Preparation method of L-ascorbic acid
By replacing sodium carbonate with sodium hydroxide or aqueous potassium hydroxide solution and adding it to the fermentation broth by adding it to the cumbersome and low efficiency of the existing L-ascorbic acid fermentation production, it has solved the problem of cumbersome and low efficiency of alkali addition methods in the existing L-ascorbic acid fermentation production, and achieved a more efficient and lower cost production process.
Patent Information
- Application Number
- CN202411954430.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-27
- Publication Date
- 2025-03-25
AI Technical Summary
In the existing two-step fermentation production of L-ascorbic acid, the commonly used alkali addition method has problems such as cumbersome preparation, disinfection needs, poor environment, large labor consumption, low solubility, easy crystallization, large foam caused by carbon dioxide, and high risk of spills.
Sodium hydroxide or potassium hydroxide aqueous solution is used as an alkaline solution to replace sodium carbonate, and added to the fermentation broth by flow addition, controlling the pH between 6.5 and 7.5, reducing carbon dioxide production, reducing foam volume, and simplifying the operation process.
Improve production efficiency, reduce production costs and fermentation broth impurities, shorten the fermentation cycle, reduce operating steps, and improve the operating environment.
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Figure BDA0005215543160000091 
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biological fermentation, and in particular to a method for preparing L-ascorbic acid. Background Art
[0002] The fermentation production of L-ascorbic acid is mainly achieved through biological fermentation, among which the most commonly used method is the two-step biological fermentation method. This method was jointly developed by the Institute of Microbiology, Chinese Academy of Sciences and Beijing Pharmaceutical Factory. By screening high-efficiency strains, it realizes the biotransformation from L-sorbose solution to 2-keto-L-gulonic acid, replacing the original chemical transformation process. It not only saves toxic and harmful reagents, but also greatly reduces production costs. L-ascorbic acid has multiple physiological functions in the human body, including participating in the synthesis of collagen, enhancing immunity, and promoting iron absorption. It is an important water-soluble vitamin that is widely present in fruits and vegetables and is essential to human health.
[0003] In the current two-step fermentation production of L-ascorbic acid, the commonly used method of adding alkali is to add sodium carbonate solution, but this method of adding alkali has many problems, such as the complicated preparation of sodium carbonate solution, the need for separate disinfection, the poor working environment, the need for additional manpower, the low solubility of sodium carbonate, and the easy crystallization and precipitation at low temperatures in winter, causing pipe blockage. At the same time, carbon dioxide is generated when sodium carbonate solution is added, resulting in large foam in the fermentation liquid and the risk of overflow. The generated carbon dioxide is not conducive to the aerobic fermentation of the fermentation bacteria. Summary of the invention
[0004] The object of the present invention is to overcome the shortcomings of the prior art and provide a method for preparing L-ascorbic acid. The preparation method of the present invention uses sodium hydroxide or potassium hydroxide aqueous solution as an alkaline solution to replace sodium carbonate. The alkaline solution has high solubility, is not easy to crystallize, can be used without separate disinfection, is added to the fermentation liquid without additional carbon dioxide generation, is conducive to aerobic fermentation, has less foam in the fermentation liquid, does not need to add an additional defoamer, can further reduce production costs, reduce impurities in the fermentation liquid, shorten the fermentation cycle, and reduce the number of operating steps.
[0005] The present invention provides a method for preparing L-ascorbic acid, comprising the following steps:
[0006] (1) inoculating the seed liquid into a fermentation medium containing L-sorbose liquid for fermentation culture to obtain a fermentation liquid, wherein the initial sugar content of the fermentation liquid is 10 to 20 g / L;
[0007] (2) adding L-sorbose solution and alkaline solution to the fermentation broth, controlling the residual sugar in the fermentation broth to be 25-35 g / L by adding L-sorbose solution, and controlling the pH of the fermentation broth to be 6.5-7.5 by adding alkaline solution;
[0008] (3) until the residual sugar concentration in the fermentation broth is less than 0.4 mg / mL, i.e., the fermentation endpoint is reached;
[0009] Wherein, the alkaline solution is selected from sodium hydroxide aqueous solution and / or potassium hydroxide aqueous solution, and the mass content of alkaline substances in the alkaline solution is 20-50%.
