CN110885860A - Method for efficiently producing L-lactic acid - Google Patents
Method for efficiently producing L-lactic acid Download PDFInfo
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Abstract
The invention discloses a method for efficiently producing L-lactic acid, which couples cell growth and cell non-growth to produce L-lactic acid and comprises the following steps: fermentation in the first stage: comprises the step of utilizing lactobacillus casei to grow and metabolize glucose to generate L-lactic acid; and (3) second-stage fermentation: comprises the step of utilizing resting cells to ferment and metabolize glucose to quickly generate L-lactic acid; the resting cells are lactobacillus casei cells in a stationary phase obtained by centrifugal collection after the first-stage fermentation step. According to the method for efficiently producing the L-lactic acid, the L-lactic acid is produced by coupling the cell growth and the cell non-growth of the lactobacillus casei, the conversion of the cells to the substrate can be effectively increased, the substrate glucose conversion rate is improved, the volume yield of the reaction tank and the overall L-lactic acid production efficiency in the fermentation process are further improved, and the aim of improving the production efficiency is finally achieved.
Description
Technical Field
The invention relates to the technical field of biological fermentation, in particular to a method for efficiently producing L-lactic acid.
Background
Lactic acid is a naturally occurring organic acid, which is widely used in industries such as food, medicine, chemical industry, cosmetics, and the like, and particularly, with the increasing enhancement of global environmental protection and the increasing exhaustion of petroleum resources, environmentally friendly polylactic acid materials are receiving more and more attention from people. However, compared to the price of traditional plastics, the cost of polylactic acid products is always a major factor that limits its overall replacement of fossil raw material products. Therefore, how to reduce the production cost of the lactic acid monomer, i.e., lactic acid, and achieve efficient production of the fermentation process has become a hot spot of current research.
Therefore, it is desirable to provide a method for efficiently producing L-lactic acid to solve the above problems.
Disclosure of Invention
The invention aims to improve the production efficiency of L-lactic acid (L-lactic acid), provides a method for efficiently producing L-lactic acid by coupling cell growth and cell non-growth, can effectively increase the conversion of cells to substrates, improves the conversion rate of the substrate Glucose (Glucose), further improves the volume yield of a reaction tank and the efficiency of integrally producing L-lactic acid in a fermentation process, and realizes the efficient production of the fermentation process.
In order to achieve the above object, the present invention provides a method for efficiently producing L-lactic acid by coupling cell growth and cell non-growth, comprising the steps of:
fermentation in the first stage: comprises the step of utilizing lactobacillus casei to grow and metabolize glucose to generate L-lactic acid;
and (3) second-stage fermentation: comprises the step of utilizing resting cells to ferment and metabolize glucose to quickly generate L-lactic acid;
the resting cells are lactobacillus casei cells in a stationary phase obtained by centrifugal collection after the first-stage fermentation step.
Further, the resting cells maintain a very high metabolic activity of the cells.
Further, the step of the second stage fermentation comprises: inoculating the resting cell sap into a fermentation culture medium, wherein the cell concentration is 2.35-18.55 g/L, and adding glucose for fermentation culture to obtain a fermentation broth; the concentration of initial glucose in the culture system is 60-140 g/L, and a pH regulator is added in the culture process to maintain the pH value of the system to be 5.8-6.2.
Further, the concentration of the initial glucose in the second stage of fermentation is 80-120 g/L. Further, the initial glucose concentration in the second stage fermentation was 100 g/L.
Further, the pH regulator is 8-12 g/L calcium carbonate or 20-30% (w/w) ammonia water solution. Further, the pH regulator is 10g/L calcium carbonate or 25% (w/w) ammonia solution.
Further, the resting cell sap is obtained by the following steps: and (3) culturing the lactobacillus casei cells in the first-stage fermentation step in a fermentation tank to obtain a fermentation culture solution, centrifuging the obtained fermentation culture solution, and resuspending the separated thalli with a glucose solution to obtain a resting cell liquid.
Further, the resting cell sap is obtained by the following steps: and culturing the lactobacillus casei cells in the first-stage fermentation step in a fermentation tank for 12-20 hours to obtain a fermentation culture solution, centrifuging the obtained fermentation culture solution at 0 ℃ at a rotating speed of 8000-12000 r/min for 2-5 min, and re-suspending the separated thalli with a glucose solution to obtain resting cell liquid.
