A method for preparing vitamin C

Abstract

The present invention provides a method for preparing vitamin C, belonging to the technical field of vitamin C; the preparation method includes the steps of preparing 2-keto-L-gulonic acid, preparing a catalyst, and a reaction step; the step of preparing the catalyst is to perform steam treatment on ZMS-5 molecular sieve, then place it in a calcination furnace, calcine it at 500-520 °C for 3.5-5.0 h, naturally restore it to room temperature, then put it into deionized water, add γ-polyglutamic acid and citric acid, stir evenly, and then filter and dry to obtain a pretreated molecular sieve; put the pretreated molecular sieve into a phosphoric acid solution for soaking, and dry it after the soaking is completed to obtain a catalyst; the vitamin C prepared by the preparation method of the present invention has high purity and yield, and the content of 2-keto-L-gulonic acid obtained by fermentation is high, and the fermentation period is short.

Description

Technical Field

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

[0002] Vitamin C, also known as L-ascorbic acid, is a water-soluble vitamin that is abundant in fruits and vegetables; it plays a regulatory role in redox metabolic reactions, and deficiency of it 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 arteries, thereby effectively preventing arteriosclerosis; vitamin C can promote the absorption of iron in the intestine and improve the utilization rate of iron by the liver, which is beneficial to the treatment of iron-deficiency anemia; vitamin C can enhance the body's immunity, increase the elasticity of capillaries, promote the healing of wounds and surgical incisions, promote growth and development, and prevent cancer, heart disease, stroke, etc. It is widely used in the fields of medicine, food and beverage, chemical industry, feed, etc.;

[0003] With the increasing demand for vitamin C in industries such as food, medicine, and chemical industry, higher requirements have been put forward for the preparation process of vitamin C.

[0004] In traditional processes, the main methods for preparing vitamin C are the acid method and the base method; in the base method, 2-keto-L-gulonic acid is first esterified with methanol under the catalysis of a strong acid (such as concentrated hydrochloric acid, concentrated sulfuric acid, etc.), and then the base conversion is carried out under alkaline conditions (sodium bicarbonate) to complete the lactonization reaction to generate sodium ascorbate, and the vitamin C product is obtained through purification; however, the above methods have the disadvantages of cumbersome processes, harsh reaction conditions, and high production costs, and are not suitable for the large-scale production of vitamin C.

[0005] The acid method uses 2-keto-L-gulonic acid as the raw material and an acid as the catalyst to directly produce vitamin C in one step. It has the advantages of simple process, low cost, and no pollution, and 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 period is long, the conversion rate is low, and the yield is low, which severely limits the fermentation production of vitamin C; and in the acid-catalyzed conversion process, usually high-concentration hydrochloric acid or sulfuric acid is used, the process has strong corrosiveness, extremely high requirements for equipment, and a long reaction period, which reduces the production efficiency of vitamin C.

[0006] CN116589439A discloses a method for preparing vitamin C. Specifically, the fermentation method of 2-keto-L-gulonic acid is as follows: Add a fermentation medium into a fermenter, inoculate a mixed seed solution of Pantoea ananatis and Gluconobacter oxydans according to the inoculation amount, dropwise add a sorbitol solution, control the sorbitol concentration in the fermenter to be between 15 - 20 g / L, dropwise add sodium carbonate, control the pH to be 6.7 - 6.8, ferment for 20 - 30 hours, and maintain 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 - 15 g / L, urea 4 - 7 g / L, magnesium sulfate 0.6 - 1 g / L, calcium carbonate 8 - 15 g / L, prepared with water; the method for preparing vitamin C is to dissolve 2-keto-L-gulonic acid in water to obtain a keto-gulonic acid solution, add a catalyst to the keto-gulonic acid solution, the catalyst consists of palladium acetate, citric acid and L-alanine, and react at 30 - 35 °C for 1 - 2 hours, heat to 50 - 55 °C, after reacting for 20 hours, stop heating and discharge the material 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;

[0007] Using this method to ferment and prepare 2-keto-L-gulonic acid, 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%; using this method to prepare vitamin C, the yield of 2-keto-L-gulonic acid is still low, and the yield of vitamin C is not high and the purity is not good. Summary of the Invention

[0008] In order to solve the technical problems existing in the prior art, the present invention provides a method for preparing vitamin C. The acid method is used to prepare vitamin C, with a high yield of 2-keto-L-gulonic acid, a high yield of vitamin C, and good product purity.