[0010] L-ascorbic acid two-step fermentation belongs to neutral aerobic fermentation, therefore, pH needs to be stably controlled between 6.5 and 7.5. If a large amount of alkaline solution is added at one time, the pH value of the fermentation liquid will cause huge fluctuations, which will cause fatal damage to the production strains and is not conducive to the fermentation production. In the present invention, if the concentration of the alkaline solution is too low, the volume of the fermentation liquid will increase, the concentration will become thinner, and the production strain will have a hypotonic effect, which is not conducive to its acid production, and is also not conducive to the flow addition of L-sorbose liquid, affecting the total acid production of a single tank; if the concentration of the alkaline solution is too high, the risk of crystallization of the alkaline solution increases, and at the same time, the alkaline solution with too high a concentration also has a certain harmful effect on the production strains, which is not conducive to the fermentation production.
[0011] If a large amount of L-sorbose solution is added at one time, the production strain will produce hypertonic dehydration and overnutrition, which will affect the reproduction of the strain, slow down the acid production of the strain, reduce the efficiency, prolong the fermentation cycle, and reduce the acid production. Therefore, adding L-sorbose solution at one time is not conducive to fermentation production. The present invention avoids the inhibition of acid production of the strain by adding L-sorbose solution at one time by first adding a part of L-sorbose solution to the culture medium and then gradually adding the remaining L-sorbose solution according to the fermentation situation, and also avoids the low sugar concentration in the early fermentation liquid, which is not conducive to the reproduction of the strain. Specifically, in the preparation method of the present invention, the initial sugar content in the fermentation broth is 10-20 g / L (i.e., the sugar concentration in the culture medium containing L-sorbose solution is 10-20 g / L), and L-sorbose solution is gradually added in the middle stage of fermentation to control the residual sugar content to 25-35 g / L to ensure the nutrient supply during the acid production and reproduction period of the bacteria. In the late stage of fermentation, according to the target acid production value and acid production situation (i.e., the acid concentration is greater than 140 mg / mL), the addition of sugar is stopped, and only alkali solution is added to maintain the pH value of the fermentation broth stable at 6.5 to 7.5.
[0012] In some embodiments, the mass content of the alkaline substance in the alkaline solution is 20%, 25%, 30%, 35%, 40%, 45%, 50% or any value therebetween.
[0013] In some embodiments, the alkaline solution is an aqueous solution of sodium hydroxide.
[0014] In some embodiments, the mass content of the alkaline substance in the alkaline solution is 30-40%, for example, 30%, 32%, 34%, 36%, 38%, 40% or any value therebetween.
[0015] In some embodiments, the fermentation medium includes pure water, a carbon source, a nitrogen source, and trace elements.
[0016] In some embodiments, the fermentation medium comprises: yeast extract, corn steep liquor, potassium dihydrogen phosphate, magnesium sulfate, urea and calcium carbonate.
[0017] In some embodiments, the fermentation medium comprises, by mass percentage, 0.05-0.3% yeast extract, 0.1-0.5% corn steep liquor, 0.05-0.2% potassium dihydrogen phosphate, 0.01-0.05% magnesium sulfate, 0.05-0.2% urea, and 0.1-0.5% calcium carbonate.
[0018] In some embodiments, the fermentation medium comprises, by mass percentage, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3% or any value therebetween of yeast extract.
[0019] In some embodiments, the fermentation medium comprises, by mass percentage, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5% or any value therebetween of corn steep liquor.
[0020] In some embodiments, the fermentation medium comprises, by mass percentage, 0.05%, 0.1%, 0.15%, 0.2% or any value therebetween of potassium dihydrogen phosphate.
[0021] In some embodiments, the fermentation medium comprises, by mass percentage, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05% or any value therebetween of magnesium sulfate.
[0022] In some embodiments, the fermentation medium comprises, by mass percentage, 0.05%, 0.1%, 0.15%, 0.2% urea or any value therebetween.
[0023] In some embodiments, the fermentation medium comprises, by mass percentage, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5% calcium carbonate or any value therebetween.
