A method for preparing 2-keto-D-gluconic acid by fermentation

Abstract

The present invention discloses a method for preparing 2-keto-D-gluconic acid by fermentation, and belongs to the field of fermentation engineering. The method comprises the following steps: mixing Pseudomonas fluorescens seed liquid, Acetobacter aceti seed liquid and Arthrobacter sphericalus seed liquid to obtain a mixed seed liquid; inoculating the mixed seed liquid into a fermentation medium, and fermenting and culturing to obtain a fermentation liquid containing 2-keto-D-gluconic acid. The present invention uses Pseudomonas fluorescens, Acetobacter aceti and Arthrobacter sphericalus to ferment and prepare 2-keto-D-gluconic acid, which has a high conversion rate, and the concentration of the obtained product is higher than that of a single bacterial fermentation. After the present invention mixes the three bacteria, the mixed bacteria fermentation prepares 2-keto-D-gluconic acid, reduces the inhibitory effect of the substrate glucose on the growth and fermentation of a single microorganism, and makes up for the deficiency of a single Pseudomonas fluorescens fermentation.

Description

Technical Field

[0001] The invention belongs to the field of fermentation engineering, and specifically relates to a method for preparing 2-keto-D-gluconic acid by fermentation. Background Art

[0002] D-Sodium erythrosorbate, also known as sodium erythrosinate, is a new type of biological food antioxidant, preservative and color-preserving agent. It can prevent the formation of carcinogens in pickled foods, namely nitrosamines, and eliminate undesirable phenomena such as discoloration, odor and turbidity of food and beverages. It is widely used in the preservation and color-preservation of meat, fish, vegetables, fruits, alcohol, beverages and canned foods. The antioxidant capacity of sodium erythrosorbate far exceeds that of sodium vitamin C. It has no effect of strengthening vitamins, but it will not hinder the body's absorption and utilization of sodium ascorbate. At the same time, sodium erythrosorbate ingested by the human body can be converted into vitamin C in the human body.

[0003] 2-Keto-D-gluconic acid (2-KGA) is an important organic acid with a wide range of uses. It can be used as a food additive, cement plasticizer, detergent and photo developer. Its main use is as a precursor of the food additive D-isoascorbic acid and its sodium salt.

[0004] There is no report on the distribution of D-isoascorbic acid and its sodium salt in nature, and the D-isoascorbic acid and its sodium salt on the market are all artificially synthesized. In the prior art, the indirect fermentation method is still the most commonly used method in industrial production. The process of producing sodium D-isoascorbate by indirect fermentation is: using microorganisms to ferment glucose into 2-keto-D-gluconic acid, and then using 2-keto-D-gluconate calcium as the starting material, and synthesizing sodium D-isoascorbate products through esterification, acidification and other processes. Therefore, the quality of 2-KGA prepared by fermentation directly determines the quality of the final product sodium D-isoascorbate.

[0005] In the prior art, the common preparation methods of 2-KGA include enzymatic method, chemical synthesis method and microbial fermentation method. Among them, the raw materials required for enzymatic method and chemical synthesis method are extremely expensive, so the preferred method at home and abroad is the microbial fermentation method with simple operation and low cost. At present, the main bacteria that can ferment and prepare 2-KGA include Pseudomonas, Erwinia, Serratia, Gluconobacter, Arthrobacter, Alcaligenes and Acetobacter, etc. The industrial production in my country is mainly based on Pseudomonas fluorescens. The document "Optimization of the Fermentation Process of 2-Keto-D-Gluconic Acid Produced by Gluconobacter oxydans" mentions that the use of Pseudomonas fluorescens alone still has problems such as the inhibitory effect of high concentrations of glucose on microbial fermentation. Therefore, the document explores the method of fermenting 2-keto-D-gluconic acid by Gluconobacter oxydans; In addition, the following prior art studies the types of strains: Invention patent CN105695520A discloses a method for increasing the production of 2-keto-D-gluconic acid, which uses Serratia ATCC 39006 to ferment and prepare 2-keto-D-gluconic acid; Invention patent CN105603018B uses Serratia marcescens to ferment and produce 2-keto-D-gluconic acid, thereby preparing sodium D-isoascorbate. There is no report on the use of mixed strains to ferment and produce 2-keto-D-gluconic acid, thereby preparing sodium D-isoascorbate. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a method for preparing 2-keto-D-gluconic acid by fermentation. The present invention uses glucose as a substrate, and uses Pseudomonas fluorescens, Acetobacter aceti and Arthrobacter sphaericus to co-ferment and prepare 2-keto-D-gluconic acid. Compared with fermentation by a single strain, the obtained 2-keto-D-gluconic acid has a higher concentration, and reduces the inhibitory effect of high-concentration glucose substrate on strain growth and fermentation, and gives full play to the synergistic effect between the three strains.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] A method for preparing 2-keto-D-gluconic acid by fermentation comprises the following steps:

[0009] (1) mixing a Pseudomonas fluorescens seed solution, an Acetobacter aceti seed solution, and an Arthrobacter sphaeroides seed solution to obtain a mixed seed solution;

[0010] (2) The mixed seed liquid is inoculated into a fermentation medium, and fermentation is performed to obtain a fermentation liquid containing 2-keto-D-gluconic acid.

