A method for increasing lactic acid production

Abstract

The invention discloses a method for improving lactic acid production, and belongs to the technical field of lactic acid production. The method comprises the following steps: soaking, embedding and fixing, fermenting, and post-processing; wherein the soaking comprises mixing an amino resin with a calcium chloride aqueous solution, stirring at 25-40°C, filtering, and collecting a filter residue to obtain the soaked amino resin; the embedding and fixing comprises activating and seed-culturing Escherichia coli, centrifuging the seed liquid, and collecting wet bacterial cells; the wet bacterial cells are mixed with physiological saline, stirring at 20-35°C, adding a sodium alginate aqueous solution and a sodium carbonate aqueous solution, continuing to stir, adding the soaked amino resin, continuing to stir, adding a calcium chloride aqueous solution, continuing to stir, filtering, and collecting a filter residue to obtain the embedded and fixed Escherichia coli; and the present invention can improve the yield of D-lactic acid when lignocellulose biomass is used as a raw material and the Escherichia coli is used to produce D-lactic acid.

Description

Technical Field

[0001] The present invention relates to the technical field of lactic acid production, and in particular to a method for increasing lactic acid production. Background Art

[0002] Lactic acid, also known as 2-hydroxypropionic acid, is a widely found organic acid in nature with applications in food, agriculture, medicine, feed, and the chemical industry. Due to the presence of a chiral carbon atom in the lactic acid molecule, lactic acid has two isomers: D-lactic acid and L-lactic acid. D-lactic acid is widely used in the production of chiral pharmaceuticals, pesticide intermediates, and polylactic acid (PLA). Because PLA is biodegradable, has high strength, high modulus, and low toxicity, it is considered an ideal material to replace traditional bioplastics. Due to the strong interactions between the poly (D-lactic acid) and poly (L-lactic acid) molecular chains in the stereocomplex, it has a higher melting point, approximately 50°C higher than either poly (D-lactic acid) or poly (L-lactic acid). It also has higher impact strength than either poly (D-lactic acid) or poly (L-lactic acid). This has led to a significant increase in the production demand for D-lactic acid.

[0003] D-lactic acid can be produced by chemical synthesis, enzymatic methods, and fermentation. Chemical synthesis offers high efficiency but is also costly and toxic. Enzymatic methods can produce D-lactic acid of high optical purity, but are complex and expensive. Fermentation methods offer mild production conditions and minimal environmental pollution. Currently, the majority of D-lactic acid on the market is produced through fermentation.

[0004] Fermentation involves the synthesis of D-lactic acid by microorganisms using substances such as sugars as a carbon source, along with nitrogen and other nutrients. Based on the metabolic pathways and products produced by microorganisms, D-lactic acid fermentation can be divided into three types: homolactic fermentation, heterolactic fermentation, and mixed acid fermentation. In homolactic fermentation, the fermentation pathway used is the glycolysis pathway, in which lactic acid is the only metabolic product of glucose. Through this pathway, 1 mol of glucose can be converted into 2 mol of lactic acid, with a theoretical conversion rate of 100%. However, due to other physiological activities of microorganisms during the fermentation process, the actual conversion rate cannot reach 100%. When the actual conversion rate is above 80%, it is considered to be homolactic fermentation; in heterolactic fermentation, the fermentation pathway used is the pentose phosphate pathway, in which glucose is fermented to produce lactic acid, carbon dioxide and acetic acid (or ethanol). Through this pathway, 1 mol of glucose can be converted into 1 mol of lactic acid; mixed acid fermentation is a fermentation mode of microorganisms that perform homolactic fermentation under special circumstances. When the glucose concentration is limited, the fermentation temperature is low or the pH value changes, the microorganism will perform mixed acid fermentation. In mixed acid fermentation, the fermentation pathway used is still the glycolysis pathway, but the metabolic pathway will change, and the metabolic byproducts generated are mainly small molecule organic acids. Currently, a wide variety of fermentation strains are used to produce D-lactic acid via fermentation. The most commonly used fermentation strains include Sporolactobacillus, Lactobacillus, and Escherichia coli. Both Sporolactobacillus and Lactobacillus are microorganisms that perform homolactic fermentation, offering advantages such as low fermentation energy consumption and high product concentration. However, wild-type Sporolactobacillus and Lactobacillus have limited lactic acid production capacity. Using molecular methods to engineer bacterial strains offers the advantages of strong targeting and the ability to amplify large quantities of genes encoding the target product. However, Sporolactobacillus and Lactobacillus engineered using molecular methods require high-nutrient culture conditions during fermentation, and the production of L-lactate dehydrogenase or lactate isomerase during fermentation can affect the optical purity of the produced D-lactic acid. Escherichia coli is one of the most commonly used engineered strains in the biological field. Its genome has been fully sequenced, and it also boasts a short fermentation cycle and low nutritional requirements. It is often used in gene cloning and vector construction in the field of biological genetic engineering. Research has shown that molecular methods can be used to generate Escherichia coli with high lactic acid production capacity.

[0005] Lignocellulosic biomass is widely available and inexpensive. Furthermore, it is rich in polysaccharides such as cellulose and hemicellulose. Microbial fermentation can convert lignocellulosic biomass into high-value-added chemicals, which is of great significance for alleviating the global energy crisis and solving environmental pollution problems. The main components of lignocellulosic hydrolysate are sugars such as glucose, xylose, and arabinose, of which approximately two-thirds are hexoses and approximately one-third are pentoses. However, most existing microorganisms cannot utilize pentoses or have a very low utilization rate for pentoses, which affects the application of lignocellulosic biomass. Studies have shown that Escherichia coli obtained through molecular means can simultaneously utilize pentoses and hexoses to produce D-lactic acid. Furthermore, when producing D-lactic acid, both low production costs and high production capacity are achieved.

