Preparation method of vitamin C

Abstract

The invention provides a preparation method of vitamin C, and belongs to the technical field of vitamin C. The preparation method comprises the following steps: preparing 2-keto-L-gulonic acid, preparing a catalyst and reacting. The preparation method of the catalyst comprises the following steps: carrying out water vapor treatment on a ZMS-5 molecular sieve, then putting the ZMS-5 molecular sieve into a calcining furnace, calcining at 500-520 DEG C for 3.5-5.0 h, naturally recovering to room temperature, putting the ZMS-5 molecular sieve into deionized water, adding gamma-polyglutamic acid and citric acid, uniformly stirring, filtering and drying to obtain a pretreated molecular sieve; the pretreated molecular sieve is put into a phosphoric acid solution to be soaked, drying is conducted after soaking is completed, and a catalyst is prepared; the vitamin C prepared by the preparation method disclosed by the invention is relatively high in purity and yield, and the 2-keto-L-gulonic acid prepared by fermentation is high in content and short in fermentation period.

Description

Technical Field

[0001] The invention belongs to the technical field of vitamin C, and particularly relates to a method for preparing vitamin C. Background Art

[0002] Vitamin C, also known as L-ascorbic acid, is a water-soluble vitamin that is rich in fruits and vegetables. It plays a regulatory role in redox metabolic reactions, and its lack can cause scurvy. Vitamin C can promote the synthesis of collagen. Vitamin C can promote the excretion of cholesterol and prevent cholesterol from depositing on the inner wall of the artery, thereby effectively preventing arteriosclerosis. Vitamin C can promote the intestinal absorption of iron, improve the liver's utilization of iron, and is beneficial for the treatment of iron deficiency anemia. Vitamin C can improve the body's immunity, increase capillary elasticity, promote wound and surgical incision healing, promote growth and development, and prevent cancer, heart disease, stroke, etc. It is widely used in medicine, food and beverage, chemical industry, feed and other fields. With the increasing demand for vitamin C in the food, medicine, chemical and other industries, higher requirements are also placed on the preparation process of vitamin C.

[0003] In traditional processes, the preparation methods of vitamin C are mainly acid method and alkaline method; the alkaline method is to first esterify 2-keto-L-gulonic acid with methanol under the catalytic action of a strong acid (such as concentrated hydrochloric acid, concentrated sulfuric acid, etc.), and then carry out alkaline conversion under alkaline conditions (sodium bicarbonate) to complete the lactonization reaction to generate vitamin C sodium salt, and then obtain the vitamin C product through refining; however, the above method has the advantages of complicated process, harsh reaction conditions, and high production cost, and is not suitable for mass production of vitamin C.

[0004] The acid method uses 2-keto-L-gulonic acid as a raw material and acid as a catalyst to produce vitamin C in one step. It has the advantages of simple process, low cost, and no pollution. It is the main method for industrial production of vitamin C at present. In the preparation process, L-sorbose is first oxidized by fermentation to prepare 2-keto-L-gulonic acid, but the fermentation cycle is long, the conversion rate is low, and the yield is low, which seriously limits the fermentation production of vitamin C. In addition, in the acid-catalyzed conversion process, a higher concentration of hydrochloric acid or sulfuric acid is usually used. The process is highly corrosive, the requirements for equipment are extremely harsh, and the reaction cycle is long, which reduces the production efficiency of vitamin C.

[0005] CN116589439A discloses a method for preparing vitamin C, specifically discloses a fermentation method of 2-keto-L-gulonic acid, which comprises adding a fermentation medium into a fermentation tank, inoculating a mixed seed solution of Pantoea ananatis and Gluconobacter oxydans according to the inoculation amount, dripping a sorbitol solution, controlling the sorbitol concentration in the fermentation tank to be between 15-20 g / L, dripping sodium carbonate, controlling the pH to be between 6.7-6.8, fermenting for 20-30 hours, and maintaining a constant temperature of 30°C; the formula of the fermentation medium is: sorbitol 20-25 g / L, yeast extract 2-3 g / L, corn steep liquor 10-15g / L, urea 4-7g / L, magnesium sulfate 0.6-1g / L, calcium carbonate 8-15g / L, prepared with water; the preparation method of vitamin C is: dissolving 2-keto-L-gulonic acid in water to obtain a ketogulonic acid solution, adding a catalyst to the ketogulonic acid solution, wherein the catalyst is composed of palladium acetate, citric acid and L-alanine, reacting at 30-35 degrees Celsius for 1-2 hours, heating to 50-55°C, and reacting for 20 hours, stopping heating and unloading to obtain a solid liquid containing vitamin C; the solid liquid containing vitamin C is cooled and crystallized at room temperature for 4-6 hours, and then centrifuged and purified to obtain vitamin C; The method is used to ferment and prepare 2-keto-L-gulonic acid, and the molar conversion rate of 2-keto-L-gulonic acid is 88.1-92.3%, and the conversion rate of vitamin C is 88.9-94.7%. When vitamin C is prepared by this method, the yield of 2-keto-L-gulonic acid is still low, and the yield of vitamin C is not high and the purity is poor. Summary of the invention