Further, the time for culturing in the fermentation tank to reach a stationary phase is 12-16 h. Further, the centrifugation is: centrifuging at a rotation speed of 10000r/min for 2-5 min at the temperature of 0 ℃.
Further, the step of the first stage fermentation comprises:
seed culture: suspending thalli in the inclined plane of an eggplant bottle by using sterile water, taking a proper amount of suspension to inoculate in a seed culture medium, wherein the inoculation amount is 17-18%, and culturing in an incubator at 35-39 ℃ and 100-120 r/min for 10-14 h to obtain a seed culture solution;
and (3) shake flask fermentation culture: inoculating the seed culture solution into a fermentation culture medium, wherein the inoculation amount is 18-22%, and performing shake-flask fermentation culture in an incubator at 35-39 ℃ and 100-120 r/min for 22-26 h to obtain a pre-fermentation culture solution;
tank fermentation culture: inoculating the pre-fermentation culture solution into a fermentation culture medium, wherein the inoculation amount is 18-22%, and culturing in a fermentation tank at the temperature of 35-39 ℃ and the rotation speed of 130-170 rpm, and the ventilation amount is 0.11-0.14 vvm to obtain the fermentation culture solution.
Further, the tank working volume during fermentation was 4/5 (V/V).
Further, in the first stage fermentation step, the pH is adjusted to be within the range of 5.8-6.2 during the shake flask fermentation culture and the tank fermentation culture.
Further, in the step of the first stage fermentation, the pH is adjusted to 6.0 during the shake flask fermentation culture and the tank fermentation culture.
Further, the step of the first stage fermentation comprises:
seed culture: suspending thalli in the inclined plane of an eggplant bottle by using sterile water, taking the suspension to inoculate in a seed culture medium, wherein the inoculum size is 17-18%, and culturing for 12 hours in an incubator at 37 ℃ and 110r/min to obtain a seed culture solution;
and (3) shake flask fermentation culture: inoculating the seed culture solution into a fermentation culture medium with the inoculation amount of 20%, and performing shake-flask fermentation culture in an incubator at 37 ℃ and 110r/min for 24h to obtain a pre-fermentation culture solution; adding a certain amount of calcium carbonate to adjust the pH value in the process at the beginning of fermentation;
tank fermentation culture: inoculating the pre-fermentation culture solution into a fermentation culture medium, wherein the inoculation amount is 20%, culturing in a fermentation tank at 37 ℃ and 150rpm, and the ventilation amount is 0.125vvm to obtain the fermentation culture solution; the process pH was maintained at 6.0 using 25% (w/w) aqueous ammonia.
Further, the seed medium comprises: glucose, peptone, beef extract, yeast extract, diammonium hydrogen citrate, disodium hydrogen phosphate, anhydrous sodium acetate, magnesium sulfate, manganese sulfate, sodium chloride, ferric sulfate, calcium carbonate and tween-80. Wherein the glucose is prepared separately from other components, and a glucose solution is prepared separately.
Further, the seed medium comprises: 35-45 g/L glucose, 8-12 g/L peptone, 8-12 g/L beef extract, 9-11 g/L yeast extract, 1.8-2.2 g/L diammonium hydrogen citrate, 1.8-2.2 g/L disodium hydrogen phosphate, 3.6-4.3 g/L anhydrous sodium acetate, 0.18-0.22 g/L magnesium sulfate, 0.18-0.22 g/L manganese sulfate, 0.02-0.04 g/L sodium chloride, 0.008-0.012 g/L ferric sulfate, 22-28 g/L calcium carbonate and 0.8-1.5 mL/L Tween-80.
Further, the initial pH value of the seed culture medium is 5.8-6.2. Further, the initial pH of the seed medium is 6.0; the pH value was adjusted with sodium hydroxide.
Further, the seed medium comprises: 40g/L glucose, 10g/L peptone, 10g/L beef extract, 10g/L yeast extract, 2g/L diammonium hydrogen citrate, 2g/L disodium hydrogen phosphate, 4g/L anhydrous sodium acetate, 0.2g/L magnesium sulfate, 0.2g/L manganese sulfate, 0.03g/L sodium chloride, 0.01g/L ferric sulfate, 25g/L calcium carbonate, and 1mL/L Tween-80.
Further, the fermentation medium comprises: glucose, peptone, yeast extract, anhydrous sodium acetate, magnesium sulfate, manganese sulfate, sodium chloride and ferric sulfate. Wherein the glucose is prepared separately from other components, and a glucose solution is prepared separately.