[0006] However, when using lignocellulosic biomass as raw material and using Escherichia coli obtained by molecular means to produce D-lactic acid, the following problems exist: First, since lignocellulosic hydrolyzate contains both hexose and pentose, Escherichia coli will give priority to using hexose during fermentation, and will start using pentose after the hexose is completely used up. Moreover, there will be a stagnation period before using pentose. Compared with using only hexose and pentose, the utilization rate is slow, and some pentose will not be fully utilized, which further affects the production of D-lactic acid. production; second, the fermentation activity of Escherichia coli is sensitive to the concentration of glucose. If the glucose concentration is too high, it will affect the osmotic pressure of Escherichia coli, resulting in a decrease in the fermentation activity of Escherichia coli, and further affecting the production of D-lactic acid. However, if the glucose concentration is low, it is necessary to control the concentration of lignocellulosic biomass, which will also lead to a low production of D-lactic acid; third, as the concentration of D-lactic acid produced during fermentation increases, it will inhibit the fermentation activity of Escherichia coli, and further affect the production of D-lactic acid.

[0007] In response to the first problem mentioned above, the most commonly used method is to optimize the fermentation conditions and extend the fermentation time. However, since the optimal fermentation temperature of Escherichia coli is 35-37°C, this optimal fermentation temperature is also suitable for the growth of various miscellaneous bacteria. As the fermentation time increases, the number of miscellaneous bacteria increases, which will affect the fermentation activity of Escherichia coli. Further, it leads to little effect on increasing the production of D-lactic acid. In response to the second problem mentioned above, the most commonly used method is to use calcium alginate hydrogel beads for embedding and fixation. Specifically, the calcium alginate aqueous solution is mixed with Escherichia coli, and then added dropwise to the calcium chloride aqueous solution. The calcium ions in the calcium chloride aqueous solution diffuse into the droplets to form solid calcium alginate hydrogel beads. However, due to the gel structure of the calcium alginate hydrogel beads, nutrients and fermentation substrates are not easily transferred to the interior of the calcium alginate hydrogel beads, resulting in the fermentation activity of Escherichia coli inside the calcium alginate hydrogel beads being affected, and further, resulting in little effect on increasing the production of D-lactic acid; in response to the third problem mentioned above, the most commonly used method is to use calcium hydroxide as a neutralizer in fermentation. Calcium hydroxide reacts with the produced lactic acid to generate calcium lactate. The calcium ions in calcium lactate can also promote the growth of Escherichia coli, but the lactate ions in calcium lactate will also have a certain inhibitory effect on the fermentation activity of Escherichia coli, and further, resulting in little effect on increasing the production of D-lactic acid. Summary of the Invention

[0008] In view of the shortcomings of the existing technology, the present invention provides a method for increasing lactic acid production, which can increase the yield of D-lactic acid when lignocellulosic biomass is used as raw material and Escherichia coli is used to produce D-lactic acid.

[0009] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0010] A method for increasing lactic acid production, comprising: soaking, embedding and fixing, fermenting, and post-processing;

[0011] The soaking step comprises mixing the amino resin with a calcium chloride aqueous solution, stirring at 25-40° C. for 5-6 hours, filtering, and collecting the filter residue to obtain the soaked amino resin;

[0012] During the soaking, the ratio of the amino resin to the calcium chloride aqueous solution is 1 g: 7-9 mL;

[0013] The manufacturer of the amino resin is Xi'an Lanxiao Technology New Materials Co., Ltd., and the model is LX1000EA;

[0014] The mass concentration of the calcium chloride aqueous solution is 5%;

[0015] When storing the amino resin after soaking, the storage temperature is 4°C;

[0016] After the embedding and fixation, the Escherichia coli is activated and seed cultured, the seed liquid is centrifuged to collect wet cells; the wet cells are mixed with physiological saline, stirred at 20-35° C. for 10-20 minutes, a sodium alginate aqueous solution and a sodium carbonate aqueous solution are added, and stirring is continued for 30-40 minutes, the soaked amino resin is added, stirring is continued for 2-3 hours, a calcium chloride aqueous solution is added, stirring is continued for 2-3 hours, and filtering is performed to obtain a filter residue to obtain the embedded and fixed Escherichia coli;

[0017] In the embedding and fixation, the Escherichia coli was donated by Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, with the deposit number CGMCC 7679;

[0018] The activation is performed by using an inoculating loop to take Escherichia coli stored in a -80°C glycerol tube and streaking it on the activation medium, and then incubating it upside down at 37°C for 18 hours to obtain activated colonies;

[0019] The activation culture medium comprises: 10 g / L tryptone, 5 g / L yeast powder, 10 g / L sodium chloride, 20 g / L xylose, 20 g / L agar, the solvent is water, and the pH value is 7.0;

[0020] The seed culture is carried out by taking a single colony from the activated colony and inoculating it into 50 mL of seed culture medium, and shaking and culturing it at 37° C. and 100-150 rpm for 15 hours, maintaining anaerobic conditions during the shaking culture without any air, to obtain a seed solution;

[0021] The seed culture medium comprises: 20 g / L xylose, 0.87 g / L ammonium dihydrogen phosphate, 2.63 g / L diammonium hydrogen phosphate, 0.18 g / L magnesium sulfate heptahydrate, 0.15 g / L betaine hydrochloride, 1.5 μg / L ferric chloride hexahydrate, 0.1 μg / L cobalt chloride hexahydrate, 0.1 μg / L copper chloride dihydrate, 0.1 μg / L zinc chloride, 0.1 μg / L sodium molybdate dihydrate, 0.2 μg / L manganese chloride tetrahydrate, 0.05 μg / L boric acid, the solvent is water, and the pH value is natural;