[0006] In order to solve the technical problems existing in the prior art, the present invention provides a method for preparing vitamin C, which adopts an acid method to prepare vitamin C, has a high yield of 2-keto-L-gulonic acid, a high yield of vitamin C, and good product purity.

[0007] In view of the above technical problems, the present invention adopts the following technical solutions: A method for preparing vitamin C includes preparing 2-keto-L-gulonic acid, preparing a catalyst and reaction steps, which are specifically as follows: 1. Preparation of 2-keto-L-gulonic acid (1) Activation culture Placing Gluconobacter oxidans and Bacillus megaterium in an activation culture medium for activation culture at a temperature of 28-30° C. for a time of 23.5-24.5 hours, and obtaining an activated bacterial solution after the culture is completed; The Gluconobacter oxidans and Bacillus megaterium were purchased from the market, and the mass ratio of Gluconobacter oxidans to Bacillus megaterium was 2.2-2.8:1; The activation medium comprises 5.3-5.8 g / L of L-sorbose, 2.6-3.0 g / L of tryptone, 4.8-5.2 g / L of yeast extract, 0.10-0.15 g / L of urea, 0.7-1.0 g / L of potassium dihydrogen phosphate, and 0.13-0.17 g / L of magnesium sulfate, and the pH value is 6.7-6.9; (2) Seed cultivation The activated bacterial liquid is inoculated into the seed culture medium at a mass ratio of 4.5-5.5% for seed culture at a temperature of 28-30° C. for a time of 19.0-20.5 h. After the culture is completed, a seed culture liquid is obtained; The components of the seed culture medium are: L-sorbose 18-22 g / L, beef extract 2.7-3.2 g / L, yeast extract 2.6-3.0 g / L, soy peptone 9.8-10.2 g / L, urea 0.8-1.1 g / L, magnesium sulfate 1.6-2.0 g / L, potassium dihydrogen phosphate 0.8-1.2 g / L, aspartic acid 0.67-0.72 g / L, ferrous sulfate heptahydrate 0.58-0.63 g / L; (3) Fermentation culture The seed culture solution is inoculated into a fermentation medium at an inoculum amount of 8-12% by mass for fermentation culture at a culture temperature of 29-31° C. for a culture time of 37.5-38.5 h. During the fermentation process, a 9.5-10.5wt% L-sorbitol solution is added to control the sorbitol concentration to 17-20 g / L, and a 2.8-3.2wt% sodium carbonate solution is added to control the pH value to 6.7-7.0 to obtain a fermentation culture solution, and the fermentation culture solution is centrifuged, separated, concentrated and crystallized to obtain 2-keto-L-gulonic acid; The fermentation medium comprises 24.0-26.0 g / L of L-sorbose, 11.5-12.5 g / L of fermentation aid, 5.8-6.2 g / L of urea, 1.0-1.4 g / L of potassium dihydrogen phosphate, 0.8-1.2 g / L of magnesium sulfate, 2.3-2.7 g / L of calcium carbonate, 0.016-0.023 g / L of manganese sulfate, 0.4-0.6 μg / L of zinc sulfate, 0.42-0.48 g / L of active agent, and 1.8-2.2 μg / L of ammonium molybdate; The preparation method of the fermentation aid comprises the following steps: adding corn steep liquor powder to deionized water, stirring evenly, adding folic acid and L-cysteine, and stirring at 380-420 rpm for 33-36 minutes to obtain the fermentation aid; In the fermentation aid, the mass ratio of the deionized water, corn steep liquor powder, folic acid and L-cysteine ​​is 48-52:9.6-10.3:0.8-1.2:1.0-1.5; The active auxiliary agent is a mixture of rhamnolipid and Span 80, and the mass ratio of the rhamnolipid to Span 80 is 1.2-1.5:1.0.