[0009] In view of the above technical problems, the present invention adopts the following technical solutions:

[0010] A method for preparing vitamin C includes preparing 2-keto-L-gulonic acid, preparing a catalyst, and a reaction step, specifically as follows:

[0011] 1. Preparation of 2-keto-L-gulonic acid

[0012] (1) Activation culture

[0013] Place Gluconobacter oxydans and Bacillus megaterium in an activation medium for activation culture. The culture temperature is 28 - 30 °C, the culture time is 23.5 - 24.5 h. After the culture is completed, an activated bacterial solution is obtained;

[0014] Both Gluconobacter oxydans and Bacillus megaterium are obtained by purchasing from the market, and the mass ratio of Gluconobacter oxydans to Bacillus megaterium is 2.2 - 2.8:1;

[0015] The activated medium consists of L - sorbose 5.3 - 5.8 g / L, tryptone 2.6 - 3.0 g / L, yeast extract 4.8 - 5.2 g / L, urea 0.10 - 0.15 g / L, potassium dihydrogen phosphate 0.7 - 1.0 g / L, magnesium sulfate 0.13 - 0.17 g / L, and the pH value is 6.7 - 6.9;

[0016] (2)Seed culture

[0017] The activated bacterial liquid is inoculated into the seed medium for seed culture according to an inoculation amount of 4.5 - 5.5% by mass. The culture temperature is 28 - 30 °C, and the culture time is 19.0 - 20.5 h. After the culture is completed, the seed culture solution is obtained;

[0018] The seed medium consists of 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;

[0019] (3)Fermentation culture

[0020] The seed culture solution is inoculated into the fermentation medium for fermentation culture according to an inoculation amount of 8 - 12% by mass. The culture temperature is 29 - 31 °C, and the culture time is 37.5 - 38.5 h. During the fermentation process, a 9.5 - 10.5 wt% L - sorbose solution is added dropwise to control the sorbitol concentration at 17 - 20 g / L, and a 2.8 - 3.2 wt% sodium carbonate solution is added dropwise to control the pH value at 6.7 - 7.0. The fermentation culture solution is obtained. The fermentation culture solution is centrifuged, separated, concentrated and crystallized to obtain 2 - keto - L - gulonic acid;

[0021] The fermentation medium consists of L - sorbose 24.0 - 26.0 g / L, fermentation aid 11.5 - 12.5 g / L, urea 5.8 - 6.2 g / L, potassium dihydrogen phosphate 1.0 - 1.4 g / L, magnesium sulfate 0.8 - 1.2 g / L, calcium carbonate 2.3 - 2.7 g / L, manganese sulfate 0.016 - 0.023 g / L, zinc sulfate 0.4 - 0.6 μg / L, active aid 0.42 - 0.48 g / L, ammonium molybdate 1.8 - 2.2 μg / L;

[0022] The preparation method of the fermentation aid is as follows: add corn steep liquor dry powder to deionized water, stir evenly, then add folic acid and L-cysteine, and stir at 380 - 420 rpm for 33 - 36 min to obtain the fermentation aid;

[0023] In the fermentation aid, the mass ratio of deionized water, corn steep liquor dry powder, folic acid and L-cysteine is 48 - 52:9.6 - 10.3:0.8 - 1.2:1.0 - 1.5;

[0024] The active aid is a mixture of rhamnolipid and span 80, and the mass ratio of rhamnolipid to span 80 is 1.2 - 1.5:1.0.