[0022] The mass concentration of the sodium alginate aqueous solution is 1%;

[0023] The mass concentration of the sodium carbonate aqueous solution is 5%;

[0024] The mass concentration of the calcium chloride aqueous solution is 5%;

[0025] The ratio of wet bacteria to normal saline is 1g:9-10mL;

[0026] The ratio of wet bacteria to sodium alginate aqueous solution and sodium carbonate aqueous solution is 1g:18-10mL:18-21mL;

[0027] The ratio of wet cells to the amino resin after soaking is 1g:2-2.3g;

[0028] The ratio of wet bacteria to calcium chloride aqueous solution is 1g:65-70mL;

[0029] When the embedded and fixed Escherichia coli is stored, the storage temperature is 4°C;

[0030] The fermentation comprises adding the embedded and fixed Escherichia coli to a fermentation medium, culturing the culture at 37° C. and 100-150 rpm with shaking, adding a calcium hydroxide aqueous solution to maintain the pH at 7, maintaining anaerobic conditions during the shaking culture without any air flow, and monitoring the total reducing sugar concentration in real time. When the total reducing sugar concentration is lower than 3 g / L, the fermentation is terminated to obtain a fermentation product.

[0031] During the fermentation, the ratio of the embedded and fixed Escherichia coli to the fermentation medium is 2 g:30-32 mL;

[0032] The molar concentration of the calcium hydroxide aqueous solution is 3 mol / L;

[0033] The fermentation medium comprises: 0.87 g / L ammonium dihydrogen phosphate, 2.63 g / L diammonium hydrogen phosphate, 0.18 g / L magnesium sulfate heptahydrate, 0.15 g / L betaine hydrochloride, 2.4 μg / L ferric chloride hexahydrate, 0.3 μg / L cobalt chloride hexahydrate, 0.15 μg / L copper chloride dihydrate, 0.3 μg / L zinc chloride, 0.3 μg / L sodium molybdate dihydrate, 0.5 μg / L manganese chloride tetrahydrate, 0.072 μg / L boric acid, the solvent is rice straw hydrolyzate, and the pH value is natural;

[0034] The rice straw hydrolyzate preparation method comprises: washing, drying, and crushing the rice straw, and passing the mixture through a 20-30 mesh sieve to obtain rice straw powder; mixing the rice straw powder with a 2% by mass sulfuric acid aqueous solution, and hydrolyzing the mixture at 121° C. for 1-1.5 hours; cooling the mixture, adding sodium hydroxide to adjust the pH to 4.8-5; adding a Trichoderma reesei cellulase solution; and stirring the mixture at 50-55° C. for 75-80 hours. During the stirring process, the pH is monitored and maintained at 4.8-5. After the stirring is completed, the mixture is filtered, the filtrate is collected, calcium hydroxide is added to the filtrate to adjust the pH to 10, the mixture is stirred at 90° C. for 30-40 minutes, filtered, the filtrate is collected, and the sulfuric acid aqueous solution is added to adjust the pH to 7 to obtain the rice straw hydrolyzate;

[0035] In the preparation of the rice straw hydrolyzate, the ratio of rice straw powder to 2% sulfuric acid aqueous solution is 1 g:10 mL;

[0036] The ratio of rice straw powder to Trichoderma reesei cellulase solution is 1g:7-9mL;

[0037] The enzyme activity of the Trichoderma reesei cellulase solution is 1 FPU / mL;

[0038] The mass concentration of the sulfuric acid aqueous solution is 10%;

[0039] The post-treatment includes: ceramic membrane filtration, nanofiltration membrane refining, and ion exchange;

[0040] The ceramic membrane filtration: after converting calcium lactate in the fermentation product into lactic acid, filtering it with a ceramic membrane, and taking the filtrate as the ceramic membrane filtrate;

[0041] The filtration accuracy of the ceramic membrane is 50nm;

[0042] The nanofiltration membrane refining: using a nanofiltration membrane to perform nanofiltration refining on the ceramic membrane filtrate, and taking the nanofiltration clear phase as the nanofiltrate;

[0043] The molecular weight cut-off of the nanofiltration membrane is 200Da;

[0044] The ion exchange is carried out by sequentially using D001 strongly acidic styrene-based cation exchange resin and D301 weakly basic acrylic acid-based anion exchange resin to perform ion exchange on the nanofiltrate, and then evaporating and concentrating the solution to obtain a lactic acid solution.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] (1) In a mixed sugar, E. coli will preferentially utilize glucose, which is easily metabolized, and will only begin to utilize another sugar after glucose is exhausted and after a period of stagnation. This means that there is catabolite repression. The generation of catabolite repression is related to the carbohydrate phosphotransferase system. The carbohydrate phosphotransferase system can specifically transport glucose from the extracellular membrane into the cell and phosphorylate glucose into glucose-6-phosphate, and then enter the glycolysis pathway. Therefore, the existing technology mainly reduces catabolite repression by inhibiting the glucose phosphorylation transport process. In the present invention, by encapsulating and fixing E. coli on the surface of the amino resin, the amino resin can fix the extracellular enzymes of E. coli, thereby increasing the activity of the extracellular enzymes, further promoting the transport of glucose by E. coli, accelerating the glucose phosphorylation transport process, and further accelerating the subsequent absorption of pentoses. In addition, by encapsulating and fixing E. coli, the generation of miscellaneous bacteria can also be avoided;