[0024] In some embodiments, the fermentation medium includes, by mass percentage, 0.15% yeast extract, 0.2% corn steep liquor, 0.1% potassium dihydrogen phosphate, 0.02% magnesium sulfate, 0.1% urea, and 0.2% calcium carbonate.
[0025] In some embodiments, the fermentation culture temperature is 20-40°C, for example, 20°C, 25°C, 30°C, 35°C, 40°C or any value therebetween.
[0026] In some embodiments, the ventilation ratio of the fermentation culture is 0.3 to 0.5 m 3 / m 3 min, for example 0.3m 3 / m 3 ·min、0.35m 3 / m 3 min, 0.4m 3 / m 3 min, 0.45m 3 / m 3 min, 0.5m 3 / m 3 min or any value in between.
[0027] In some embodiments, the fermentation culture period is 25-30 h, for example, 30 h, 29 h, 28 h, 27 h, 26 h, 25 h or any value therebetween.
[0028] In some embodiments, the fermentation culture is carried out in a fermentor.
[0029] In some embodiments, the fermentation medium is pumped into the fermenter through a continuous elimination system.
[0030] In some embodiments, during the fermentation process, the pH of the fermentation broth is controlled at 6.5-7.5 (when the pH is lower than 6.5, the alkaline solution is added, and when the pH is higher than 7.5, the addition of the alkaline solution is stopped), and the residual sugar is 25-35 g / L (when the residual sugar concentration is lower than 25 g / L, L-sorbose solution is added, and when the residual sugar concentration is higher than 35 g / L, the addition of L-sorbose is stopped), until the total content of acid and residual sugar reaches the desired target content, that is, greater than 140 mg / mL, the addition of L-sorbose solution is stopped, and the addition of alkaline solution is continued, until the residual sugar is detected to be <0.4 mg / mL, that is, the fermentation end point is reached.
[0031] In some embodiments, the acid concentration after the fermentation is 120-150 mg / mL, for example, 120 mg / mL, 125 mg / mL, 130 mg / mL, 135 mg / mL, 140 mg / mL, 145 mg / mL, 150 mg / mL or any value therebetween.
[0032] In some embodiments, the acid concentration after the fermentation is completed is 140-150 mg / mL.
[0033] In some embodiments, the bacterial species used in the fermentation culture include macrobacteria and microbacteria.
[0034] In some embodiments, the macrobacterium includes Bacillus megaterium, and the microbacterium includes Gluconobacter oxydans.
[0035] In some embodiments, the method for preparing the seed solution comprises:
[0036] 1. Activation of bacterial strains: Wash the bacterial strains in the frozen tube into a 250 mL Erlenmeyer flask, culture on a shaking table for 24 to 36 hours at a temperature of 29.5°C to obtain the isolated seed solution;
[0037] 2. Dilute the isolated seed solution obtained in step 1 to 10 -6 , isolated single colonies of Bacillus megaterium (hereinafter referred to as large bacteria) and Gluconobacter oxydans (hereinafter referred to as small bacteria), the large bacteria were cultured for 2 to 3 days, the culture medium composition was: yeast extract 0.1%, L-sorbose 0.5%, magnesium sulfate 0.01%, agar powder 1.5%, pH 6.7 to 7.0; the small bacteria were cultured for 3 to 4 days, the culture temperature was 29.5°C, the culture medium composition was: yeast extract 0.1%, corn steep liquor 0.3%, L-sorbose 2%, magnesium sulfate 0.1%, urea 0.1%, calcium carbonate 0.1%, agar powder 1.5%, pH 6.7 to 7.0;
[0038] 3. Build the slant: The slant culture medium is the same as the large bacteria culture medium. Use the single colony separated in step 2 to select 2 rings of full small bacteria and build them on the slant. Culture for 1 day. Observe that the small bacteria are normal. Then select large bacteria and dip an appropriate amount of them on the small bacteria. Culture for another 2 days at a temperature of 29.5°C.