Further, the fermentation medium comprises: 100-140 g/L glucose, 12-14 g/L peptone, 12-14 g/L yeast extract, 0.6-0.7 g/L anhydrous sodium acetate, 0.012-0.015 g/L magnesium sulfate, 0.013-0.014 g/L manganese sulfate, 0.012-0.015 g/L sodium chloride, 0.013-0.014 g/L ferric sulfate.
Further, the seed medium and the fermentation medium were sterilized at 115 ℃ for 20 min.
Further, the fermentation medium comprises: 140g/L glucose, 13.33g/L peptone, 13.33g/L yeast extract, 0.67g/L anhydrous sodium acetate, 0.0133g/L magnesium sulfate, 0.0133g/L manganese sulfate, 0.0133g/L sodium chloride and 0.0133g/L ferric sulfate.
Further, glucose in the seed medium and the fermentation medium is prepared separately from other components, and a glucose solution is prepared separately.
Further, the glucose solution was sterilized at 115 ℃ for 20 min.
In the embodiment of the invention, the Lactobacillus casei (Lactobacillus paracasei NCBIO-01) is a deposited strain of the national research center for biochemical engineering technology (Shanghai).
The materials and apparatus of the present invention are commercially available products.
In the embodiment of the invention, the cell concentration is measured by an ultraviolet-visible spectrophotometry method: diluting the fermentation liquor to a certain concentration by using dilute hydrochloric acid, and measuring the light absorption value at the wavelength of 620nm under an ultraviolet-visible spectrophotometer to ensure that the measured Optical Density (OD) is between 0.2 and 0.8. Cell biomass can be expressed by the Dry Cell Weight (DCW) in the following relationship: 1OD corresponds to 0.41g/L DCW.
In the specific embodiment of the invention, the L-lactic acid and glucose in the fermentation liquor are measured as follows: taking a certain amount of fermentation liquor in the embodiment, centrifuging for 10min under the condition of 5000r/min, discarding thalli, and diluting supernatant liquid to ensure that the concentrations of glucose and L-lactic acid are respectively below 1g/L and 0.5 g/L.
In a specific embodiment of the present invention, the L-lactate concentration and the glucose concentration are measured by an SBA-40C biosensor analyzer.
The invention has the beneficial effects that:
the method of the invention produces L-lactic acid by coupling cell growth and cell non-growth of lactobacillus casei, can effectively increase the conversion of cells to substrates, and simultaneously improves the substrate conversion rate under the condition of consuming a small amount of nutrient substances. The cells are able to grow rapidly and metabolize glucose to produce L-lactate during the growth phase. The method for coupled cell non-growth co-production of lactic acid can reduce a series of auxiliary time of fermentation tank cleaning, seed preparation, cell growth and the like, and further improve the volume yield of the reaction tank and the production efficiency of the L-lactic acid in the whole fermentation process.
The method utilizes the lactobacillus casei to rapidly grow and metabolize glucose to generate the L-lactic acid, and simultaneously utilizes resting cells in the stationary phase to keep very high cell metabolic activity within a certain time range after the fermentation is finished, and continuously metabolizes the glucose to generate the L-lactic acid. The method can ensure that the lactobacillus casei cells continue to produce the L-lactic acid with high density and high efficiency after the conventional fermentation is finished, the production rate is about 2.4 times of that of the conventional fermentation stage, and the conversion rate of the L-lactic acid is obviously improved and can reach 0.985g/g, which is very close to the theoretical value (1.0 g/g).
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a graph showing data on glucose consumption rate and L-lactic acid production rate in second-stage fermentations in examples 3-6 of the present invention.
FIG. 2 shows the specific L-lactic acid production rates in the second stage fermentations in examples 7 to 14 of the present invention.
FIG. 3 is a graph showing production data of L-lactic acid by cells coupled with growth and non-growth of the cells in Experimental example 3 of the present invention.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
This example provides a method for efficiently producing L-lactic acid by coupling cell growth and cell non-growth, comprising the steps of:
fermentation in the first stage: comprises the step of utilizing lactobacillus casei to grow and metabolize glucose to generate L-lactic acid;
and (3) second-stage fermentation: comprises the step of utilizing resting cells to ferment and metabolize glucose to quickly generate L-lactic acid; the resting cells are lactobacillus casei cells in a stationary phase obtained by centrifugal collection after the first-stage fermentation step.