[0008] 2. Preparation of Catalyst Treat the ZMS-5 molecular sieve with steam at a space velocity of 1.8-2.2h -1 , control the heating rate to be 8-12℃ / min, raise the temperature to 310-330℃, treat for 38-42min, wait for it to naturally return to room temperature, place it in a calcining furnace, raise the temperature to 500-520℃ at a rate of 2.8-3.2℃ / min, and calcine for 3.5-5.0h. After the calcination is completed, naturally return to room temperature, put it into deionized water, add γ-polyglutamic acid and citric acid, raise the temperature to 40-45℃, stir for 0.8-1.2h, after the stirring is completed, filter and dry to obtain a pretreated molecular sieve; put the pretreated molecular sieve into a phosphoric acid solution for soaking, the soaking time is 1.8-2.2h, the soaking temperature is 50-55℃, after the soaking is completed, dry it at 78-82℃ for 9.5-10.5h to obtain a catalyst; The particle size of the ZSM-5 molecular sieve is 120-140 nm, and the silicon-aluminum ratio is 48-52; The mass ratio of the ZMS-5 molecular sieve, deionized water, γ-polyglutamic acid and citric acid is 9.5-10.6:98-105:0.3-0.5:0.8-1.2; The mass ratio of the pretreated molecular sieve to the phosphoric acid solution is 1:4-6; The mass concentration of the phosphoric acid solution is 20-24%.

[0009] 3. Reaction Mix 2-keto-L-gulonic acid, n-butanol and a catalyst, stir evenly, react at 40-45°C for 2.6-3.0h, then heat to 52-57°C at a rate of 0.4-0.6°C / min, stir and react for 4.3-4.8h to obtain a vitamin C solution, crystallize, centrifuge, wash and dry the vitamin C solution to obtain a vitamin C product; The mass ratio of the 2-keto-L-gulonic acid, n-butanol and catalyst is 9.8-10.2:21.0-21.8:2.5-3.0.

[0010] Compared with the prior art, the present invention has achieved the following beneficial effects: 1. The present invention uses 2-keto-L-gulonic acid as a reaction raw material to prepare vitamin C. First, Gluconobacter oxidans and Bacillus megaterium are fermented to prepare 2-keto-L-gulonic acid. Specifically, a specific aspartic acid component is added to the seed culture medium. Aspartic acid can provide energy during seed culture, promote the growth and reproduction of microorganisms, and improve the utilization rate of nutrients by microorganisms. In the fermentation culture step, specific fermentation aids and active aids are added. Folic acid in the fermentation aid can promote cell growth and improve the metabolic activity of microorganisms. L-cysteine ​​effectively increases the growth rate of microorganisms and increases the production efficiency of 2-keto-L-gulonic acid. It is combined with the active aid to improve the permeability of cells and improve the utilization rate of microorganisms below the nutrient level. The utilization rate of materials is improved, the yield of products is increased, the conversion rate of strains is increased, and the generation of by-products is reduced, thereby promoting the synthesis of vitamin C; in the process of catalyst preparation, ZSM-5 acidic molecular sieve is used, and it is first treated with water vapor to remove impurities in the molecular sieve, combined with calcination treatment to improve the stability of the molecular sieve, and then treated with citric acid and polyglutamic acid to enhance the dispersibility of the molecular sieve and improve the catalytic performance of the molecular sieve. Under the immersion of phosphoric acid solution, the phosphoric acid solution is fully adsorbed into the pores of the molecular sieve to obtain a catalyst. The catalyst has many active sites, good binding properties with 2-keto-L-gulonic acid, and good stability. In the reaction step, it effectively promotes the conversion of 2-keto-L-gulonic acid and improves the yield and purity of vitamin C. 2. The method of the present invention is used to prepare vitamin C with a yield of 94.0-94.6% and a purity of 98.96-99.45%; 3. Vitamin C is prepared by the method of the present invention, and the content of 2-keto-L-gulonic acid in the obtained fermentation culture medium is 64.8-66.5 g / L. DETAILED DESCRIPTION

[0011] In order to more clearly understand the technical features, purposes and effects of the present invention, specific embodiments of the present invention are now described.