[0011] Preferably, the strain number of Pseudomonas fluorescens is CCTCC AB 93075; the strain number of Acetobacter aceti is CCTCC CB 20082244; and the strain number of Arthrobacter sphaeroides is CCTCC AB 206780.

[0012] Preferably, the weight ratio of Pseudomonas fluorescens seed solution, Acetobacter aceti seed solution and Arthrobacter sphaeroides seed solution in the mixed seed solution is 5:(0.5-1.2):(2-4).

[0013] Preferably, the number of viable bacteria in the mixed seed solution is ≥3.0×10 8 cfu / mL.

[0014] Preferably, the fermentation medium comprises the following components:

[0015] Glucose 100-140g / L, corn syrup powder 20-25g / L, calcium carbonate 0.1-0.2g / L, magnesium sulfate 0.2-0.3g / L, ammonium sulfate 1-1.5g / L, dipotassium hydrogen phosphate 0.2-0.3g / L, pH 6.8-7.0.

[0016] Preferably, the inoculation amount of the mixed seed solution is 10-15% of the volume of the fermentation medium.

[0017] Preferably, the fermentation culture method is a batch fed fermentation, and when the glucose concentration in the fermentation medium drops to 30-60 g / L, glucose is added to maintain the glucose concentration at 100-120 g / L.

[0018] Preferably, the fermentation culture temperature is 30-35°C, the gas flux is 0.2-1.5v / v / m, the oxygen dissolution is 26-28, and the fermentation time is 24-28h.

[0019] Beneficial effects of the present invention:

[0020] (1) The present invention uses Pseudomonas fluorescens, Acetobacter aceti and Arthrobacter sphaeroides to co-ferment and prepare 2-keto-D-gluconic acid, which has a high conversion rate and a higher concentration of the obtained product than that of single bacteria fermentation.

[0021] (2) The present invention mixes three kinds of bacteria and ferments them to prepare 2-keto-D-gluconic acid, thereby reducing the inhibitory effect of the substrate glucose on the growth and fermentation of a single microorganism and making up for the deficiency of fermentation of a single fluorescent Pseudomonas.

[0022] (3) The fermentation cycle of the present invention is short, the cost is low, and it is easy to promote industrialization. DETAILED DESCRIPTION

[0023] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0024] As described in the background technology, 2-keto-D-gluconic acid is an important organic acid and an important precursor for preparing D-isoascorbic acid and its sodium salt. At present, the main bacteria that can ferment and prepare 2-KGA include Pseudomonas, Erwinia, Serratia, Gluconobacter, Arthrobacter, Alcaligenes and Acetobacter, etc., and the most commonly used strain is Pseudomonas fluorescens, but the use of this strain still has problems such as inhibition of high concentrations of glucose, so screening out new strains to ferment and prepare 2-keto-D-gluconic acid has become a hot topic in the field, but only the fermentation of a single strain has been explored in the prior art, and there has been no relevant report on mixed bacterial fermentation.

[0025] Based on this, the present invention provides a method for preparing 2-keto-D-gluconic acid by fermentation. The method uses mixed fermentation of three microorganisms. Compared with fermentation of a single strain, the method has a higher conversion rate, a higher concentration of the obtained product, and reduces the inhibitory effect of high-concentration glucose substrate on strain growth and fermentation.

[0026] The strains used in the present invention are Pseudomonas fluorescens, Acetobacter aceti and Arthrobacter sphaeroides. Among them, the inventors screened other strains capable of fermenting and producing 2-keto-D-gluconic acid based on the most widely used Pseudomonas fluorescens and according to the fermentation and growth conditions of Pseudomonas fluorescens as the standard, and found in the experiment that when Pseudomonas fluorescens, Acetobacter aceti and Arthrobacter sphaeroides were mixed for fermentation culture, the number of live bacteria in the fermentation liquid after the fermentation was completed was much higher than that of the live bacteria after fermentation of a single strain, indicating that the three strains have a synergistic effect of mutually promoting each other. Based on this, the inventors further investigated the ability of the three strains to prepare 2-keto-D-gluconic acid, and found that the use of mixed strains for fermentation can effectively improve the concentration and conversion rate of the product.