[0025] 2. Preparation of the catalyst

[0026] Perform steam treatment on ZMS-5 molecular sieve with an airspeed of 1.8 - 2.2 h -1 , control the heating rate at 8 - 12 °C / min, raise the temperature to 310 - 330 °C, and treat for 38 - 42 min. Wait for it to naturally return to room temperature, then place it in a calcination furnace and heat it to 500 - 520 °C at a rate of 2.8 - 3.2 °C / min for calcination for 3.5 - 5.0 h. After the calcination is completed, let it naturally return to room temperature, put it into deionized water, add γ-polyglutamic acid and citric acid, raise the temperature to 40 - 45 °C, and stir for 0.8 - 1.2 h. After the stirring is completed, filter and dry to obtain the pretreated molecular sieve; put the pretreated molecular sieve into a phosphoric acid solution for soaking, the soaking time is 1.8 - 2.2 h, the soaking temperature is 50 - 55 °C. After the soaking is completed, dry it at 78 - 82 °C for 9.5 - 10.5 h to obtain the catalyst;

[0027] The particle size of the ZSM-5 molecular sieve is 120 - 140 nm, and the silica-alumina ratio is 48 - 52;

[0028] 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;

[0029] The mass ratio of the pretreated molecular sieve to the phosphoric acid solution is 1:4 - 6;

[0030] The mass concentration of the phosphoric acid solution is 20 - 24%.

[0031] 3. Reaction

[0032] Mix 2-keto-L-gulonic acid, n-butanol and a catalyst, stir evenly, react at 40 - 45 °C for 2.6 - 3.0 h, then heat up to 52 - 57 °C at a rate of 0.4 - 0.6 °C / min, stir and react for 4.3 - 4.8 h to obtain a vitamin C solution, and crystallize, centrifuge, wash and dry the vitamin C solution to obtain a vitamin C product;

[0033] The mass ratio of the 2-keto-L-gulonic acid, n-butanol and the catalyst is 9.8 - 10.2:21.0 - 21.8:2.5 - 3.0.

[0034] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0035] 1. The present invention uses 2-keto-L-gulonic acid as a reaction raw material to prepare vitamin C. First, Gluconobacter oxydans and Bacillus megaterium are fermented to prepare 2-keto-L-gulonic acid. Specifically, in the seed culture medium, a specific aspartic acid component is added. Aspartic acid can provide energy during seed culture, promote the growth and reproduction of microorganisms, improve the utilization rate of nutrients by microorganisms. In the fermentation culture step, specific fermentation aids and activity aids are added. Folic acid in the fermentation aids 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. Combined with the activity aids, it improves the cell permeability, improves the utilization rate of nutrients by microorganisms, increases the product yield, improves the conversion rate of the strain, and reduces the generation of by-products, thereby promoting the synthesis of vitamin C; In the catalyst preparation process, ZSM-5 acidic molecular sieve is used. First, it is treated with water vapor to remove impurity components in the molecular sieve, combined with calcination treatment to improve the stability of the molecular sieve. Then, it is 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 soaking of phosphoric acid solution, the phosphoric acid solution is fully adsorbed into the pores of the molecular sieve to prepare the catalyst. The catalyst has many active sites, good binding property with 2-keto-L-gulonic acid, and good stability, effectively promoting the conversion of 2-keto-L-gulonic acid in the reaction step, and improving the yield and purity of vitamin C;

[0036] 2. Using the method of the present invention to prepare vitamin C, the yield is 94.0 - 94.6%, and the purity is 98.96 - 99.45%;

[0037] 3. Using the method of the present invention to prepare vitamin C, the content of 2-keto-L-gulonic acid in the prepared fermentation culture solution is 64.8 - 66.5 g / L. Specific Embodiments

[0038] In order to more clearly understand the technical features, objectives and effects of the present invention, the specific embodiments of the present invention are now described.