[0039] 4. Inoculation: The composition of the seed liquid culture medium: 0.1% yeast extract, 0.3% corn syrup, 2% L-sorbose, 0.1% magnesium sulfate, 0.1% urea, 0.2% calcium carbonate, pH 6.7 to 7.0. Use an eggplant bottle obtained in step 3 to connect a bottle of seed liquid, use a blank seed liquid to wash the moss and access to a 220mL / 750mL thorn bottle, shake and culture for 18 to 24h, culture temperature 29.5℃, 220 rpm, this is the first-level seed liquid; the first-level seed liquid is inoculated with 15% inoculation amount and accessed to 220ml / 750ml thorn bottle, each bottle can connect to 6 bottles, shake and culture for 16 to 18h, culture temperature 29.5℃, 220 rpm, this is the second-level seed liquid. If qualified, it can be put into the tank;
[0040] 5. Seed selection: Use the slant strain from step 3 to inoculate a 250ml Erlenmeyer flask containing 20ml of seed solution, incubate on a shaker at 29.5℃ for 20-24h, 220 rpm, and prepare to inoculate the fermentation bottle after microscopic examination and pH value are qualified;
[0041] 3 mL of seed solution was drawn from a 250 mL triangular flask and added to the fermentation bottle. The culture was shaken at 29.5°C for 60 h, 220 rpm. When the acid production reached a certain level, the seed solution bottle with high conversion rate and high acid resistance was selected for the next separation and culture. This achieved the optimization and iteration of the strain.
[0042] Compared with the prior art, the present invention has the following beneficial effects:
[0043] 1. Sodium hydroxide or potassium hydroxide has high solubility, is not easy to crystallize, and is not easy to block the pipe. Using a higher concentration of sodium hydroxide or potassium hydroxide solution can reduce the volume of the fermentation liquid and improve production efficiency;
[0044] 2. There are finished alkaline solutions for sale, which do not need to be prepared. At the same time, since sodium hydroxide has strong alkalinity, it can be used without disinfection, which improves the working environment, saves manpower, and reduces energy consumption;
[0045] 3. Adding sodium hydroxide or potassium hydroxide solution to the fermentation liquid does not produce additional carbon dioxide, which is conducive to aerobic fermentation. Since no additional carbon dioxide is produced, the fermentation liquid has less foam, the risk of overflow is reduced, and no additional defoaming agent is required, which can further reduce production costs and impurities in the fermentation liquid;
[0046] 4. The method of the present invention adopts a flow-addition method to add alkaline solution and L-sorbose solution. This method combines the advantages of batch fermentation and continuous fermentation. It can not only gradually add relevant substances during the fermentation process, but also reduce the impact of the fermentation environment on microorganisms in continuous fermentation. DETAILED DESCRIPTION
[0047] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation to the present invention.
[0048] The endpoints and any values of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, the endpoint values of each range and the individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.
[0049] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the raw materials, instruments and equipment used in the following examples, etc., can all be purchased on the market or can be obtained by existing methods; the reagent amounts, unless otherwise specified, are the reagent amounts used in conventional experimental operations; the experimental methods, unless otherwise specified, are all conventional methods.
[0050] Unless otherwise specified, "%" in the present invention refers to mass percentage.
[0051] Unless otherwise specified, "sterilization" in the present invention refers to steam sterilization at 85°C for 20 minutes.
[0052] In the embodiments of the present invention, some raw materials are from the following sources:
[0053] L-sorbose solution was produced in our factory (content ≥ 250 mg / mL);
[0054] Yeast extract, biochemical reagent grade (nitrogen content ≥ 7%);
[0055] Corn steep liquor (dry matter ≥ 35%, total nitrogen ≥ 6%);
[0056] Potassium dihydrogen phosphate, food grade (content ≥96%);
[0057] Magnesium sulfate, technical grade (content ≥98%);
[0058] Urea, technical grade (nitrogen content > 46%);
[0059] Calcium carbonate, food grade (content > 98.5%);
[0060] Sodium carbonate, food grade (content > 99%);
[0061] Liquid alkali, food grade (sodium hydroxide or potassium hydroxide content 20% to 50%).
[0062] The bacterial species used for fermentation culture in the following examples and comparative examples of the present invention are all mixed bacteria of Bacillus megaterium (large bacteria) and Gluconobacter oxydans (small bacteria).
