%, Tween 80 0.3%, sodium acetate 1.5%, triammonium citrate 0.6%, dipotassium hydrogen phosphate 0.6%, magnesium sulfate heptahydrate 0.06%, and manganese sulfate monohydrate 0.015%; ② Addition of hydrolyzed starch milk: Mix the basic nutrient culture medium with the hydrolyzed starch milk at a volume ratio of 2:1 to obtain the starch saccharification culture medium.

[0033] First, sterile culture medium A is introduced into the first fermenter, and the volume is adjusted to 50%-60%. Then, the seed culture is inoculated into the first fermenter at an inoculum rate of 8%-10%. Fermentation is carried out under the following conditions: rotation speed of 150 r / min, temperature of 55℃, pH set to 6.8, and calcium hydroxide solution is used as a neutralizing agent. After the strain in the fermenter has proliferated to the stationary phase, sterile starch saccharification medium is introduced into the feed inlet of the first fermenter. At the same time, the valve on the connecting pipe between the first and second fermenters is opened, allowing the first fermenter to start fermenting. The fermentation broth in the first fermenter overflows into the second fermenter through a connecting pipe. As the sterile starch saccharification medium is continuously added to the first fermenter, the liquid in the second fermenter will continuously increase until the fermentation broth overflows into the third fermenter through the connecting pipe connecting the first and second fermenters. Similarly, the third fermenter overflows into the fourth fermenter, and so on, until it overflows into the Nth fermenter. When the residual sugar in the fermentation broth at the overflow point of the Nth fermenter is less than 0.5 g / L, fermentation can be considered complete.

[0034] There are two feeding methods for the starch saccharification medium. One method is to mix the basic nutrient medium and the starch saccharification solution and then feed them into the first fermenter. The other method is to feed the basic nutrient medium and the hydrolyzed starch saccharification solution directly into the first fermenter according to the preparation ratio. After experiments, it was found that these two feeding methods have no effect on the continuous fermentation of the strain.

[0035] The lactic acid content in the fermentation broth overflowing from the Nth fermenter was measured at 231.58 g / L, indicating that starch saccharification can be used as a continuous fermentation medium, saving raw material costs for enterprises. Furthermore, traditional processes require water dilution when preparing the medium using starch saccharification. This excess water occupies storage space and needs to be evaporated in the subsequent extraction stage. Therefore, by directly preparing the medium using starch saccharification, enterprises can reduce raw material costs while saving over 20% on water and steam consumption costs.

[0036] Example 4:

[0037] The effect of feed rate on the number of tanks in series N fermenters are connected in series by connecting pipes, with the connecting pipes connecting adjacent fermenters at 80%-85% of their volume. The connecting pipes are equipped with on / off valves.

[0038] First, introduce sterile culture medium A into the first fermenter, bringing the volume to 50%-60%. Then, inoculate the seed culture into the first fermenter at an inoculum rate of 8%-10%. Fermentation is carried out under the following conditions: rotation speed 150 r / min, temperature 55℃, pH 6.8, using calcium hydroxide solution as a neutralizing agent. Once the bacterial strain in the fermenter has reached the stationary phase, introduce sterile culture medium B through the inlet of the first fermenter. Simultaneously, open the valve on the connecting pipe between the first and second fermenters, allowing the first fermenter to... The fermentation broth in the first fermenter overflows into the second fermenter through the connecting pipe. As the sterile culture medium B is continuously added to the first fermenter, the liquid in the second fermenter will continuously increase until the fermentation broth in the second fermenter overflows into the third fermenter through the connecting pipe connecting the first and second fermenters. Similarly, the third fermenter overflows into the fourth fermenter, and so on, until it overflows into the Nth fermenter. When the residual sugar in the fermentation broth at the overflow point of the Nth fermenter is less than 0.5 g / L, the fermentation can be considered complete.

[0039] The sterile culture medium A consists of: 8-12% glucose, 3% ammonium sulfate, 0.1% Tween 80, 0.5% sodium acetate, 0.2% triammonium citrate, 0.2% dipotassium hydrogen phosphate, 0.02% magnesium sulfate heptahydrate, and 0.005% manganese sulfate monohydrate. The culture medium for sterile medium B is: 30% glucose, 3% ammonium sulfate, 0.1% Tween 80, 0.5% sodium acetate, 0.2% triammonium citrate, 0.2% dipotassium hydrogen phosphate, 0.02% magnesium sulfate heptahydrate, and 0.005% manganese sulfate monohydrate.

[0040] The feeding rate of sterile culture medium B was adjusted to (based on the ratio of the volume of sterile culture medium B per hour to the volume of the first fermenter) as follows: 1:8, 1:9, 1:10, 1:11, 1:12. That is, when the flow rate of sterile culture medium B is 1:8, assuming the volume of the fermenter is 350m³. 3 Therefore, the flow acceleration of sterile culture medium B is 43.75 m. 3 / h; Similarly, when the flow rate of sterile culture medium B is 1:9, assuming the volume of the fermenter is 350m³, 3 Therefore, the flow rate of sterile culture medium B is 38.89 m / s. 3 / h; Similarly, when the flow rate of sterile culture medium B is 1:10, that is, the flow rate is 35m. 3 / h; When the flow rate of sterile culture medium B is 1:11, that is, the flow rate is 31.82m. 3 / h; When the flow rate of sterile culture medium B is 1:12, that is, the flow rate is 29.17m. 3 / h; The residual sugar in the overflow fermentation liquid of each fermenter is tested. When the residual sugar in the overflow fermentation liquid is less than 0.5 g / L, the fermentation can be judged to be complete.