[0039] Example 1

[0040] 1. Preparation of 2-keto-L-gulonic acid

[0041] (1)Activation culture

[0042] Gluconobacter oxydans and Bacillus megaterium are placed in an activation medium for activation culture. The culture temperature is 29 °C and the culture time is 24.0 h. After the culture is completed, an activated bacterial solution is obtained;

[0043] Both Gluconobacter oxydans and Bacillus megaterium are purchased from the market, and the mass ratio of Gluconobacter oxydans to Bacillus megaterium is 2.5:1;

[0044] The components of the activation medium are 5.5 g / L of L-sorbose, 2.8 g / L of tryptone, 5.0 g / L of yeast extract, 0.12 g / L of urea, 0.8 g / L of potassium dihydrogen phosphate, 0.15 g / L of magnesium sulfate, and the pH value is 6.8;

[0045] (2)Seed culture

[0046] The activated bacterial solution is inoculated into a seed medium at an inoculation amount of 5% by mass for seed culture. The culture temperature is 29 °C and the culture time is 20.0 h. After the culture is completed, a seed culture solution is obtained;

[0047] The components of the seed medium are 20 g / L of L-sorbose, 3.0 g / L of beef extract, 2.8 g / L of yeast extract, 10.0 g / L of soy peptone, 1.0 g / L of urea, 1.8 g / L of magnesium sulfate, 1.0 g / L of potassium dihydrogen phosphate, 0.70 g / L of aspartic acid, and 0.6 g / L of ferrous sulfate heptahydrate;

[0048] (3)Fermentation culture

[0049] The seed culture solution is inoculated into a fermentation medium at an inoculation amount of 10% by mass for fermentation culture. The culture temperature is 30 °C and the culture time is 38.0 h. During the fermentation process, a 10 wt% L-sorbose solution is added dropwise to control the sorbitol concentration at 18 g / L, and a 3.0 wt% sodium carbonate solution is added dropwise to control the pH value at 6.8. A fermentation culture solution is obtained. The fermentation culture solution is centrifuged, separated, concentrated and crystallized to obtain 2-keto-L-gulonic acid;

[0050] The components of the fermentation medium are as follows: 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;

[0051] 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, then add 1.0 g of folic acid and 1.2 g of L-cysteine, and stir at 400 rpm for 35 min to obtain the fermentation aid;

[0052] The active aid is a mixture of rhamnolipid and span 80, and the mass ratio of rhamnolipid to span 80 is 1.3:1.0.

[0053] 2. Preparation of catalyst

[0054] Perform steam treatment on 10.0 g of ZMS-5 molecular sieve, with an airspeed of 2.0 h -1 , control the heating rate at 10 °C / min, raise the temperature to 320 °C, treat for 40 min, wait for it to naturally return to room temperature, place it in a calcination furnace, raise the temperature to 510 °C at a rate of 3.0 °C / min and calcine for 4.0 h. After the calcination is completed, let it naturally return to room temperature, put it into 100 g of deionized water, add 0.4 g of γ-polyglutamic acid and 1.0 g of citric acid, raise the temperature to 43 °C, stir for 1.0 h. After the stirring is completed, filter and dry to obtain the pretreated molecular sieve; put the pretreated molecular sieve into 5 times its mass of 22 wt% phosphoric acid solution for soaking, with a soaking time of 2.0 h and a soaking temperature of 52 °C. After the soaking is completed, dry at 80 °C for 10.0 h to obtain the catalyst;

[0055] The particle size of the ZSM-5 molecular sieve is 130 nm, and the silica-alumina ratio is 50.

[0056] 3. Reaction

[0057] Mix 10.0 g of 2-keto-L-gulonic acid, 21.3 g of n-butanol and 2.7 g of catalyst, stir evenly, react at 42 °C for 2.8 h, then raise the temperature to 55 °C at a rate of 0.5 °C / min and stir and react for 4.5 h to obtain the vitamin C solution. Crystallize, centrifuge, wash and dry the vitamin C solution to obtain the vitamin C product.

[0058] In the fermentation culture solution of Example 1, the content of 2-keto-L-gulonic acid is 66.5 g / L, the yield of vitamin C is 94.6%, and the purity is 99.45%.