[0012] Example 1 1. Preparation of 2-keto-L-gulonic acid (1) Activation culture Placing Gluconobacter oxidans and Bacillus megaterium in an activation culture medium for activation culture at a temperature of 29° C. for 24.0 h. After the culture is completed, an activated bacterial solution is obtained. The Gluconobacter oxidans and Bacillus megaterium were purchased from the market, and the mass ratio of Gluconobacter oxidans to Bacillus megaterium was 2.5:1; The activation medium comprises 5.5 g / L L-sorbose, 2.8 g / L tryptone, 5.0 g / L yeast extract, 0.12 g / L urea, 0.8 g / L potassium dihydrogen phosphate, and 0.15 g / L magnesium sulfate, and the pH value is 6.8; (2) Seed cultivation The activated bacterial liquid was inoculated into the seed culture medium at a mass ratio of 5% for seed culture at a culture temperature of 29° C. for 20.0 h. After the culture was completed, a seed culture liquid was obtained; The components of the seed culture medium are: 20 g / L L-sorbose, 3.0 g / L beef extract, 2.8 g / L yeast extract, 10.0 g / L soy peptone, 1.0 g / L urea, 1.8 g / L magnesium sulfate, 1.0 g / L potassium dihydrogen phosphate, 0.70 g / L aspartic acid, and 0.6 g / L ferrous sulfate heptahydrate; (3) Fermentation culture The seed culture solution was inoculated into a fermentation medium at an inoculum amount of 10% by mass for fermentation culture at a culture temperature of 30° C. for 38.0 h. During the fermentation process, a 10wt% L-sorbitol solution was added to control the sorbitol concentration to 18 g / L, and a 3.0wt% sodium carbonate solution was added to control the pH value to 6.8 to obtain a fermentation culture solution. The fermentation culture solution was centrifuged, separated, concentrated and crystallized to obtain 2-keto-L-gulonic acid. The fermentation medium comprises 25.0 g / L of L-sorbose, 12.0 g / L of fermentation aid, 6.0 g / L of urea, 1.2 g / L of potassium dihydrogen phosphate, 1.0 g / L of magnesium sulfate, 2.5 g / L of calcium carbonate, 0.020 g / L of manganese sulfate, 0.5 μg / L of zinc sulfate, 0.45 g / L of active aid, and 2.0 μg / L of ammonium molybdate; The preparation method of the fermentation aid is as follows: add 10.0 g of corn steep liquor powder to 50 g of deionized water, stir evenly, add 1.0 g of folic acid and 1.2 g of L-cysteine, stir at 400 rpm for 35 min, and obtain the fermentation aid; The active auxiliary agent is a mixture of rhamnolipid and Span 80, and the mass ratio of the rhamnolipid to Span 80 is 1.3:1.0.

[0013] 2. Preparation of Catalyst 10.0 g ZMS-5 molecular sieve was treated with steam at a space velocity of 2.0 h -1, control the heating rate to be 10℃ / min, raise the temperature to 320℃, treat for 40min, wait until it naturally returns to room temperature, place in a calcining furnace, heat to 510℃ at a rate of 3.0℃ / min and calcine for 4.0h, after the calcination is completed, naturally return to room temperature, put into 100g deionized water, add 0.4g γ-polyglutamic acid and 1.0g citric acid, raise the temperature to 43℃, stir for 1.0h, after the stirring is completed, filter and dry to obtain a pretreated molecular sieve; put the pretreated molecular sieve into 5 times the mass of 22wt% phosphoric acid solution for immersion, the immersion time is 2.0h, the immersion temperature is 52℃, after the immersion is completed, dry at 80℃ for 10.0h to obtain a catalyst; The particle size of the ZSM-5 molecular sieve is 130 nm, and the silicon-aluminum ratio is 50.

[0014] 3. Reaction Mix 10.0 g of 2-keto-L-gulonic acid, 21.3 g of n-butanol and 2.7 g of a catalyst, stir evenly, react at 42°C for 2.8 h, then heat to 55°C at a rate of 0.5°C / min, stir and react for 4.5 h to obtain vitamin C liquid. The vitamin C liquid is crystallized, centrifuged, washed and dried to obtain a vitamin C product.

[0015] In the fermentation broth of Example 1, the content of 2-keto-L-gulonic acid was 66.5 g / L, the yield of vitamin C was 94.6%, and the purity was 99.45%.