[0027] Note: In the following examples and comparative examples, the strain number of Pseudomonas fluorescens used is CCTCC AB93075, the strain number of Acetobacter aceti is CCTCC CB 20082244, and the strain number of Arthrobacter sphaeroides is CCTCC AB206780. All of the above strains were purchased from China Center for Type Culture Collection.

[0028] Wherein, the Pseudomonas fluorescens seed solution is prepared by the following method:

[0029] After activating Pseudomonas fluorescens, 10% (volume ratio) of the inoculation amount was inoculated into a Pseudomonas fluorescens seed culture medium, and cultured at 30° C. for 24 hours to obtain a Pseudomonas fluorescens seed solution.

[0030] Pseudomonas fluorescens seed culture medium: by weight percentage, 5% lactose, 2% glucose, 1% peptone, 0.2% potassium chloride, 0.1% magnesium sulfate heptahydrate, and the balance is sterile water.

[0031] The acetobacter aceti seed solution is prepared by the following method:

[0032] After activating Acetobacter aceti, 12% (volume ratio) of the inoculation amount was inoculated into the Acetobacter aceti seed culture medium, and the culture was carried out at 30° C. for 36 hours to obtain the Acetobacter aceti seed liquid.

[0033] Acetobacter aceti seed culture medium: 100g glucose, 10g yeast extract powder, 20g calcium carbonate, 20ml anhydrous ethanol, 15g agar, 1000ml distilled water.

[0034] Arthrobacter sphaeroides seed solution was prepared by the following method:

[0035] After activating Arthrobacter sphaeroides, 10% (volume ratio) of the inoculum was inoculated into the Arthrobacter sphaeroides seed culture medium, and cultured at 30° C. for 12 h to obtain the Arthrobacter sphaeroides seed solution.

[0036] Arthrobacter sphaeroides seed culture medium: 5g peptone, 3g beef extract powder, 5g sodium chloride, 15g agar, 1000ml distilled water.

[0037] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0038] Example 1: Preparation of 2-keto-D-gluconic acid by fermentation.

[0039] (1) mixing a Pseudomonas fluorescens seed solution, a Acetobacter aceti seed solution, and a Sphaeroides seed solution in a weight ratio of 5:1:3 to obtain a mixed seed solution;

[0040] The number of live bacteria in the mixed seed solution was 3.0×10 8 cfu / mL.

[0041] (2) The mixed seed liquid is inoculated into a fermentation medium, and the inoculation amount is 12% of the volume of the fermentation medium, and fermentation is performed to obtain a fermentation liquid containing 2-keto-D-gluconic acid.

[0042] The fermentation method is batch feeding fermentation. When the glucose concentration in the fermentation medium drops to 50 g / L, glucose is added to maintain the glucose concentration at 110 g / L. The fermentation temperature is 32°C, the gas flux is 1.0 v / v / m, the oxygen dissolved is 27, and the fermentation time is 24 h.

[0043] Example 2: Preparation of 2-keto-D-gluconic acid by fermentation.

[0044] (1) mixing a Pseudomonas fluorescens seed solution, a Acetobacter aceti seed solution, and a Sphaeroides seed solution in a weight ratio of 5:0.5:2 to obtain a mixed seed solution;

[0045] The number of live bacteria in the mixed seed solution was 3.0×10 8 cfu / mL.

[0046] (2) The mixed seed liquid is inoculated into a fermentation medium, with the inoculation amount being 10% of the volume of the fermentation medium, and fermentation is performed to obtain a fermentation liquid containing 2-keto-D-gluconic acid.

[0047] The fermentation method is batch feeding fermentation. When the glucose concentration in the fermentation medium drops to 30 g / L, glucose is added to maintain the glucose concentration at 100 g / L. The fermentation temperature is 30°C, the gas flux is 0.2 v / v / m, the oxygen dissolved is 26, and the fermentation time is 24 h.

[0048] Example 3: Preparation of 2-keto-D-gluconic acid by fermentation.

[0049] (1) mixing a Pseudomonas fluorescens seed solution, a Acetobacter aceti seed solution, and a Sphaeroides seed solution in a weight ratio of 5:1.2:4 to obtain a mixed seed solution;

[0050] The number of live bacteria in the mixed seed solution was 3.0×10 8 cfu / mL.

[0051] (2) The mixed seed liquid is inoculated into a fermentation medium, and the inoculation amount is 15% of the volume of the fermentation medium, and fermentation is performed to obtain a fermentation liquid containing 2-keto-D-gluconic acid.

[0052] The fermentation method is batch feeding fermentation. When the glucose concentration in the fermentation medium drops to 60 g / L, glucose is added to maintain the glucose concentration at 120 g / L. The fermentation temperature is 35°C, the gas flux is 1.5 v / v / m, the oxygen dissolved is 28, and the fermentation time is 28 h.