[0059] Example 2

[0060] 1. Preparation of 2-keto-L-gulonic acid

[0061] (1)Activation culture

[0062] Gluconobacter oxydans and Bacillus megaterium are placed in an activation medium for activation culture at a culture temperature of 28 °C for 23.5 h. After the culture, an activated bacterial liquid is obtained;

[0063] Both Gluconobacter oxydans and Bacillus megaterium are purchased from the market, and the mass ratio of Gluconobacter oxydans to Bacillus megaterium is 2.2:1;

[0064] The components of the activation medium are 5.3 g / L of L-sorbose, 2.6 g / L of tryptone, 4.8 g / L of yeast extract, 0.10 g / L of urea, 0.7 g / L of potassium dihydrogen phosphate, 0.13 g / L of magnesium sulfate, and the pH value is 6.7;

[0065] (2)Seed culture

[0066] The activated bacterial liquid is inoculated into a seed medium at an inoculation amount of 4.5% by mass for seed culture at a culture temperature of 28 °C for 19.0 h. After the culture, a seed culture solution is obtained;

[0067] The components of the seed medium are 18 g / L of L-sorbose, 2.7 g / L of beef extract, 2.6 g / L of yeast extract, 9.8 g / L of soy peptone, 0.8 g / L of urea, 1.6 g / L of magnesium sulfate, 0.8 g / L of potassium dihydrogen phosphate, 0.67 g / L of aspartic acid, 0.58 g / L of ferrous sulfate heptahydrate;

[0068] (3)Fermentation culture

[0069] The seed culture solution is inoculated into a fermentation medium at an inoculation amount of 8% by mass for fermentation culture at a culture temperature of 29 °C for 37.5 h. During the fermentation, a 9.5 wt% L-sorbose solution is added dropwise to control the sorbitol concentration at 17 g / L, and a 2.8 wt% sodium carbonate solution is added dropwise to control the pH value at 6.7 to obtain a fermentation culture solution. The fermentation culture solution is centrifuged, separated, concentrated and crystallized to obtain 2-keto-L-gulonic acid;

[0070] The components of the fermentation medium are 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, 0.42 g / L of active aid, 1.8 μg / L of ammonium molybdate;

[0071] The preparation method of the fermentation aid is as follows: Add 9.6 g of corn steep liquor powder to 48 g of deionized water, stir evenly, then add 0.8 g of folic acid and 1.0 g of L-cysteine, and stir at 380 rpm for 33 min to obtain the fermentation aid;

[0072] The active aid is a mixture of rhamnolipid and span 80, and the mass ratio of rhamnolipid to span 80 is 1.2:1.0.

[0073] 2. Preparation of the catalyst

[0074] Perform steam treatment on 9.5 g of ZSM-5 molecular sieve with an airspeed of 1.8 h -1 , control the heating rate at 8 °C / min, raise the temperature to 310 °C, treat for 38 min, wait for it to naturally return to room temperature, place it in a calcination furnace, raise the temperature to 500 °C at a rate of 2.8 °C / min and calcine for 5.0 h. After the calcination is completed, let it naturally return to room temperature, put it into 98 g of deionized water, add 0.3 g of γ-polyglutamic acid and 0.8 g of citric acid, raise the temperature to 40 °C, and stir for 0.8 h. After the stirring is completed, filter and dry to obtain the pretreated molecular sieve; Put the pretreated molecular sieve into a 20 wt% phosphoric acid solution with 4 times the mass for soaking, the soaking time is 1.8 h, the soaking temperature is 50 °C, and after the soaking is completed, dry at 78 °C for 10.5 h to obtain the catalyst;

[0075] The particle size of the ZSM-5 molecular sieve is 120 nm, and the silica-alumina ratio is 48.