[0016] Example 2 1. Preparation of 2-keto-L-gulonic acid (1) Activation culture Placing Gluconobacter oxidans and Bacillus megaterium in an activation culture medium for activation culture at a temperature of 28° C. for 23.5 hours, and obtaining an activated bacterial solution after the culture is completed; The Gluconobacter oxidans and Bacillus megaterium were purchased from the market, and the mass ratio of Gluconobacter oxidans to Bacillus megaterium was 2.2:1; The activation medium comprises 5.3 g / L L-sorbose, 2.6 g / L tryptone, 4.8 g / L yeast extract, 0.10 g / L urea, 0.7 g / L potassium dihydrogen phosphate, and 0.13 g / L magnesium sulfate, and the pH value is 6.7; (2) Seed cultivation The activated bacterial liquid was inoculated into the seed culture medium at a mass ratio of 4.5% for seed culture at a temperature of 28° C. for 19.0 h. After the culture was completed, a seed culture liquid was obtained; The components of the seed culture medium are: L-sorbose 18 g / L, beef extract 2.7 g / L, yeast extract 2.6 g / L, soy peptone 9.8 g / L, urea 0.8 g / L, magnesium sulfate 1.6 g / L, potassium dihydrogen phosphate 0.8 g / L, aspartic acid 0.67 g / L, ferrous sulfate heptahydrate 0.58 g / L; (3) Fermentation culture The seed culture solution was inoculated into a fermentation medium at an inoculum amount of 8% by mass for fermentation culture at a culture temperature of 29° C. for 37.5 h. During the fermentation process, a 9.5wt% L-sorbitol solution was added to control the sorbitol concentration to 17 g / L, and a 2.8wt% sodium carbonate solution was added to control the pH value to 6.7 to obtain a fermentation culture solution. The fermentation culture solution was centrifuged, separated, concentrated and crystallized to obtain 2-keto-L-gulonic acid. The fermentation medium comprises 24.0 g / L of L-sorbose, 11.5 g / L of fermentation aid, 5.8 g / L of urea, 1.0 g / L of potassium dihydrogen phosphate, 0.8 g / L of magnesium sulfate, 2.3 g / L of calcium carbonate, 0.016 g / L of manganese sulfate, 0.4 μg / L of zinc sulfate, 0.4 μg / L of active aid, and 1.8 μg / L of ammonium molybdate; The preparation method of the fermentation aid is as follows: 9.6 g of corn steep liquor powder is added to 48 g of deionized water, and after stirring evenly, 0.8 g of folic acid and 1.0 g of L-cysteine ​​are added, and the mixture is stirred at 380 rpm for 33 min to obtain the fermentation aid; The active auxiliary agent is a mixture of rhamnolipid and Span 80, and the mass ratio of the rhamnolipid to Span 80 is 1.2:1.0.

[0017] 2. Preparation of Catalyst 9.5 g ZMS-5 molecular sieve was treated with steam at a space velocity of 1.8 h -1 , control the heating rate to 8℃ / min, raise the temperature to 310℃, treat for 38min, wait until it naturally returns to room temperature, place in a calcining furnace, heat to 500℃ at a rate of 2.8℃ / min and calcine for 5.0h, after calcination, naturally return to room temperature, put into 98g deionized water, add 0.3g γ-polyglutamic acid and 0.8g citric acid, raise the temperature to 40℃, stir for 0.8h, after stirring, filter and dry to obtain a pretreated molecular sieve; put the pretreated molecular sieve into 4 times the mass of 20wt% phosphoric acid solution for immersion, the immersion time is 1.8h, the immersion temperature is 50℃, after immersion, dry at 78℃ for 10.5h to obtain a catalyst; The particle size of the ZSM-5 molecular sieve is 120 nm, and the silicon-aluminum ratio is 48.

[0018] 3. Reaction Mix 9.8 g of 2-keto-L-gulonic acid, 21.0 g of n-butanol and 2.5 g of a catalyst, stir evenly, react at 40°C for 3.0 h, then heat to 52°C at a rate of 0.4°C / min, stir and react for 4.8 h to obtain vitamin C liquid. The vitamin C liquid is crystallized, centrifuged, washed and dried to obtain a vitamin C product.

[0019] In the fermentation broth of Example 2, the content of 2-keto-L-gulonic acid was 64.8 g / L, the yield of vitamin C was 94.0%, and the purity was 98.96%.