[0063] The steps for preparing the seed solution in the following examples and comparative examples of the present invention are as follows:
[0064] 1. Activation of bacterial strains: Wash the bacterial strains in the frozen tube into a 250 mL Erlenmeyer flask, culture on a shaking table for 24 to 36 hours at a temperature of 29.5°C to obtain the isolated seed solution;
[0065] 2. Dilute the isolated seed solution obtained in step 1 to 10 -6, isolated single colonies of Bacillus megaterium (hereinafter referred to as large bacteria) and Gluconobacter oxydans (hereinafter referred to as small bacteria), the large bacteria were cultured for 2 to 3 days, the culture medium composition was: yeast extract 0.1%, L-sorbose 0.5%, magnesium sulfate 0.01%, agar powder 1.5%, pH 6.7 to 7.0; the small bacteria were cultured for 3 to 4 days, the culture temperature was 29.5°C, the culture medium composition was: yeast extract 0.1%, corn steep liquor 0.3%, L-sorbose 2%, magnesium sulfate 0.1%, urea 0.1%, calcium carbonate 0.1%, agar powder 1.5%, pH 6.7 to 7.0;
[0066] 3. Build the slant: The slant culture medium is the same as the large bacteria culture medium. Use the single colony separated in step 2 to select 2 rings of full small bacteria and build them on the slant. Culture for 1 day. Observe that the small bacteria are normal. Then select large bacteria and dip an appropriate amount of them on the small bacteria. Culture for another 2 days at a temperature of 29.5°C.
[0067] 4. Inoculation: The composition of the seed liquid culture medium: 0.1% yeast extract, 0.3% corn syrup, 2% L-sorbose, 0.1% magnesium sulfate, 0.1% urea, 0.2% calcium carbonate, pH 6.7 to 7.0. Use an eggplant bottle obtained in step 3 to connect a bottle of seed liquid, use a blank seed liquid to wash the moss and connect it to a 220mL / 750mL thorn bottle, shake and culture for 18 to 24 hours, the culture temperature is 29.5℃, 220 rpm, this is the first-level seed liquid; the first-level seed liquid is connected to a 220mL / 750mL thorn bottle at a 15% inoculation amount, each bottle can connect 6 bottles, shake and culture for 16 to 18 hours, the culture temperature is 29.5℃, 220 rpm, this is the second-level seed liquid. If it is qualified, it can be put into the tank;
[0068] 5. Seed selection: Use the slant strain from step 3 to inoculate a 250mL Erlenmeyer flask containing 20mL of seed solution, incubate on a shaker at 29.5℃ for 20-24h, 220 rpm, and prepare to inoculate the fermentation bottle after microscopic examination and pH are qualified;
[0069] Pipette 3 mL of seed solution from a 250 mL Erlenmeyer flask and add it to the fermentation bottle. Cultivate it on a shaker at 29.5°C, 220 rpm for 60 h. When the acid production reaches a certain level, select the seed solution bottle with high conversion rate and high acid resistance for the next separation and cultivation.
[0070] The D-sorbitol is converted into L-sorbose by aerobic fermentation with Gluconobacter nigromatus, thus obtaining the L-sorbose solution used in the present invention.
[0071] The product obtained after the first fermentation is subjected to a second fermentation.
[0072] The culture medium used in the second fermentation process in the following examples and comparative examples of the present invention is as follows:
[0073] Preparation and sterilization of culture medium: 0.15% yeast extract, 0.2% corn steep liquor, 0.1% potassium dihydrogen phosphate, 0.02% magnesium sulfate, 0.1% urea, 0.2% calcium carbonate, add water to dissolve, adjust pH to 6.5-7.5, sterilize continuously to fermentation tank, add sterilized L-sorbose solution (steam sterilization 85°C, 20min, 5kg (pure)), culture medium preparation is complete, wherein the initial sugar content is 10-20g / L. The following examples are all implemented using the above culture medium.