[0076] 3. Reaction

[0077] Mix 9.8 g of 2-keto-L-gulonic acid, 21.0 g of n-butanol and 2.5 g of the catalyst, stir evenly, react at 40 °C for 3.0 h, then raise the temperature to 52 °C at a rate of 0.4 °C / min and stir and react for 4.8 h to obtain the vitamin C solution. Crystallize, centrifuge, wash and dry the vitamin C solution to obtain the vitamin C product.

[0078] In the fermentation culture medium of Example 2, the content of 2-keto-L-gulonic acid is 64.8 g / L, the yield of vitamin C is 94.0%, and the purity is 98.96%.

[0079] Example 3

[0080] 1. Preparation of 2-keto-L-gulonic acid

[0081] (1)Activation culture

[0082] Place Gluconobacter oxydans and Bacillus megaterium in the activation medium for activation culture at a culture temperature of 30 °C and a culture time of 24.5 h. After the culture is completed, obtain the activated bacterial liquid;

[0083] Both Gluconobacter oxydans and Bacillus megaterium were purchased from the market, and the mass ratio of Gluconobacter oxydans to Bacillus megaterium is 2.8:1;

[0084] The activated medium consists of L-sorbose 5.8 g / L, tryptone 3.0 g / L, yeast extract 5.2 g / L, urea 0.15 g / L, potassium dihydrogen phosphate 1.0 g / L, magnesium sulfate 0.17 g / L, and the pH value is 6.9;

[0085] (2) Seed culture

[0086] The activated bacterial liquid was inoculated into the seed medium at an inoculation amount of 5.5% by mass for seed culture. The culture temperature was 30 °C and the culture time was 20.5 h. After the culture was completed, a seed culture solution was obtained;

[0087] The seed medium consists of 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;

[0088] (3) Fermentation culture

[0089] The seed culture solution was inoculated into the fermentation medium at an inoculation amount of 12% by mass for fermentation culture. The culture temperature was 31 °C and the culture time was 38.5 h. During the fermentation process, a 10.5 wt% L-sorbose solution was added dropwise to control the sorbitol concentration at 20 g / L, and a 3.2 wt% sodium carbonate solution was added dropwise to control the pH value at 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;

[0090] The fermentation medium consists of L-sorbose 26.0 g / L, fermentation aid 12.5 g / L, urea 6.2 g / L, potassium dihydrogen phosphate 1.4 g / L, magnesium sulfate 1.2 g / L, calcium carbonate 2.7 g / L, manganese sulfate 0.023 g / L, zinc sulfate 0.6 μg / L, active aid 0.48 g / L, ammonium molybdate 2.2 μg / L;

[0091] 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, then add 1.2 g of folic acid and 1.5 g of L-cysteine, and stir at 420 rpm for 36 min to obtain the fermentation aid;

[0092] The active aid is a mixture of rhamnolipid and span 80, and the mass ratio of rhamnolipid to span 80 is 1.5:1.0.

[0093] 2. Preparation of catalyst

[0094] Perform steam treatment on 10.6 g of ZMS-5 molecular sieve with a space velocity of 2.2 h -1 , control the heating rate at 12 °C / min, raise the temperature to 330 °C, treat for 42 min, wait for it to naturally return to room temperature, place it in a calcination furnace, raise the temperature to 520 °C at a rate of 3.2 °C / min and calcine for 3.5 h. After the calcination is completed, let it naturally return to room temperature, put it into 105 g of deionized water, add 0.5 g of γ-polyglutamic acid and 1.2 g of citric acid, raise the temperature to 45 °C, stir for 1.2 h. After the stirring is completed, filter and dry to obtain the pretreated molecular sieve; put the pretreated molecular sieve into 6 times the mass of 24 wt% phosphoric acid solution for soaking, the soaking time is 2.2 h, the soaking temperature is 55 °C, after the soaking is completed, dry at 82 °C for 9.5 h to obtain the catalyst;

[0095] The particle size of the ZSM-5 molecular sieve is 140 nm and the silica-alumina ratio is 52.