The step of the first stage fermentation comprises:
seed culture: suspending thalli in the inclined plane of an eggplant bottle by using sterile water, taking a proper amount of suspension to inoculate in a seed culture medium, wherein the inoculum size is 17-18%, and culturing in an incubator at 35-39 ℃ and 100-120 r/min to obtain a seed culture solution;
and (3) shake flask fermentation culture: inoculating the seed culture solution into a fermentation culture medium, wherein the inoculation amount is 18-22%, and performing shake-flask fermentation culture in an incubator at 37 ℃ and 110r/min to obtain a pre-fermentation culture solution;
tank fermentation culture: inoculating the pre-fermentation culture solution into a fermentation culture medium, wherein the inoculation amount is 18-22%, culturing in a fermentation tank at the temperature of 35-39 ℃ and the rotation speed of 130-170 rpm, and culturing in the fermentation tank with the ventilation amount of 0.11-0.14 vvm to obtain the fermentation culture solution.
In the first stage fermentation step, the pH is adjusted to be within the range of 5.8-6.2 in the processes of shake flask fermentation culture and tank fermentation culture.
The second stage fermentation step comprises:
inoculating the resting cell sap into a fermentation culture medium, wherein the cell concentration is 2.35-18.55 g/L, and adding glucose for fermentation culture to obtain a fermentation broth; the concentration of initial glucose in the culture system is 60-140 g/L, and a pH regulator is added in the culture process to maintain the pH value of the system to be 5.8-6.2.
The pH regulator is 8-12 g/L calcium carbonate or 20-30% (w/w) ammonia water solution.
The resting cell sap is obtained by the following steps: and (3) culturing the lactobacillus casei cells in the first-stage fermentation step in a fermentation tank for 12-20 hours to obtain a fermentation culture solution, centrifuging the obtained fermentation culture solution at 0 ℃ at a rotating speed of 8000-12000 r/min for 2-5 min, and re-suspending the obtained thalli with a glucose solution to obtain resting cell liquid.
The seed culture medium comprises: 35-45 g/L glucose, 8-12 g/L peptone, 8-12 g/L beef extract, 9-11 g/L yeast extract, 1.8-2.2 g/L diammonium hydrogen citrate, 1.8-2.2 g/L disodium hydrogen phosphate, 3.6-4.3 g/L anhydrous sodium acetate, 0.18-0.22 g/L magnesium sulfate, 0.18-0.22 g/L manganese sulfate, 0.02-0.04 g/L sodium chloride, 0.01g/L ferric sulfate, 22-28 g/L calcium carbonate and 0.8-1.5 mL/L Tween-80. The initial pH value of the seed culture medium is 5.8-6.2.
The fermentation medium comprises: 100-140 g/L glucose, 12-14 g/L peptone, 12-14 g/L yeast extract, 0.6-0.7 g/L anhydrous sodium acetate, 0.012-0.015 g/L magnesium sulfate, 0.013-0.014 g/L manganese sulfate, 0.012-0.015 g/L sodium chloride, 0.013-0.014 g/L ferric sulfate.
The glucose in the seed medium and the fermentation medium is formulated separately from the other components.
Example 2
This example provides a method for efficiently producing L-lactic acid by coupling cell growth and cell non-growth, comprising the steps of:
fermentation in the first stage: comprises the step of utilizing lactobacillus casei to grow and metabolize glucose to generate L-lactic acid;
and (3) second-stage fermentation: comprises the step of utilizing resting cells to ferment and metabolize glucose to quickly generate L-lactic acid; the resting cells are lactobacillus casei cells in a stationary phase obtained by centrifugal collection after the first-stage fermentation step. The resting cells retain very high metabolic activity of the cells.
The step of the first stage fermentation comprises:
seed culture: suspending thallus in the inclined plane of an eggplant bottle by using sterile water, taking a proper amount of suspension to inoculate in a seed culture medium, wherein the inoculum size is 18%, and culturing for 10 hours in an incubator at 35 ℃ and 120r/min to obtain a seed culture solution;
and (3) shake flask fermentation culture: inoculating the seed culture solution into a fermentation culture medium with the inoculum size of 22%, and performing shake-flask fermentation culture in an incubator at 35 ℃ and 120r/min for 22h to obtain a pre-fermentation culture solution;
tank fermentation culture: inoculating the pre-fermentation culture solution into a fermentation culture medium with an inoculum size of 22%, culturing in a 5L fermentation tank at 35 deg.C and a rotation speed of 170rpm with an aeration rate of 0.11vvm to obtain a fermentation culture solution. The working volume of the tank in the fermentation process is 4L.