[0047] (2) When embedding and fixing Escherichia coli, the present invention first adsorbs calcium ions on the surface of the amino resin, then mixes sodium alginate and sodium carbonate with Escherichia coli, and then mixes with the amino resin after adsorbing calcium ions. The calcium ions on the surface of the amino resin can combine with alginate and carbonate, and bind Escherichia coli to the surface of the amino resin. In the subsequent process of adding calcium ions, the embedding and fixing of Escherichia coli can be promoted. The calcium ions combine with alginate to form calcium alginate to embed Escherichia coli. The calcium ions combine with the alginate to form calcium alginate to embed Escherichia coli. Carbonate ions can generate calcium carbonate to fix Escherichia coli. During the fermentation of Escherichia coli, since both calcium hydroxide and calcium carbonate can serve as neutralizers, but calcium hydroxide has a stronger neutralizing ability, the lactic acid produced preferentially combines with calcium hydroxide. However, as the fermentation progresses, when the concentration of the product on the surface of Escherichia coli is too high, it will react with calcium carbonate, causing the calcium carbonate to dissolve and release calcium ions, thereby increasing the pores and promoting the entry of nutrients and fermentation substrates, and the release of calcium lactate, thus avoiding the inhibitory effect of excessive calcium lactate concentration on Escherichia coli.

[0048] (3) According to the method of the present invention, when lignocellulosic biomass is used as raw material and used to produce D-lactic acid by Escherichia coli, the yield of D-lactic acid can be increased. For rice straw hydrolyzate with a total reducing sugar content of 32.87 g / L, wherein the concentration of glucose is 18.03 g / L, the content of xylose is 12.32 g / L, and the content of arabinose is 2.44 g / L, the fermentation time is 17.7-18.1 h when the total reducing sugar concentration is lower than 3 g / L. When the fermentation is continued, the minimum total reducing sugar concentration can reach 1.01-1.03 g / L, and the content of D-lactic acid in the fermentation product can reach 28.43-28.50 g / L. The optical purity of D-lactic acid in the lactic acid solution obtained after post-treatment is 99.81-99.87%. DETAILED DESCRIPTION

[0049] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, the specific embodiments of the present invention are now described.

[0050] Example 1 Preparation of Rice Straw Hydrolyzate

[0051] The rice straw was washed, dried, and crushed, and passed through a 20-mesh sieve to obtain rice straw powder; the rice straw powder was mixed with a 2% sulfuric acid aqueous solution at a ratio of 1 g:10 mL, and hydrolyzed at 121° C. for 1 h. After cooling, sodium hydroxide was added to adjust the pH to 4.8, and Trichoderma reesei cellulase solution was added. The ratio of rice straw powder to Trichoderma reesei cellulase solution was 1 g:8 mL. The mixture was stirred at 50° C. at a stirring speed of 200 r / min for 75 h. During the stirring process, the pH was monitored and maintained at 4.8. After the stirring was completed, the mixture was filtered, the filtrate was collected, calcium hydroxide was added to the filtrate to adjust the pH to 10, and the mixture was stirred at 90° C. at a stirring speed of 200 r / min for 30 min. The mixture was filtered, the filtrate was collected, and a sulfuric acid aqueous solution was added to adjust the pH to 7 to obtain a rice straw hydrolyzate;

[0052] The enzyme activity of the Trichoderma reesei cellulase solution is 1 FPU / mL;

[0053] The mass concentration of the sulfuric acid aqueous solution is 10%;

[0054] The total reducing sugar content in the rice straw hydrolyzate is 32.87 g / L, wherein the concentration of glucose is 18.03 g / L, the content of xylose is 12.32 g / L, and the content of arabinose is 2.44 g / L.

[0055] Example 2 Encapsulation, fermentation and post-processing

[0056] 1. Soaking: Mix the amino resin and calcium chloride aqueous solution in a ratio of 1 g:7 mL, stir at 25°C at a stirring speed of 100 r / min for 5 hours, filter, and collect the filter residue to obtain the soaked amino resin. Store the soaked amino resin at 4°C;

[0057] The manufacturer of the amino resin is Xi'an Lanxiao Technology New Materials Co., Ltd., and the model is LX1000EA;

[0058] The mass concentration of the calcium chloride aqueous solution is 5%;

[0059] 2. Embedding and fixation: Use an inoculating loop to take Escherichia coli preserved in a -80℃ glycerol tube and streak it on the activation culture medium, then invert and culture it at 37℃ for 18 hours; take a single colony and inoculate it into 50mL seed culture medium, and culture it at 37℃ and 100r / min for 15 hours. Keep anaerobic during the shaking culture without any air, and obtain seed liquid. Centrifuge the seed liquid and collect wet bacteria; mix the wet bacteria with physiological saline at a ratio of 1g:9mL, stir it at 20℃ and 20r / min for 10 minutes, and add sea salt. The sodium alginate aqueous solution and the sodium carbonate aqueous solution are used in a ratio of 1 g:18 mL:18 mL for wet bacteria to the sodium alginate aqueous solution and the sodium carbonate aqueous solution, and the stirring is continued for 30 min. The amino resin after soaking is added, and the ratio of the wet bacteria to the amino resin after soaking is 1 g:2 g. The stirring is continued for 2 h. The calcium chloride aqueous solution is added, and the ratio of the wet bacteria to the calcium chloride aqueous solution is 1 g:65 mL. The stirring is continued for 2 h. The solution is filtered and the filter residue is collected to obtain the embedded and fixed Escherichia coli. The embedded and fixed Escherichia coli is stored at 4°C.