[0020] Example 3 1. Preparation of 2-keto-L-gulonic acid (1) Activation culture Placing Gluconobacter oxidans and Bacillus megaterium in an activation culture medium for activation culture at a temperature of 30° C. for 24.5 hours, and obtaining an activated bacterial solution after the culture is completed; The Gluconobacter oxidans and Bacillus megaterium were purchased from the market, and the mass ratio of Gluconobacter oxidans to Bacillus megaterium was 2.8:1; The activation medium comprises 5.8 g / L L-sorbose, 3.0 g / L tryptone, 5.2 g / L yeast extract, 0.15 g / L urea, 1.0 g / L potassium dihydrogen phosphate, and 0.17 g / L magnesium sulfate, and the pH value is 6.9; (2) Seed cultivation The activated bacterial liquid was inoculated into the seed culture medium at a mass ratio of 5.5% for seed culture at a temperature of 30°C for 20.5 hours. After the culture was completed, a seed culture liquid was obtained. The components of the seed culture medium are: L-sorbose 22 g / L, beef extract 3.2 g / L, yeast extract 3.0 g / L, soy peptone 10.2 g / L, urea 1.1 g / L, magnesium sulfate 2.0 g / L, potassium dihydrogen phosphate 1.2 g / L, aspartic acid 0.72 g / L, ferrous sulfate heptahydrate 0.63 g / L; (3) Fermentation culture The seed culture solution was inoculated into a fermentation medium at an inoculum amount of 12% by mass for fermentation culture at a culture temperature of 31° C. for 38.5 h. During the fermentation process, 10.5 wt % L-sorbitol solution was added to control the sorbitol concentration to 20 g / L, and 3.2 wt % sodium carbonate solution was added to control the pH value to 7.0 to obtain a fermentation culture solution. The fermentation culture solution was centrifuged, separated, concentrated and crystallized to obtain 2-keto-L-gulonic acid. The fermentation medium comprises 26.0 g / L of L-sorbose, 12.5 g / L of fermentation aid, 6.2 g / L of urea, 1.4 g / L of potassium dihydrogen phosphate, 1.2 g / L of magnesium sulfate, 2.7 g / L of calcium carbonate, 0.023 g / L of manganese sulfate, 0.6 μg / L of zinc sulfate, 0.48 g / L of active aid, and 2.2 μg / L of ammonium molybdate; The preparation method of the fermentation aid is as follows: add 10.3 g of corn steep liquor powder to 52 g of deionized water, stir evenly, add 1.2 g of folic acid and 1.5 g of L-cysteine, stir at 420 rpm for 36 min, and obtain the fermentation aid; The active auxiliary agent is a mixture of rhamnolipid and Span 80, and the mass ratio of the rhamnolipid to Span 80 is 1.5:1.0.

[0021] 2. Preparation of Catalyst 10.6 g ZMS-5 molecular sieve was treated with steam at a space velocity of 2.2 h -1 , control the heating rate to be 12℃ / min, raise the temperature to 330℃, treat for 42min, wait until it naturally returns to room temperature, place in a calcining furnace, heat to 520℃ at a rate of 3.2℃ / min and calcine for 3.5h, after the calcination is completed, naturally return to room temperature, put into 105g deionized water, add 0.5g γ-polyglutamic acid and 1.2g citric acid, raise the temperature to 45℃, stir for 1.2h, after the stirring is completed, filter and dry to obtain a pretreated molecular sieve; put the pretreated molecular sieve into 6 times the mass of 24wt% phosphoric acid solution for immersion, the immersion time is 2.2h, the immersion temperature is 55℃, after the immersion is completed, dry at 82℃ for 9.5h to obtain a catalyst; The particle size of the ZSM-5 molecular sieve is 140 nm, and the silicon-aluminum ratio is 52.

[0022] 3. Reaction Mix 10.2 g of 2-keto-L-gulonic acid, 21.8 g of n-butanol and 3.0 g of a catalyst, stir evenly, react at 45°C for 2.6 h, then heat to 57°C at a rate of 0.6°C / min, stir and react for 4.3 h to obtain vitamin C liquid. The vitamin C liquid is crystallized, centrifuged, washed and dried to obtain a vitamin C product.

[0023] In the fermentation broth of Example 3, the content of 2-keto-L-gulonic acid was 65.3 g / L, the yield of vitamin C was 94.4%, and the purity was 99.27%.

[0024] Comparative Example 1 Based on Example 1, the changes are as follows: in the step of preparing 2-keto-L-gulonic acid, (1) In the seed culture step, the aspartic acid component in the seed culture medium is omitted; (2) In the fermentation culture step, fermentation aids and active auxiliary ingredients in the fermentation medium are omitted; The rest of the operations are the same.

[0025] In the fermentation culture broth of Comparative Example 1, the content of 2-keto-L-gulonic acid was 51.5 g / L, the yield of vitamin C was 91.2%, and the purity was 96.68%.

[0026] Comparative Example 2 Based on Example 1, the changes are as follows: In the reaction step, the catalyst was replaced with phosphoric acid in equal amounts; The rest of the operations are the same.

[0027] The product obtained in Comparative Example 2 had a vitamin C yield of 86.32% and a purity of 92.35%.