In the first stage fermentation step, the pH is adjusted to be within the range of 5.8-6.2 in the processes of shake flask fermentation culture and tank fermentation culture.
The second stage fermentation step comprises: inoculating the resting cell sap into a fermentation culture medium, wherein the cell concentration is 16.47g/L, and adding glucose for fermentation culture to obtain a fermentation broth; the concentration of initial glucose in the culture system is 60g/L, and a pH regulator is added in the culture process to maintain the pH value of the system to be 5.8-6.2. The pH regulator is 25% (w/w) ammonia water solution.
The resting cell sap is obtained by the following steps: and (3) culturing the lactobacillus casei cells in the first-stage fermentation step in a fermentation tank for 12 hours to obtain a fermentation culture solution, placing the obtained fermentation culture solution at the temperature of 0 ℃ and centrifuging at the rotating speed of 12000r/min for 2min, and then re-suspending the obtained thalli with a glucose solution to obtain a resting cell solution.
The seed culture medium comprises: 45g/L glucose, 8g/L peptone, 12g/L beef extract, 9g/L yeast extract, 2.2g/L diammonium hydrogen citrate, 1.8g/L disodium hydrogen phosphate, 4.3g/L anhydrous sodium acetate, 0.18g/L magnesium sulfate, 0.22g/L manganese sulfate, 0.02g/L sodium chloride, 0.012g/L ferric sulfate, 22g/L calcium carbonate, and 1.5mL/L Tween-80; wherein the glucose is prepared separately from other components, and a glucose solution is prepared separately. The initial pH value of the seed culture medium is 5.8-6.2.
The fermentation medium comprises: 140g/L glucose, 12g/L peptone, 14g/L yeast extract, 0.6g/L anhydrous sodium acetate, 0.015g/L magnesium sulfate, 0.013g/L manganese sulfate, 0.015g/L sodium chloride, 0.013g/L ferric sulfate; wherein the glucose is prepared separately from other components, and a glucose solution is prepared separately.
Example 3
This example provides a method for efficiently producing L-lactic acid by coupling cell growth and cell non-growth, comprising the steps of:
fermentation in the first stage: comprises the step of utilizing lactobacillus casei to grow and metabolize glucose to generate L-lactic acid;
and (3) second-stage fermentation: comprises the step of utilizing resting cells to ferment and metabolize glucose to quickly generate L-lactic acid;
the resting cells are lactobacillus casei cells in a stationary phase obtained by centrifugal collection after the first-stage fermentation step. The resting cells retain very high metabolic activity of the cells.
The step of the first stage fermentation comprises:
seed culture: suspending thalli in the inclined plane of an eggplant bottle by using 50mL of sterile water, taking 15mL of suspension, inoculating the suspension into 85mL of seed culture medium, and culturing for 12h in an incubator at 37 ℃ and 110r/min to obtain a seed culture solution;
and (3) shake flask fermentation culture: inoculating 20mL of seed culture solution into 80mL of fermentation medium, wherein the inoculation amount is 20%, and performing shake-flask fermentation culture in an incubator at 37 ℃ and 110r/min for 24h to obtain a pre-fermentation culture solution; adding a proper amount of calcium carbonate at the beginning of fermentation to adjust the pH value in the process to be 5.8-6.2;
tank fermentation culture: inoculating the pre-fermentation culture solution into a fermentation culture medium with an inoculum size of 20%, culturing in a 5L fermentation tank at 37 deg.C and 150rpm with ventilation of 0.125vvm to obtain a fermentation culture solution; the working volume of the tank in the fermentation process is 4L; the process pH was maintained at 6.0 using 25% (w/w) aqueous ammonia.
The second stage fermentation step comprises: inoculating the resting cell sap into a fermentation culture medium, wherein the cell concentration is 18.55g/L, and adding glucose for fermentation culture to obtain a fermentation broth; the initial glucose concentration in the culture system is 100g/L, and a pH regulator is added in the culture process to maintain the pH value of the system to be 6.0. The pH regulator is 25% (w/w) ammonia water solution.