[0060] The Escherichia coli was donated by Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, with the deposit number CGMCC 7679;

[0061] The activation culture medium comprises: 10 g / L tryptone, 5 g / L yeast powder, 10 g / L sodium chloride, 20 g / L xylose, 20 g / L agar, the solvent is water, and the pH value is 7.0;

[0062] The seed culture medium comprises: 20 g / L xylose, 0.87 g / L ammonium dihydrogen phosphate, 2.63 g / L diammonium hydrogen phosphate, 0.18 g / L magnesium sulfate heptahydrate, 0.15 g / L betaine hydrochloride, 1.5 μg / L ferric chloride hexahydrate, 0.1 μg / L cobalt chloride hexahydrate, 0.1 μg / L copper chloride dihydrate, 0.1 μg / L zinc chloride, 0.1 μg / L sodium molybdate dihydrate, 0.2 μg / L manganese chloride tetrahydrate, 0.05 μg / L boric acid, the solvent is water, and the pH value is natural;

[0063] The mass concentration of the sodium alginate aqueous solution is 1%;

[0064] The mass concentration of the sodium carbonate aqueous solution is 5%;

[0065] The mass concentration of the calcium chloride aqueous solution is 5%;

[0066] 3. Fermentation: The immobilized Escherichia coli was added to a fermentation medium at a ratio of 2 g of immobilized Escherichia coli to 30 mL of fermentation medium. The culture was shaken at 37°C and 100 rpm. A calcium hydroxide aqueous solution was added to maintain the pH at 7. The shaking culture was maintained anaerobic without any air flow. The total reducing sugar concentration was monitored in real time. Fermentation was terminated when the total reducing sugar concentration was less than 3 g / L to obtain a fermentation product.

[0067] The fermentation medium comprises: 0.87 g / L ammonium dihydrogen phosphate, 2.63 g / L diammonium hydrogen phosphate, 0.18 g / L magnesium sulfate heptahydrate, 0.15 g / L betaine hydrochloride, 2.4 μg / L ferric chloride hexahydrate, 0.3 μg / L cobalt chloride hexahydrate, 0.15 μg / L copper chloride dihydrate, 0.3 μg / L zinc chloride, 0.3 μg / L sodium molybdate dihydrate, 0.5 μg / L manganese chloride tetrahydrate, and 0.072 μg / L boric acid; the solvent is the rice straw hydrolyzate obtained in Example 1, and the pH value is natural;

[0068] The molar concentration of the calcium hydroxide aqueous solution is 3 mol / L;

[0069] 4. Post-processing:

[0070] (1) Ceramic membrane filtration: After converting calcium lactate in the fermentation product into lactic acid, it is filtered using a ceramic membrane, and the filtrate is taken as the ceramic membrane filtrate;

[0071] The filtration accuracy of the ceramic membrane is 50nm;

[0072] (2) Nanofiltration membrane refining: Use nanofiltration membrane to purify the ceramic membrane filtrate, and take the nanofiltration clear phase as the nanofiltrate;

[0073] The molecular weight cut-off of the nanofiltration membrane is 200Da;

[0074] (3) Ion exchange: The nanofiltrate was subjected to ion exchange using D001 strong acid styrene-based cation exchange resin and D301 weak base acrylic anion exchange resin in sequence, and then evaporated and concentrated to obtain a lactic acid solution.

[0075] Example 3 Encapsulation, fermentation and post-processing

[0076] 1. Soaking: Mix the amino resin and calcium chloride aqueous solution in a ratio of 1 g:9 mL, stir at 40°C at a stirring speed of 300 r / min for 6 hours, filter, and collect the filter residue to obtain the soaked amino resin. Store the soaked amino resin at 4°C;

[0077] The manufacturer of the amino resin is Xi'an Lanxiao Technology New Materials Co., Ltd., and the model is LX1000EA;

[0078] The mass concentration of the calcium chloride aqueous solution is 5%;

[0079] 2. Embedding and fixation: Use an inoculating loop to take Escherichia coli preserved in a -80℃ glycerol tube and streak it on the activation culture medium, then invert and culture it at 37℃ for 18 hours; take a single colony and inoculate it into 50mL seed culture medium, and culture it at 37℃ and 150r / min for 15 hours. Keep anaerobic during the shaking culture without any air, and obtain seed liquid. Centrifuge the seed liquid and collect wet bacteria; mix the wet bacteria with physiological saline at a ratio of 1g:10mL, stir it at 35℃ and 60r / min for 20 minutes, and add sea salt. Sodium alginate aqueous solution, sodium carbonate aqueous solution, the amount ratio of wet bacteria to sodium alginate aqueous solution, sodium carbonate aqueous solution is 1g:10mL:21mL, continue stirring for 40min, add the soaked amino resin, the amount ratio of wet bacteria to the soaked amino resin is 1g:2.3g, continue stirring for 3h, add calcium chloride aqueous solution, the amount ratio of wet bacteria to calcium chloride aqueous solution is 1g:70mL, continue stirring for 3h, filter, take the filter residue, and obtain the embedded and fixed Escherichia coli, and store the embedded and fixed Escherichia coli at 4°C;

[0080] The Escherichia coli was donated by Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, with the deposit number CGMCC 7679;

[0081] The activation culture medium comprises: 10 g / L tryptone, 5 g / L yeast powder, 10 g / L sodium chloride, 20 g / L xylose, 20 g / L agar, the solvent is water, and the pH value is 7.0;

[0082] The seed culture medium comprises: 20 g / L xylose, 0.87 g / L ammonium dihydrogen phosphate, 2.63 g / L diammonium hydrogen phosphate, 0.18 g / L magnesium sulfate heptahydrate, 0.15 g / L betaine hydrochloride, 1.5 μg / L ferric chloride hexahydrate, 0.1 μg / L cobalt chloride hexahydrate, 0.1 μg / L copper chloride dihydrate, 0.1 μg / L zinc chloride, 0.1 μg / L sodium molybdate dihydrate, 0.2 μg / L manganese chloride tetrahydrate, 0.05 μg / L boric acid, the solvent is water, and the pH value is natural;

[0083] The mass concentration of the sodium alginate aqueous solution is 1%;

[0084] The mass concentration of the sodium carbonate aqueous solution is 5%;

[0085] The mass concentration of the calcium chloride aqueous solution is 5%;

[0086] 3. Fermentation: The immobilized Escherichia coli was added to a fermentation medium at a ratio of 2 g of immobilized Escherichia coli to 32 mL of fermentation medium. The culture was shaken at 37°C and 150 rpm. A calcium hydroxide aqueous solution was added to maintain the pH at 7. The shaking culture was maintained anaerobic without any air flow. The total reducing sugar concentration was monitored in real time. Fermentation was terminated when the total reducing sugar concentration was less than 3 g / L to obtain a fermentation product.