[0074] Example 1
[0075] The above culture medium was used for inoculation, the fermentation temperature was 32°C, and the ventilation ratio was 0.4m 3 / m 3 ·min, the initial pH of the fermentation liquid was 7.0, and according to the fermentation situation (when the sugar concentration dropped to 25g / L, L-sorbose liquid was added, and when it was higher than 35g / L, the addition of L-sorbose liquid was stopped, and when the pH of the fermentation liquid was lower than 6.5, alkaline solution was added, and when it was higher than 7.5, the addition was stopped), alkaline solution (sodium hydroxide aqueous solution, mass concentration 20%) and L-sorbose liquid were added in a feeding manner to control the residual sugar content in the fermentation liquid to be 25-35g / L, and according to the target acid production value (acid concentration greater than 140mg / mL) and the acid production situation, the addition of L-sorbose was stopped, and only alkaline solution was added to maintain the pH of the fermentation liquid stable at 6.5 to 7.5. The total amount of L-sorbose liquid added during the fermentation process was 35kg, and the residual sugar was detected to be less than 0.4mg / mL after 30h, reaching the fermentation end point. At this time, the acid concentration of the fermentation tank was 142mg / mL, as shown in Table 1.
[0076] Example 2
[0077] The difference from Example 1 is that the alkaline solution added is a potassium hydroxide aqueous solution with a mass concentration of 50%, and the residual sugar detected after 28 hours is less than 0.4 mg / mL, reaching the fermentation end point, and the acid concentration in the fermentation tank is 140 mg / mL. The other steps are the same as Example 1.
[0078] Example 3
[0079] The inoculation amount and the total amount of L-sorbose solution added were the same as those in Example 1, and the difference from Example 1 was that the alkaline solution added was a sodium hydroxide aqueous solution with a mass concentration of 35%, and the residual sugar detected after 25 hours was less than 0.4 mg / mL, reaching the fermentation end point, and the acid concentration in the fermentation tank was 149 mg / mL. The other steps were the same as those in Example 1.
[0080] Example 4
[0081] The difference from Example 1 is that the alkaline solution added is a 50% sodium hydroxide aqueous solution, and after 27 hours, the residual sugar detected is less than 0.4 mg / mL, reaching the fermentation end point, and the acid concentration in the fermentation tank is 135 mg / mL. The other steps are the same as Example 1.
[0082] Example 5
[0083] The difference from Example 1 is that the alkaline solution added is a 10% sodium hydroxide aqueous solution, and after 30 hours, the residual sugar detected is less than 0.4 mg / mL, reaching the fermentation end point, and the acid concentration in the fermentation tank is 115 mg / mL. The other steps are the same as Example 1.
[0084] Example 6
[0085] The difference from Example 1 is that the alkaline solution added is a 20% potassium hydroxide aqueous solution, and after 29 hours, the residual sugar detected is less than 0.4 mg / mL, reaching the fermentation end point, and the acid concentration in the fermentation tank is 121 mg / mL. The other steps are the same as in Example 1.
[0086] Example 7
[0087] The difference from Example 1 is that during the fermentation process, L-sorbose solution was added to control the residual sugar content in the fermentation solution to be 25-35 g / L, the total amount of L-sorbose solution added during the fermentation process was 30 kg, and the residual sugar detected after 25 hours was less than 0.4 mg / mL, reaching the fermentation endpoint, and the acid concentration in the fermentation tank was 132 mg / mL. The other steps were the same as in Example 1.
[0088] Comparative Example 1
[0089] The difference from Example 1 is that the culture medium used in Comparative Example 1 includes: 0.15% yeast extract, 0.2% corn syrup, 0.1% potassium dihydrogen phosphate, 0.02% magnesium sulfate, 0.1% urea, 0.2% calcium carbonate, dissolved in water, adjusted to pH 6.5-7.5, and continuously disinfected to the fermentation tank, and the culture medium is prepared. After inoculation, L-sorbose is added once to control the residual sugar content in the fermentation liquid to 25-35g / L. The total amount of L-sorbose liquid added during the fermentation process is 40kg. The other steps are the same as in Example 1. After 37h, the residual sugar is detected to be less than 0.4mg / mL, reaching the fermentation end point, and the acid concentration of the fermentation tank is 120mg / mL.
[0090] Comparative Example 2
[0091] The difference from Example 1 is that the alkaline solution added is a sodium carbonate aqueous solution with a mass concentration of 30% (prepared and sterilized separately, and sterilized by steam at 115° C. for 15 min), and since the fermentation liquid has large foam, a small amount of defoamer (polyether defoamer) needs to be added, and the residual sugar is less than 0.4 mg / mL after 35 hours, reaching the fermentation end point, and the acid concentration of the tank is 118 mg / mL. The other steps are the same as those in Example 1.