The resting cell sap is obtained by the following steps: and (3) culturing the lactobacillus casei cells in the first-stage fermentation step in a fermentation tank for 14 hours to obtain a fermentation culture solution, placing the obtained fermentation culture solution at the temperature of 0 ℃ and centrifuging at the rotating speed of 10000r/min for 4min, and then re-suspending the obtained thalli with a glucose solution to obtain a resting cell solution.
The seed culture medium comprises: 40g/L glucose, 10g/L peptone, 10g/L beef extract, 10g/L yeast extract, 2g/L diammonium hydrogen citrate, 2g/L disodium hydrogen phosphate, 4g/L anhydrous sodium acetate, 0.2g/L magnesium sulfate, 0.2g/L manganese sulfate, 0.03g/L sodium chloride, 0.01g/L ferric sulfate, 25g/L calcium carbonate, and 1mL/L Tween-80; wherein the glucose is prepared separately from other components, and a glucose solution is prepared separately. The initial pH value of the seed culture medium is 5.8-6.2.
The fermentation medium comprises: 140g/L glucose, 13.33g/L peptone, 13.33g/L yeast extract, 0.67g/L anhydrous sodium acetate, 0.0133g/L magnesium sulfate, 0.0133g/L manganese sulfate, 0.0133g/L sodium chloride and 0.0133g/L ferric sulfate; wherein the glucose is prepared separately from other components, and a glucose solution is prepared separately.
Examples 4 to 6
Examples 4 to 6 differ from example 3 only in that: the initial glucose concentrations in the second stage fermentation steps described in examples 4-6 were 80g/L, 120g/L and 140g/L, respectively, and the other conditions were exactly the same as in example 3.
Example 7
This example provides a method for efficiently producing L-lactic acid by coupling cell growth and cell non-growth, comprising the steps of:
fermentation in the first stage: comprises the step of utilizing lactobacillus casei to grow and metabolize glucose to generate L-lactic acid;
and (3) second-stage fermentation: comprises the step of utilizing resting cells to ferment and metabolize glucose to quickly generate L-lactic acid; the resting cells are lactobacillus casei cells in a stationary phase obtained by centrifugal collection after the first-stage fermentation step. The resting cells retain very high metabolic activity of the cells.
The step of the first stage fermentation comprises:
seed culture: suspending thallus in the inclined plane of an eggplant bottle by using sterile water, taking a proper amount of suspension to inoculate in a seed culture medium, wherein the inoculum size is 17%, and culturing for 14 hours in an incubator at 39 ℃ and 100r/min to obtain a seed culture solution;
and (3) shake flask fermentation culture: inoculating the seed culture solution into a fermentation culture medium with the inoculation amount of 18%, and performing shake-flask fermentation culture in an incubator at 39 ℃ and 100r/min for 26h to obtain a pre-fermentation culture solution;
tank fermentation culture: inoculating the pre-fermentation culture solution into a fermentation culture medium with an inoculum size of 18%, culturing in a 5L fermentation tank at 39 deg.C and 130rpm with ventilation of 0.14vvm to obtain a fermentation culture solution. The working volume of the tank in the fermentation process is 4L.
In the first stage fermentation step, the pH is adjusted to be within the range of 5.8-6.2 in the processes of shake flask fermentation culture and tank fermentation culture.
The second stage fermentation step comprises: inoculating the resting cell sap into a fermentation culture medium, wherein the cell concentration is 2.35g/L, and adding glucose for fermentation culture to obtain a fermentation broth; the concentration of initial glucose in the culture system is 140g/L, and a pH regulator is added in the culture process to maintain the pH value of the system to be 5.8-6.2. The pH regulator is 10g/L calcium carbonate.
The resting cell sap is obtained by the following steps: and (3) culturing the lactobacillus casei cells in the first-stage fermentation step in a fermentation tank for 20 hours to obtain a fermentation culture solution, placing the obtained fermentation culture solution at the temperature of 0 ℃ and centrifuging for 5 minutes at the rotating speed of 8000r/min, and then re-suspending the obtained thalli with a glucose solution to obtain a resting cell solution.
The seed culture medium comprises: 35g/L glucose, 12g/L peptone, 8g/L beef extract, 11g/L yeast extract, 1.8g/L diammonium hydrogen citrate, 2.2g/L disodium hydrogen phosphate, 3.6g/L anhydrous sodium acetate, 0.22g/L magnesium sulfate, 0.18g/L manganese sulfate, 0.04g/L sodium chloride, 0.008g/L ferric sulfate, 28g/L calcium carbonate, and 0.8mL/L Tween-80; wherein the glucose is prepared separately from other components, and a glucose solution is prepared separately. The initial pH value of the seed culture medium is 5.8-6.2.