[0087] The fermentation medium comprises: 0.87 g / L ammonium dihydrogen phosphate, 2.63 g / L diammonium hydrogen phosphate, 0.18 g / L magnesium sulfate heptahydrate, 0.15 g / L betaine hydrochloride, 2.4 μg / L ferric chloride hexahydrate, 0.3 μg / L cobalt chloride hexahydrate, 0.15 μg / L copper chloride dihydrate, 0.3 μg / L zinc chloride, 0.3 μg / L sodium molybdate dihydrate, 0.5 μg / L manganese chloride tetrahydrate, and 0.072 μg / L boric acid; the solvent is the rice straw hydrolyzate obtained in Example 1, and the pH value is natural;

[0088] The molar concentration of the calcium hydroxide aqueous solution is 3 mol / L;

[0089] 4. Post-processing:

[0090] (1) Ceramic membrane filtration: After converting calcium lactate in the fermentation product into lactic acid, it is filtered using a ceramic membrane, and the filtrate is taken as the ceramic membrane filtrate;

[0091] The filtration accuracy of the ceramic membrane is 50nm;

[0092] (2) Nanofiltration membrane refining: Use nanofiltration membrane to purify the ceramic membrane filtrate, and take the nanofiltration clear phase as the nanofiltrate;

[0093] The molecular weight cut-off of the nanofiltration membrane is 200Da;

[0094] (3) Ion exchange: The nanofiltrate was subjected to ion exchange using D001 strong acid styrene-based cation exchange resin and D301 weak base acrylic anion exchange resin, followed by evaporation and concentration to obtain a lactic acid solution.

[0095] Comparative Example 1

[0096] This comparative example is modified based on the technical solution of Example 2, and the specific changes are as follows:

[0097] In the first soaking step, an equal volume of deionized water was used instead of the aqueous calcium chloride solution.

[0098] The remaining technical solutions are the same as those in Example 2.

[0099] Comparative Example 2

[0100] This comparative example is modified based on the technical solution of Example 2, and the specific changes are as follows:

[0101] The soaking step in step 1 is omitted, and the addition of the amino resin after soaking is omitted in the embedding and fixing step in step 2. Specifically, the embedding and fixing step in step 2 is changed to:

[0102] Using an inoculating loop, streak the Escherichia coli preserved in a -80°C glycerol tube on an activated culture medium, and then invert and culture at 37°C for 18 hours; take a single colony and inoculate it into 50 mL of seed culture medium, and culture it at 37°C and 100 r / min for 15 hours, maintaining anaerobicity during the shaking culture without any air, to obtain a seed solution, centrifuge the seed solution, and collect wet bacteria; mix the wet bacteria with physiological saline at a ratio of 1 g:10 mL, stir at 20°C and 20 r / min for 10 minutes, add sodium alginate aqueous solution and sodium carbonate aqueous solution, the ratio of wet bacteria to sodium alginate aqueous solution and sodium carbonate aqueous solution is 1 g:18 mL:18 mL, continue stirring for 30 minutes, add calcium chloride aqueous solution, the ratio of wet bacteria to calcium chloride aqueous solution is 1 g:65 mL, continue stirring for 2 hours, filter, and take the filter residue to obtain the embedded and fixed Escherichia coli, and store the embedded and fixed Escherichia coli at 4°C;

[0103] The Escherichia coli was donated by Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, with the deposit number CGMCC 7679;

[0104] The activation culture medium comprises: 10 g / L tryptone, 5 g / L yeast powder, 10 g / L sodium chloride, 20 g / L xylose, 20 g / L agar, the solvent is water, and the pH value is 7.0;

[0105] The seed culture medium comprises: 20 g / L xylose, 0.87 g / L ammonium dihydrogen phosphate, 2.63 g / L diammonium hydrogen phosphate, 0.18 g / L magnesium sulfate heptahydrate, 0.15 g / L betaine hydrochloride, 1.5 μg / L ferric chloride hexahydrate, 0.1 μg / L cobalt chloride hexahydrate, 0.1 μg / L copper chloride dihydrate, 0.1 μg / L zinc chloride, 0.1 μg / L sodium molybdate dihydrate, 0.2 μg / L manganese chloride tetrahydrate, 0.05 μg / L boric acid, the solvent is water, and the pH value is natural;

[0106] The mass concentration of the sodium alginate aqueous solution is 1%;

[0107] The mass concentration of the sodium carbonate aqueous solution is 5%;

[0108] The mass concentration of the calcium chloride aqueous solution is 5%.

[0109] The remaining technical solutions are the same as those in Example 2.