[0028] The present invention adopts 2-keto-L-gulonic acid as a reaction raw material to prepare vitamin C. First, Gluconobacter oxidans and Bacillus megaterium are fermented to prepare 2-keto-L-gulonic acid. Specifically, a specific aspartic acid component is added to a seed culture medium. Aspartic acid can provide energy during seed culture, promote the growth and reproduction of microorganisms, and improve the utilization rate of nutrients by microorganisms. In the fermentation culture step, a specific fermentation aid and an active aid are added. Folic acid in the fermentation aid can promote cell growth and improve the metabolic activity of microorganisms. L-cysteine ​​effectively improves the growth rate of microorganisms and increases the production efficiency of 2-keto-L-gulonic acid. The combination of the active aid and the oxidative stress enhances the permeability of cells and improves the utilization rate of microorganisms below nutrients. The utilization rate of the material is improved, the output of the product is increased, the conversion rate of the strain is increased, and the generation of by-products is reduced, thereby promoting the synthesis of vitamin C; in the process of catalyst preparation, ZSM-5 acidic molecular sieve is used, and it is first treated with water vapor to remove impurities in the molecular sieve, combined with calcination treatment to improve the stability of the molecular sieve, and then treated with citric acid and polyglutamic acid to enhance the dispersibility of the molecular sieve and improve the catalytic performance of the molecular sieve. Under the immersion of phosphoric acid solution, the phosphoric acid solution is fully adsorbed into the pores of the molecular sieve to obtain a catalyst. The catalyst has many active sites, good binding with 2-keto-L-gulonic acid, and good stability. It effectively promotes the conversion of 2-keto-L-gulonic acid in the reaction step, and improves the yield and purity of vitamin C.

[0029] In Comparative Example 1, the aspartic acid component was omitted from the seed culture medium, and the fermentation aid and active aid were omitted from the fermentation medium, resulting in incomplete transformation of the strain, low content of 2-keto-L-gulonic acid, and more by-products, which affected the formation of vitamin C and reduced the yield and purity of the product; in Comparative Example 2, phosphoric acid was directly used as a catalyst, the reaction process was unstable, and the reaction with 2-keto-L-gulonic acid was incomplete, resulting in poor product quality and low yield.

[0030] Unless otherwise specified, all percentages used in the present invention are by mass.

[0031] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing vitamin C, characterized in that: The method comprises the steps of preparing 2-keto-L-gulonic acid, preparing a catalyst and reacting; The step of preparing 2-keto-L-gulonic acid includes activation culture, seed culture and fermentation culture steps; In the seed culture step, the components of the seed culture medium are: L-sorbose 18-22 g / L, beef extract 2.7-3.2 g / L, yeast extract 2.6-3.0 g / L, soy peptone 9.8-10.2 g / L, urea 0.8-1.1 g / L, magnesium sulfate 1.6-2.0 g / L, potassium dihydrogen phosphate 0.8-1.2 g / L, aspartic acid 0.67-0.72 g / L, ferrous sulfate heptahydrate 0.58-0.63 g / L; In the fermentation culture step, the fermentation medium comprises 24.0-26.0 g / L of L-sorbose, 11.5-12.5 g / L of fermentation aid, 5.8-6.2 g / L of urea, 1.0-1.4 g / L of potassium dihydrogen phosphate, 0.8-1.2 g / L of magnesium sulfate, 2.3-2.7 g / L of calcium carbonate, 0.016-0.023 g / L of manganese sulfate, 0.4-0.6 μg / L of zinc sulfate, 0.42-0.48 g / L of active agent, and 1.8-2.2 μg / L of ammonium molybdate; The preparation method of the fermentation aid comprises the following steps: adding corn steep liquor powder to deionized water, stirring evenly, adding folic acid and L-cysteine, and stirring at 380-420 rpm for 33-36 minutes to obtain the fermentation aid; The active auxiliary agent is a mixture of rhamnolipid and Span 80; The steps of preparing the catalyst are: subjecting the ZMS-5 molecular sieve to water vapor treatment, then placing it in a calcining furnace, calcining it at 500-520° C. for 3.5-5.0 hours, naturally recovering to room temperature, placing it in deionized water, adding γ-polyglutamic acid and citric acid, stirring evenly, filtering and drying to obtain a pretreated molecular sieve; placing the pretreated molecular sieve in a phosphoric acid solution for immersion, and drying after the immersion to obtain a catalyst; The reaction steps are as follows: 2-keto-L-gulonic acid, n-butanol and a catalyst are mixed, stirred evenly and reacted to obtain a vitamin C liquid; the vitamin C liquid is crystallized, centrifuged, washed and dried to obtain a vitamin C product.