[0096] 3. Reaction

[0097] Mix 10.2 g of 2-keto-L-gulonic acid, 21.8 g of n-butanol and 3.0 g of catalyst, stir evenly, react at 45 °C for 2.6 h, then raise the temperature to 57 °C at a rate of 0.6 °C / min and stir and react for 4.3 h to obtain a vitamin C solution. Crystallize, centrifuge, wash and dry the vitamin C solution to obtain vitamin C product.

[0098] In the fermentation culture broth of Example 3, the content of 2-keto-L-gulonic acid is 65.3 g / L, the yield of vitamin C is 94.4%, and the purity is 99.27%.

[0099] Comparative Example 1

[0100] Based on Example 1, the changes are as follows. In the step of preparing 2-keto-L-gulonic acid,

[0101] (1) In the seed culture step, omit the aspartic acid component in the seed medium;

[0102] (2) In the fermentation culture step, omit the fermentation aid and active aid components in the fermentation medium;

[0103] The remaining operations are the same.

[0104] In the fermentation culture broth of Comparative Example 1, the content of 2-keto-L-gulonic acid is 51.5 g / L, the yield of vitamin C is 91.2%, and the purity is 96.68%.

[0105] Comparative Example 2

[0106] Based on Example 1, the changes are as follows:

[0107] In the reaction step, the catalyst was replaced with phosphoric acid in equal amounts;

[0108] All other operations were the same.

[0109] For the product obtained in Comparative Example 2, the yield of vitamin C was 86.32% and the purity was 92.35%.

[0110] The present invention uses 2-keto-L-gulonic acid as a reaction raw material to prepare vitamin C. First, Gluconobacter oxydans and Bacillus megaterium are fermented to prepare 2-keto-L-gulonic acid. Specifically, in the seed medium, a specific aspartic acid component is added. 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 activity aids are added. Folic acid in the fermentation aids can promote cell growth and improve the metabolic activity of microorganisms. L-cysteine effectively increases the growth rate of microorganisms and the production efficiency of 2-keto-L-gulonic acid. In combination with the activity aids, it improves the cell permeability, increases the utilization rate of nutrients by microorganisms, increases the product yield, improves the conversion rate of the strain, and reduces the generation of by-products, thereby promoting the synthesis of vitamin C; in the catalyst preparation process, ZSM-5 acidic molecular sieve is used. First, it is treated with water vapor to remove impurity components in the molecular sieve, and combined with calcination treatment to improve the stability of the molecular sieve. Then, it is 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 soaking of phosphoric acid solution, the phosphoric acid solution is fully adsorbed into the pores of the molecular sieve to obtain the catalyst. The catalyst has many active sites, good binding property with 2-keto-L-gulonic acid, and good stability, effectively promoting the conversion of 2-keto-L-gulonic acid in the reaction step and increasing the yield and purity of vitamin C.

[0111] In Comparative Example 1, the aspartic acid component was omitted in the seed medium, and the fermentation aids and activity aids were omitted in the fermentation medium, resulting in incomplete conversion of the strain, low content of 2-keto-L-gulonic acid, and more by-products, which affected the production of vitamin C and reduced the yield and purity of the product; in Comparative Example 2, phosphoric acid was directly used as the catalyst, the reaction process was unstable, the reaction with 2-keto-L-gulonic acid was incomplete, resulting in poor product quality and low yield.

[0112] Unless otherwise specified, the percentages used in the present invention are all mass percentages.

[0113] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within 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; The activation culture step comprises placing Gluconobacter oxidans and Bacillus megaterium in an activation culture medium for activation culture to obtain an activated bacterial solution; 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 ZSM-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 mass ratio of the ZSM-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%; 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.

2. The method for preparing vitamin C according to claim 1, characterized in that: In the activation culture step, the culture temperature is 28-30°C and the culture time is 23.5-24.5h; 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 and culturing step comprises: inoculating the seed culture solution in a fermentation medium at an inoculation amount of 8-12% by mass for fermentation and culturing, the culturing temperature is 29-31° C., the culturing 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 ZSM-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.