[0110] Comparative Example 3

[0111] This comparative example is modified based on the technical solution of Example 2, and the specific changes are as follows:

[0112] In the second embedding and fixing step, the addition of sodium carbonate aqueous solution is omitted, that is, the second embedding and fixing step is changed to:

[0113] Use an inoculating loop to take Escherichia coli preserved in a -80°C glycerol tube and streak it on the activated culture medium, then invert and culture it at 37°C for 18 hours; take a single colony and inoculate it into 50 mL of seed culture medium, and culture it at 37°C and 100 r / min for 15 hours. During the shaking culture, maintain anaerobic conditions without any air, to obtain seed liquid, centrifuge the seed liquid, and collect wet cells; mix the wet cells with physiological saline at a ratio of 1 g:10 mL, and stir at 20°C and 20 r / min for 10 minutes. min, add sodium alginate aqueous solution, the ratio of wet bacteria to sodium alginate aqueous solution is 1g:18mL, continue stirring for 30min, add the immersed amino resin, the ratio of wet bacteria to the immersed amino resin is 1g:2g, continue stirring for 2h, add calcium chloride aqueous solution, the ratio of wet bacteria to calcium chloride aqueous solution is 1g:65mL, continue stirring for 2h, filter, take the filter residue to obtain the embedded and fixed Escherichia coli, and store the embedded and fixed Escherichia coli at 4°C;

[0114] The Escherichia coli was donated by Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, with the deposit number CGMCC 7679;

[0115] The activation culture medium comprises: 10 g / L tryptone, 5 g / L yeast powder, 10 g / L sodium chloride, 20 g / L xylose, 20 g / L agar, the solvent is water, and the pH value is 7.0;

[0116] The seed culture medium comprises: 20 g / L xylose, 0.87 g / L ammonium dihydrogen phosphate, 2.63 g / L diammonium hydrogen phosphate, 0.18 g / L magnesium sulfate heptahydrate, 0.15 g / L betaine hydrochloride, 1.5 μg / L ferric chloride hexahydrate, 0.1 μg / L cobalt chloride hexahydrate, 0.1 μg / L copper chloride dihydrate, 0.1 μg / L zinc chloride, 0.1 μg / L sodium molybdate dihydrate, 0.2 μg / L manganese chloride tetrahydrate, 0.05 μg / L boric acid, the solvent is water, and the pH value is natural;

[0117] The mass concentration of the sodium alginate aqueous solution is 1%;

[0118] The mass concentration of the calcium chloride aqueous solution is 5%.

[0119] The remaining technical solutions are the same as those in Example 2.

[0120] Comparative Example 4

[0121] This comparative example is modified based on the technical solution of Example 2, and the specific changes are as follows:

[0122] The first soaking step and the second embedding and fixing step are omitted, and in the third fermentation step, wet cells of equal mass are used instead of the embedded and fixed Escherichia coli. The preparation method of the wet cells is as follows:

[0123] Use an inoculating loop to streak Escherichia coli preserved in a -80°C glycerol tube on the activated culture medium, and then incubate it upside down at 37°C for 18 hours; take a single colony and inoculate it into 50 mL of seed culture medium, and culture it with shaking at 37°C and 100 rpm for 15 hours. Maintain anaerobic conditions during the shaking culture without passing any air to obtain seed liquid, centrifuge the seed liquid, and collect the wet bacteria.

[0124] The remaining technical solutions are the same as those in Example 2.

[0125] Test Example 1

[0126] The fermentation time in the fermentation steps of Examples 2-3 and Comparative Examples 1-4 was statistically analyzed, i.e., the fermentation time from the start of fermentation to the time when the total reducing sugar concentration was lower than 3 g / L. The statistical results are as follows:

[0127]

[0128] Test Example 2

[0129] In the fermentation steps of Examples 2-3 and Comparative Examples 1-4, after the total reducing sugar concentration was lower than 3 g / L, the fermentation was continued, and the total reducing sugar concentration was monitored in real time, and the lowest total reducing sugar concentration was recorded. The recorded results are as follows:

[0130]

[0131] After the total reducing sugar concentration no longer changes and the calcium lactate in the fermentation product is converted into lactic acid, the D-lactic acid content is tested. The test results are as follows:

[0132]

[0133] Test Example 3

[0134] The optical purity of D-lactic acid in the lactic acid solution obtained in the post-treatment steps of Examples 2-3 and Comparative Examples 1-4 was tested, and the test results were as follows:

[0135]

[0136] It can be seen from the results of Test Example 1 and Test Example 2 that, compared with Example 2, Comparative Examples 1-4 all have the problems of slow fermentation speed, incomplete fermentation, and low D-lactic acid production.

Claims

1. A method for increasing lactic acid production, characterized in that: include: Soaking, embedding, fixation, fermentation, and post-processing; The soaking step comprises mixing the amino resin with a calcium chloride aqueous solution, stirring at 25-40° C., filtering, and collecting the filter residue to obtain the soaked amino resin; The embedding and fixation process activates and cultures the Escherichia coli, centrifuges the seed solution, and collects the wet cells; mixes the wet cells with physiological saline, stirs at 20-35° C., adds a sodium alginate aqueous solution and a sodium carbonate aqueous solution, continues stirring, adds the soaked amino resin, continues stirring, adds a calcium chloride aqueous solution, continues stirring, filters, and collects the filter residue to obtain the embedded and fixed Escherichia coli; In the embedding and fixation, the Escherichia coli was donated by Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, with the deposit number CGMCC 7679; The fermentation comprises adding the embedded and fixed Escherichia coli to a fermentation medium, culturing the culture at 37° C. and 100-150 rpm with shaking, adding a calcium hydroxide aqueous solution to maintain the pH at 7, maintaining anaerobic conditions during the shaking culture without any air flow, and monitoring the total reducing sugar concentration in real time. When the total reducing sugar concentration is lower than 3 g / L, the fermentation is terminated to obtain a fermentation product. The solvent of the fermentation medium is rice straw hydrolyzate.

2. The method for increasing lactic acid production according to claim 1, wherein During the soaking, the ratio of the amino resin to the calcium chloride aqueous solution is 1 g: 7-9 mL; The manufacturer of the amino resin is Xi'an Lanxiao Technology New Materials Co., Ltd., and the model is LX1000EA; The mass concentration of the calcium chloride aqueous solution is 5%; When the amino resin is stored after soaking, the storage temperature is 4°C.