The fermentation medium comprises: 100g/L glucose, 14g/L peptone, 12g/L yeast extract, 0.7g/L anhydrous sodium acetate, 0.012g/L magnesium sulfate, 0.014g/L manganese sulfate, 0.012g/L sodium chloride, 0.014g/L ferric sulfate; wherein the glucose is prepared separately from other components, and a glucose solution is prepared separately.
Examples 8 to 14
Examples 8 to 14 differ from example 7 only in that: the resting cell concentrations in the second stage fermentation steps described in examples 8 to 14 were 4.69g/L, 7.43g/L, 9.36g/L, 11.94g/L, 14.12g/L, 16.47g/L and 18.55g/L, respectively, and the other conditions were exactly the same as in example 7.
Test example 1
In the experiment, the glucose consumption rate and the L-lactic acid production rate of the cells in the embodiments 3 to 6 of the invention are respectively detected to detect the metabolic capacity of the resting cells under different initial glucose concentrations, and the experimental results are shown in FIG. 1.
As can be seen from FIG. 1, the experimental results show that the resting cells in the stationary phase can still maintain high metabolic activity within about 6h, and the resting cells can completely exhaust all glucose. Wherein, when the initial glucose concentration is 80 and 100g/L, the thalli can consume all glucose (the consumption time is 5h and 6.4h respectively) very quickly and generate L-lactic acid, the glucose consumption rate is 16.0g/L/h and 15.8g/L/h respectively, and the L-lactic acid generation rate is 15.6g/L/h and 15.3g/L/h respectively. The invention can completely exhaust 100g/L initial glucose and generate 98 g/L-lactic acid. Therefore, the method can increase the conversion of the cells to the substrate glucose, realize the complete consumption of the glucose, improve the conversion rate of the glucose, further improve the volume yield of the reaction tank and the overall L-lactic acid production efficiency in the fermentation process, and finally realize the improvement of the production efficiency.
Test example 2
The test respectively carries out the detection of the specific production rate of the samples 7 to 14 of the invention to observe the specific production rate of different resting cell concentrations, and the detection results are shown in figure 2.
As can be seen from FIG. 2, the L-lactic acid production was linear with cell concentration in the initial 2h, which also indicates that the specific cell production rate was maintained substantially constant during this period; with time, the specific production rate was maintained at a lower cell concentration, which was consistent with the initial stage, and the specific production rate was significantly decreased at a higher cell concentration, however, it was found by analyzing the above test example 1 that this was probably because a higher cell concentration produced a higher concentration of lactic acid at the early stage, and the higher concentration of lactic acid significantly inhibited the metabolism of the cells, and thus it was found that the resting cells at a higher cell concentration still maintained a high metabolic activity, rather than decreased the metabolic activity of the cells with time.
Test example 3
The product obtained by the method for efficiently producing L-lactic acid in example 3 of the present invention was examined to observe the overall situation of the produced L-lactic acid, and two parallel tests were carried out, and the test results are shown in FIG. 3.
As can be seen from FIG. 3, in the first stage, two batches of parallel fermentation can quickly deplete about 140g/L of initial glucose and produce about 126g/L of L-lactic acid, with glucose consumption and L-lactic acid production rates of 6.3g/L/h and 5.9g/L/h, respectively, and L-lactic acid conversion of 0.940 g/g.
After the first stage is finished, the thalli of the two batches of parallel fermenters are combined by centrifugation to continue the fermentation of resting cells, and the initial glucose concentration is 102 g/L. At this time, the cell concentration is about 1.7 times of the biomass of a single fermentation tank in the first stage, the thalli can consume all glucose within 7 hours to generate 98g/L of L-lactic acid, and the glucose consumption rate, the L-lactic acid production rate and the L-lactic acid conversion rate respectively reach 14.6g/L/h, 14.4g/L/h and 0.985 g/g.