2. The method for preparing vitamin C according to claim 1, characterized in that: The activation culture step comprises placing Gluconobacter oxidans and Bacillus megaterium in an activation culture medium for activation culture at a culture temperature of 28-30° C. for a culture time of 23.5-24.5 hours. After the culture is completed, an activated bacterial solution is obtained. The Gluconobacter oxidans and Bacillus megaterium were purchased from the market, and the mass ratio of Gluconobacter oxidans to Bacillus megaterium was 2.2-2.8:1; The activation culture medium comprises 5.3-5.8 g / L of L-sorbose, 2.6-3.0 g / L of tryptone, 4.8-5.2 g / L of yeast extract, 0.10-0.15 g / L of urea, 0.7-1.0 g / L of potassium dihydrogen phosphate, and 0.13-0.17 g / L of magnesium sulfate, and the pH value is 6.7-6.

9.

3. The method for preparing vitamin C according to claim 1, characterized in that: The seed culture step comprises inoculating the activated bacterial solution into a seed culture medium at a mass ratio of 4.5-5.5% for seed culture at a culture temperature of 28-30° C. for a culture time of 19.0-20.5 h. After the culture is completed, a seed culture solution is obtained.

4. The method for preparing vitamin C according to claim 1, characterized in that: The fermentation culture step comprises: inoculating the seed culture solution in a fermentation medium at an inoculation amount of 8-12% by mass for fermentation culture, the culture temperature is 29-31° C., the culture time is 37.5-38.5 hours, during the fermentation process, adding 9.5-10.5wt% L-sorbitol solution to control the sorbitol concentration to 17-20g / L, and adding 2.8-3.2wt% sodium carbonate solution to control the pH value to 6.7-7.0, to obtain a fermentation culture solution, and subjecting the fermentation culture solution to centrifugation, separation, concentration and crystallization to obtain 2-keto-L-gulonic acid; In the fermentation aid, the mass ratio of the deionized water, corn syrup powder, folic acid and L-cysteine ​​is 48-52: 9.6-10.3:0.8-1.2:1.0-1.5； In the active auxiliary agent, the mass ratio of the rhamnolipid to Span 80 is 1.2-1.5:1.

0.

5. The method for preparing vitamin C according to claim 1, characterized in that: The catalyst preparation step is to treat the ZMS-5 molecular sieve with water vapor at a space velocity of 1.8-2.2h -1 , control the heating rate to 8-12℃ / min, increase the temperature to 310-330℃, treat for 38-42min, wait for it to naturally return to room temperature, place it in a calcining furnace, heat it to 500-520℃ at a rate of 2.8-3.2℃ / min and calcine for 3.5-5.0h. After the calcination is completed, naturally return to room temperature, put it into deionized water, add γ-polyglutamic acid and citric acid, increase the temperature to 40-45℃, stir for 0.8-1.2h, after the stirring is completed, filter and dry to obtain a pretreated molecular sieve; put the pretreated molecular sieve into a phosphoric acid solution for immersion, the immersion time is 1.8-2.2h, the immersion temperature is 50-55℃, after the immersion is completed, dry it at 78-82℃ for 9.5-10.5h to obtain a catalyst.

6. The method for preparing vitamin C according to claim 1, characterized in that: In the step of preparing the catalyst, the particle size of the ZSM-5 molecular sieve is 120-140 nm, and the silicon-aluminum ratio is 48-52; The mass ratio of the ZMS-5 molecular sieve, deionized water, γ-polyglutamic acid and citric acid is 9.5-10.6:98-105:0.3-0.5:0.8-1.2; The mass ratio of the pretreated molecular sieve to the phosphoric acid solution is 1:4-6; The mass concentration of the phosphoric acid solution is 20-24%.

7. The method for preparing vitamin C according to claim 1, characterized in that: The reaction steps are as follows: 2-keto-L-gulonic acid, n-butanol and a catalyst are mixed, stirred evenly, reacted at 40-45° C. for 2.6-3.0 hours, then heated to 52-57° C. at a rate of 0.4-0.6° C. / min, stirred and reacted for 4.3-4.8 hours to obtain a vitamin C solution, and the vitamin C solution is crystallized, centrifuged, washed and dried to obtain a vitamin C product; The mass ratio of the 2-keto-L-gulonic acid, n-butanol and catalyst is 9.8-10.2:21.0-21.8:2.5-3.0.