3. The method for increasing lactic acid production according to claim 1, wherein: During the embedding and fixation, the activation step is to use an inoculation loop to take Escherichia coli stored in a -80°C glycerol tube and streak it on the activation medium, and then incubate it upside down at 37°C for 18 hours to obtain activated colonies; The activation culture medium comprises the following components: 10 g / L tryptone, 5 g / L yeast powder, 10 g / L sodium chloride, 20 g / L xylose, 20 g / L agar, the solvent is water, and the pH value is 7.

0.

4. The method for increasing lactic acid production according to claim 1, wherein: In the embedding and fixation, the seed culture is carried out by taking a single colony from the activated colony and inoculating it into 50 mL of seed culture medium, and shaking and culturing it at 37° C. and 100-150 rpm for 15 hours. The shaking culture is maintained anaerobic without any air, to obtain a seed solution; The components of the seed culture medium include: 20g / L xylose, 0.87g / L ammonium dihydrogen phosphate, 2.63g / L diammonium hydrogen phosphate, 0.18g / L magnesium sulfate heptahydrate, 0.15g / L betaine hydrochloride, 1.5μg / L ferric chloride hexahydrate, 0.1μg / L cobalt chloride hexahydrate, 0.1μg / L copper chloride dihydrate, 0.1μg / L zinc chloride, 0.1μg / L sodium molybdate dihydrate, 0.2μg / L manganese chloride tetrahydrate, 0.05μg / L boric acid, the solvent is water, and the pH value is natural.

5. The method for increasing lactic acid production according to claim 1, wherein: In the embedding and fixation, the mass concentration of the sodium alginate aqueous solution is 1%; The mass concentration of the sodium carbonate aqueous solution is 5%; The mass concentration of the calcium chloride aqueous solution is 5%.

6. The method for increasing lactic acid production according to claim 1, wherein: During the embedding and fixation, the ratio of wet bacteria to physiological saline is 1 g: 9-10 mL; The ratio of wet bacteria to sodium alginate aqueous solution and sodium carbonate aqueous solution is 1g:18-10mL:18-21mL; The ratio of wet cells to the amino resin after soaking is 1g:2-2.3g; The ratio of wet bacteria to calcium chloride aqueous solution is 1g:65-70mL; When the embedded and fixed Escherichia coli is stored, the storage temperature is 4°C.

7. The method for increasing lactic acid production according to claim 1, wherein: During the fermentation, the ratio of the embedded and fixed Escherichia coli to the fermentation medium is 2 g:30-32 mL; The molar concentration of the calcium hydroxide aqueous solution is 3 mol / L.

8. The method for increasing lactic acid production according to claim 1, wherein: During the fermentation, the components of the fermentation medium include: 0.87 g / L ammonium dihydrogen phosphate, 2.63 g / L diamine hydrogen phosphate, 0.18 g / L magnesium sulfate heptahydrate, 0.15 g / L betaine hydrochloride, 2.4 μg / L ferric chloride hexahydrate, 0.3 μg / L cobalt chloride hexahydrate, 0.15 μg / L copper chloride dihydrate, 0.3 μg / L zinc chloride, 0.3 μg / L sodium molybdate dihydrate, 0.5 μg / L manganese chloride tetrahydrate, 0.072 μg / L boric acid, the solvent is rice straw hydrolyzate, and the pH value is natural.

9. The method for increasing lactic acid production according to claim 8, wherein: In the fermentation, the rice straw hydrolyzate is prepared by: washing, drying, and crushing the rice straw, and passing it through a 20-30 mesh sieve to obtain rice straw powder; mixing the rice straw powder with a 2% by mass sulfuric acid aqueous solution, hydrolyzing it at 121° C. for 1-1.5 hours, cooling it, adding sodium hydroxide to adjust the pH to 4.8-5, adding Trichoderma reesei cellulase solution, stirring it at 50-55° C. for 75-80 hours, monitoring the pH during the stirring process and maintaining it at 4.8-5, filtering it after the stirring is completed, taking the filtrate, adding calcium hydroxide to the filtrate to adjust the pH to 10, stirring it at 90° C. for 30-40 minutes, filtering it, taking the filtrate, and adding sulfuric acid aqueous solution to adjust the pH to 7 to obtain the rice straw hydrolyzate; In the preparation of the rice straw hydrolyzate, the ratio of rice straw powder to 2% sulfuric acid aqueous solution is 1 g:10 mL; The ratio of rice straw powder to Trichoderma reesei cellulase solution is 1g:7-9mL; The enzyme activity of the Trichoderma reesei cellulase solution is 1 FPU / mL; The mass concentration of the sulfuric acid aqueous solution is 10%.

10. The method for increasing lactic acid production according to claim 1, wherein: The post-treatment includes: ceramic membrane filtration, nanofiltration membrane refining, and ion exchange; The ceramic membrane filtration: after converting calcium lactate in the fermentation product into lactic acid, filtering it with a ceramic membrane, and taking the filtrate as the ceramic membrane filtrate; The filtration accuracy of the ceramic membrane is 50nm; The nanofiltration membrane refining: using a nanofiltration membrane to perform nanofiltration refining on the ceramic membrane filtrate, and taking the nanofiltration clear phase as the nanofiltrate; The molecular weight cut-off of the nanofiltration membrane is 200Da; The ion exchange is carried out by sequentially using D001 strongly acidic styrene-based cation exchange resin and D301 weakly basic acrylic acid-based anion exchange resin to perform ion exchange on the nanofiltrate, and then evaporating and concentrating the solution to obtain a lactic acid solution.

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