It is understood that although the time for treating resting cells in stationary phase may be about 1 hour, the production rate in the second stage is much higher than that in the first stage, and it should be noted that the conversion rate of L-lactic acid in the second stage is significantly higher than that in the first stage because of the absence of cell growth, thereby further improving the production efficiency.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method for efficiently producing L-lactic acid by coupling cell growth and cell non-growth, comprising the steps of:
fermentation in the first stage: comprises the step of utilizing lactobacillus casei to grow and metabolize glucose to generate L-lactic acid;
and (3) second-stage fermentation: comprises the step of utilizing resting cells to ferment and metabolize glucose to quickly generate L-lactic acid;
the resting cells are lactobacillus casei cells in a stationary phase obtained by centrifugal collection after the first-stage fermentation step.
2. The method for efficiently producing L-lactic acid according to claim 1, wherein the step of the second stage fermentation comprises:
inoculating the resting cell sap into a fermentation culture medium, wherein the cell concentration is 2.35-18.55 g/L, and adding glucose for fermentation culture to obtain a fermentation broth; the concentration of initial glucose in the culture system is 60-140 g/L, and a pH regulator is added in the culture process to maintain the pH value of the system to be 5.8-6.2.
3. The method for efficiently producing L-lactic acid according to claim 2, wherein the pH regulator is 8-12 g/L calcium carbonate or 20-30% ammonia solution.
4. The method for efficiently producing L-lactic acid according to claim 1 or 2, wherein the resting cell fluid is obtained by the steps of: and (3) culturing the lactobacillus casei cells in the first-stage fermentation step in a fermentation tank to obtain a fermentation culture solution after a stabilization period, centrifuging the obtained fermentation culture solution, and resuspending the separated thalli by using a glucose solution to obtain a resting cell liquid.
5. The method for efficient production of L-lactic acid according to claim 1, wherein the step of the first stage fermentation comprises:
seed culture: suspending thalli in the inclined plane of an eggplant bottle by using sterile water, taking a proper amount of suspension to inoculate in a seed culture medium, wherein the inoculum size is 17-18%, and culturing in an incubator at 35-39 ℃ and 100-120 r/min to obtain a seed culture solution;
and (3) shake flask fermentation culture: inoculating the seed culture solution into a fermentation culture medium, wherein the inoculation amount is 18-22%, and performing shake flask fermentation culture in an incubator at the temperature of 35-39 ℃ and the rotation speed of 100-120 r/min to obtain a pre-fermentation culture solution;
tank fermentation culture: inoculating the pre-fermentation culture solution into a fermentation culture medium, wherein the inoculation amount is 18-22%, culturing in a fermentation tank at the temperature of 35-39 ℃ and the rotation speed of 130-170 rpm, and culturing in the fermentation tank with the ventilation amount of 0.11-0.14 vvm to obtain the fermentation culture solution.
6. The method for efficient production of L-lactic acid according to claim 1 or 5, wherein in the first stage fermentation step, the pH is adjusted to 5.8-6.2 during the shake flask fermentation culture and the tank fermentation culture.
7. The method for efficiently producing L-lactic acid according to claim 5, wherein said seed medium comprises: 35-45 g/L glucose, 8-12 g/L peptone, 8-12 g/L beef extract, 9-11 g/L yeast extract, 1.8-2.2 g/L diammonium hydrogen citrate, 1.8-2.2 g/L disodium hydrogen phosphate, 3.6-4.3 g/L anhydrous sodium acetate, 0.18-0.22 g/L magnesium sulfate, 0.18-0.22 g/L manganese sulfate, 0.02-0.04 g/L sodium chloride, 0.008-0.012 g/L ferric sulfate, 22-28 g/L calcium carbonate and 0.8-1.5 mL/L Tween-80.
8. The method for efficiently producing L-lactic acid according to claim 5 or 7, wherein the initial pH of the seed medium is 5.8 to 6.2.
9. The method for efficiently producing L-lactic acid according to claim 5, wherein the fermentation medium comprises: 100-140 g/L glucose, 12-14 g/L peptone, 12-14 g/L yeast extract, 0.6-0.7 g/L anhydrous sodium acetate, 0.012-0.015 g/L magnesium sulfate, 0.013-0.014 g/L manganese sulfate, 0.012-0.015 g/L sodium chloride, 0.013-0.014 g/L ferric sulfate.
10. The method for efficiently producing L-lactic acid according to claim 7 or 9, wherein the glucose in the seed medium and the fermentation medium is prepared separately from other components.
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| CN101613667A (en) * | 2009-07-23 | 2009-12-30 | 中国科学院微生物研究所 | A kind of method and special bacteria agent thereof for preparing L-lactic acid |
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