[0041] Table 2. Effect of fed-batch culture rate on the number of fermenters in series

[0042] As shown in Table 2, when the flow rate of sterile culture medium B with a sugar concentration of 30% (measured as the ratio of the volume of sterile culture medium B per hour to the volume of the first fermenter) is 1:8, the residual sugar concentration in the fermentation broth overflowing from the seventh fermenter reaches 0.37 g / L, indicating that seven fermenters need to be connected in series to completely consume the sugar in the fermentation broth. When the flow rate is 1:9 to 1:12, the residual sugar concentration in the fermentation liquid overflowing from the fifth fermenter is less than 0.5 g / L, indicating that five fermenters need to be connected in series to completely consume the sugar in the fermentation liquid. As shown above, when the flow rate of sterile culture medium B with a sugar concentration of 30% (measured as the ratio of the volume of sterile culture medium B per hour to the volume of the first fermenter) is 1:9, the number of fermenters used is five, meaning the utilization rate of the fermenters can be maximized. However, in actual production, since the sugar concentration and flow rate of sterile culture medium B cannot be maintained very precisely, we set the initial sugar concentration of sterile culture medium B between 30% and 32% and the flow rate between 1:9 and 1:11 in actual production. This ensures both high utilization of the fermentation equipment and sufficient outflow rate.

[0043] As shown above, by using five fermenters connected in series for continuous and uninterrupted discharge, the liquid volume of all fermenters can reach 80%-85%, greatly improving the utilization rate of the tanks. Simultaneously, when the initial sugar concentration of the fed-batch sterile culture medium B is set at 30%-32% and the feeding rate is set between 1:9 and 1:11, the lactic acid concentration in the fermentation broth after fermentation can reach a maximum of 231.58 g / L, which is 24.79%-35.84% higher than that of single-tank fermentation. This significantly increases the product content per unit volume of the fermentation broth, thus increasing yield. Furthermore, continuous fermentation reduces the number of seed culture inoculations, the number of fermenter sterilizations, and non-fermentation time. It also significantly reduces the energy consumption, such as water for tank washing during transfer and steam for pre-transfer sterilization, thus directly or indirectly reducing labor, time, and resource costs, and greatly lowering the company's production costs.

[0044] Measurement of lactic acid production: A SBA-40C biosensor analyzer manufactured by the Institute of Biology, Shandong Academy of Sciences was used. Measurement method: Lactic acid was displaced from the fermentation broth sample using sulfuric acid, followed by centrifugation at 10,000 r / min for 10 min to remove calcium sulfate and cell residues. The supernatant was collected for determining lactic acid production.

[0045] The above description is merely a preferred embodiment of this application and an explanation of the technical principles and solutions employed. Furthermore, the scope of the invention involved in this application is not limited to technical solutions formed by specific combinations of the above-described technical features, but should also cover other technical solutions formed by arbitrary combinations of the above-described technical features or their equivalents without departing from the inventive concept. For example, technical solutions formed by substituting the above-described features with (but not limited to) technical features with similar functions disclosed in this application.

Claims

1. A process for continuous fermentation to produce lactic acid, characterized in that, Includes the following steps: 1) The first fermenter, the second fermenter, ... the Nth fermenter are connected in series by connecting pipes to form a multi-tank continuous fermentation system; 2) Sterile culture medium A and seed liquid of Lactobacillus de Germanis strain were introduced into the first fermenter for the initial fermentation culture of the strain. 3) Once the bacterial strain in the first fermenter enters the stable growth phase, the continuous fermentation system is started. Sterile culture medium B is added to the first fermenter at a steady rate. Simultaneously, when the fermentation broth in the first fermenter reaches 80%-85% of its total volume, it overflows through the connecting pipe into the second fermenter. Similarly, when the fermentation broth in the second fermenter reaches 80%-85% of its total volume, it overflows into the third fermenter, and so on, until the fermentation broth overflowing from the Nth fermenter is completely fermented, achieving continuous overflow discharge. The sugar concentration of sterile culture medium B is 30%-32%.

2. The process for continuous fermentation to produce lactic acid according to claim 1, characterized in that, Sterile culture medium B is one of glucose medium or starch saccharification medium.

3. The process for continuous fermentation to produce lactic acid according to claim 2, characterized in that, The starch saccharification medium can be directly added to the first fermenter.

4. The process for continuous fermentation to produce lactic acid according to claim 1, characterized in that, The sugar concentration of sterile culture medium A is 8%-12%.

5. The process for continuous fermentation to produce lactic acid according to claim 1, characterized in that, In the continuous fermentation system, the addition rate of sterile culture medium B is calculated as the ratio of the volume of sterile culture medium B to the volume of the first fermenter, which is 1:9-1:11.

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