[0092] Comparative Example 3
[0093] The difference from Example 1 is that the alkaline solution added is a sodium carbonate aqueous solution with a mass concentration of 15% (prepared and sterilized separately, and sterilized by steam at 115° C. for 15 min), and since the fermentation liquid has large foam, a small amount of defoamer (polyether defoamer) needs to be added, and the residual sugar detected after 38 hours is less than 0.4 mg / mL, reaching the fermentation end point, and the acid concentration of the fermentation tank is 115 mg / mL. The other steps are the same as those in Example 1.
[0094] Table 1
[0095]
[0096]
[0097] It can be seen from the above examples that, compared with Comparative Examples 1, 2, and 3, the fermentation cycle of Examples 1-7 using the preparation method of the present invention is significantly reduced, and the acid concentration in the fermentation tank is significantly increased. In addition, the alkali solution does not need to be prepared and disinfected separately, and no defoaming agent needs to be added.
[0098] The present invention shortens the fermentation cycle and increases the acid production by controlling the concentration and adding method of the alkaline solution, controlling the sugar content in the fermentation liquid, and controlling the adding method of the L-sorbose solution.
[0099] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.
Claims
1. A method for preparing L-ascorbic acid, comprising the following steps: (1) inoculating the seed liquid into a fermentation medium containing L-sorbose liquid for fermentation culture to obtain a fermentation liquid, wherein the initial sugar content of the fermentation liquid is 10 to 20 g / L; (2) adding L-sorbose solution and alkaline solution to the fermentation broth, controlling the residual sugar in the fermentation broth to be 25-35 g / L by adding L-sorbose solution, and controlling the pH of the fermentation broth to be 6.5-7.5 by adding alkaline solution; (3) until the residual sugar concentration in the fermentation broth is less than 0.4 mg / mL, i.e., the fermentation endpoint is reached; in, The alkaline solution is selected from a sodium hydroxide aqueous solution and / or a potassium hydroxide aqueous solution, and the mass content of the alkaline substance in the alkaline solution is 20-50%.
2. The preparation method according to claim 1, characterized in that: The alkaline solution is a sodium hydroxide aqueous solution; and / or the mass content of the alkaline substance in the alkaline solution is 30-40%.
3. The preparation method according to claim 1 or 2, characterized in that: The fermentation medium comprises pure water, a carbon source, a nitrogen source and trace elements; Preferably, the fermentation medium comprises: yeast extract, corn steep liquor, potassium dihydrogen phosphate, magnesium sulfate, urea and calcium carbonate; Preferably, the fermentation medium comprises, by mass percentage, 0.05-0.3% yeast extract, 0.1-0.5% corn steep liquor, 0.05-0.2% potassium dihydrogen phosphate, 0.01-0.05% magnesium sulfate, 0.05-0.2% urea, and 0.1-0.5% calcium carbonate.
4. The preparation method according to any one of claims 1 to 3, characterized in that Measured in percentage by mass, the fermentation medium comprises: 0.15% yeast extract, 0.2% corn steep liquor, 0.1% potassium dihydrogen phosphate, 0.02% magnesium sulfate, 0.1% urea, and 0.2% calcium carbonate.
5. The preparation method according to any one of claims 1 to 4, characterized in that: The temperature of the fermentation culture is 20-40°C.
6. The preparation method according to any one of claims 1 to 5, characterized in that: The ventilation ratio of the fermentation culture is 0.3-0.5m 3 / m 3 ·min.
7. The preparation method according to any one of claims 1 to 6, characterized in that The fermentation culture cycle is 25-30 hours.
8. The preparation method according to any one of claims 1 to 7, characterized in that: The fermentation culture is carried out in a fermenter; Preferably, the fermentation medium is pumped into the fermenter through a continuous elimination system.
9. The preparation method according to any one of claims 1 to 8, characterized in that: The acid concentration after the fermentation is 120-150 mg / mL, preferably 140-150 mg / mL.
10. The preparation method according to any one of claims 1 to 9, characterized in that: The bacterial species used in the fermentation culture include large bacteria and small bacteria. Preferably, the large bacteria include Bacillus megaterium, and the small bacteria include Gluconobacter oxydans.