[0053] Comparative Example 1: Preparation of 2-keto-D-gluconic acid by fermentation.

[0054] The difference between this comparative example and Example 1 is that the mixed seed solution is replaced with a Pseudomonas fluorescens seed solution, and the number of live bacteria in the Pseudomonas fluorescens seed solution is 3.0×10 8 The remaining preparation conditions were the same as those in Example 1, and a fermentation broth containing 2-keto-D-gluconic acid was obtained.

[0055] Comparative Example 2: Preparation of 2-keto-D-gluconic acid by fermentation.

[0056] The difference between this comparative example and Example 1 is that the mixed seed solution consists of Pseudomonas fluorescens seed solution and Acetobacter aceti seed solution, and the number of live bacteria in the mixed seed solution is 3.0×10 8 The remaining preparation conditions were the same as those in Example 1, and a fermentation broth containing 2-keto-D-gluconic acid was obtained.

[0057] Comparative Example 3: Preparation of 2-keto-D-gluconic acid by fermentation.

[0058] The difference between this comparative example and Example 1 is that the mixed seed solution consists of Pseudomonas fluorescens seed solution and Arthrobacter sphaeroides seed solution, and the number of viable bacteria in the mixed seed solution is 3.0×10 8 The remaining preparation conditions were the same as those in Example 1, and a fermentation broth containing 2-keto-D-gluconic acid was obtained.

[0059] Test example:

[0060] (1) Test method:

[0061] Take the fermentation broth prepared in Example 1 and Comparative Examples 1-3, and refer to the method described in the paper "Study on 2-Keto-D-Gluconic Acid Fermentation" to detect the content of 2-KGA in the fermentation broth by high performance liquid chromatography. The detection was performed using an AMINEX HPX-87H organic acid column. The detection conditions were: mobile phase 0.5 mmol / L dilute H2SO4 solution, flow rate 0.5 mL / min, column temperature 35°C, and injection volume 10 μL.

[0062] Conversion rate (%) = 2-keto-D-gluconic acid production / [(initial glucose content of medium + glucose content of feed) - glucose content of medium after fermentation]

[0063] (2) The test results are shown in Table 1:

[0064] Table 1 Concentration and conversion rate of 2-KGA

[0065]

[0066] As can be seen from the above table, compared with fermentation using a single fluorescing Pseudomonas, the mixed fermentation of fluorescing Pseudomonas, Acetobacter aceti and Arthrobacter sphaeroides used in the present invention can give full play to the synergistic effect between the strains and improve the concentration and conversion rate of 2-KGA.

[0067] The above description is only a preferred embodiment of the present invention and is not intended to limit the implementation and application of the present invention. Any modifications, equivalent substitutions, etc. made in the implementation and application within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing 2-keto-D-gluconic acid by fermentation, characterized in that: The following steps are involved: (1) mixing a Pseudomonas fluorescens seed solution, an Acetobacter aceti seed solution, and an Arthrobacter sphaeroides seed solution to obtain a mixed seed solution; (2) inoculating the mixed seed liquid into a fermentation medium, and fermenting and culturing to obtain a fermentation liquid containing 2-keto-D-gluconic acid; The strain number of the Pseudomonas fluorescens is CCTCC AB 93075; the strain number of the Acetobacter aceti is CCTCC CB 20082244; the strain number of the Arthrobacter sphaeroides is CCTCC AB 206780; The weight ratio of the Pseudomonas fluorescens seed solution, the Acetobacter aceti seed solution and the Arthrobacter sphaeroides seed solution in the mixed seed solution is 5:(0.5-1.2):(2-4).

2. The method according to claim 1, characterized in that The number of viable bacteria in the mixed seed solution is ≥3.0×10 8 cfu / mL.

3. The method according to claim 1, characterized in that The fermentation medium comprises the following components: Glucose 100-140g / L, corn syrup powder 20-25g / L, calcium carbonate 0.1-0.2g / L, magnesium sulfate 0.2-0.3g / L, ammonium sulfate 1-1.5g / L, dipotassium hydrogen phosphate 0.2-0.3g / L, pH 6.8-7.0g / L.

4. The method according to claim 1, characterized in that The inoculation amount of the mixed seed liquid is 10-15% of the volume of the fermentation medium.

5. The method according to claim 1, characterized in that The fermentation culture method is batch fed fermentation. When the glucose concentration in the fermentation medium drops to 30-60 g / L, glucose is added to maintain the glucose concentration at 100-120 g / L.

6. The method according to claim 1, characterized in that The fermentation culture temperature is 30-35° C., the gas flux is 0.2-1.5 v / v / m, the oxygen dissolution is 26-28, and the fermentation time is 24-28 hours.

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