Method for preparing acetoin through fermentation of bacillus velezensis strain
The preparation of B. velezensis AHHY-1A by fermentation of Bacillus Berellis strain B.velezensis AHHY-1A, solved the problems of extraction difficulties and chemical synthesis pollution in the prior art, and achieved efficient and low-cost Berlin production, which was suitable for industrial applications.
Patent Information
- Application Number
- CN202510356130.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-25
- Publication Date
- 2025-07-08
AI Technical Summary
The prior art is difficult to extract Essential from plants efficiently and at low cost. Chemical synthesis methods pose a risk of carcinogen pollution, and raw materials are scarce, which cannot meet market demand.
Bacillus Bacillus strain B.velezensis AHHY-1A was used to prepare the fermentation of Bacillus B.velezensis AHHY-1A. Through seed culture and shake flask fermentation, glucose was used as raw material, and the culture conditions were optimized to increase the edible yield and reduce by-products.
It has achieved efficient and low-cost production, high output, suitable for large-scale industrial applications, few by-products, and avoids the risk of environmental pollution caused by chemical synthesis.
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Figure CN120272541A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biological fermentation engineering. Specifically, it relates to a method for fermenting Bacillus velezensis strain to produce acetoin. Background Art
[0002] Acetoin, with the English name Acetoin (AC) and the chemical name 3-hydroxy-2-butanone, also known as 3-hydroxybutanone or methyl acetyl methanol, is an organic compound with the molecular formula C4H8O2 and exists in two chiral enantiomers. It has a pleasant fat, cream, and butter-like aroma. The monomer is a colorless or light yellow liquid, and the dimer is a white crystalline powder. Acetoin can be widely used in the fields of food, cosmetics, plants, medicine, and chemical engineering.
[0003] Acetoin widely exists in substances such as strawberries, raspberries, walnuts, rum, cream, candies, caramel, coconuts, coffee, and corn. Natural acetoin products can be extracted from plants such as strawberries, raspberries, and walnuts. However, as a secondary metabolite, its content in plants is extremely low, and the growth cycle of plants is long, and the growth status is restricted by various environmental factors. The amount extracted from plants purely naturally cannot meet the market demand; if chemical synthesis methods are used, carcinogenic and pathogenic compounds are easily doped in chemically synthesized acetoin products, which limits the use of acetoin as a food flavor and fragrance. The scarcity of fossil resources leads to limited raw materials, low product yields, and serious environmental pollution. Therefore, the preparation of acetoin by microbial fermentation as an alternative to chemical synthesis has received increasing wide attention. Using the microbial fermentation method to produce high-value-added acetoin from low-cost raw materials is the most potential way. Summary of the Invention
[0004] The purpose of the present invention is to provide a method for fermenting Bacillus velezensis strain to produce acetoin, so as to provide a preparation method with low cost, high efficiency, and few by-products for the synthesis of natural acetoin.
[0005] The purpose of the present invention can be achieved by the following technical solutions:
[0006] A method for fermenting Bacillus velezensis strain to produce acetoin, using glucose as a raw material and Bacillus velezensis strain (B.velezensis AHHY-1A) as the starting strain, and producing acetoin through seed culture and shake flask fermentation.
[0007] Among them, the mutagenesis, screening, and isolation method of Bacillus velezensis strain (B.velezensis AHHY-1A) is as follows:
[0008] The Bacillus velezensis strain preserved in the laboratory was cultured to the logarithmic phase. 10 mL of the bacterial liquid was taken, centrifuged at 3300 rpm for 10 min, and the supernatant was discarded. The cells were washed three times with phosphate buffer solution at pH 7.0. The washed strain was resuspended in 9 mL of phosphate buffer solution at pH 7.0, and then 1 mL of nitrosoguanidine solution was added to make the concentration of nitrosoguanidine reach 1 mg / mL. It was placed in a shaker at 37 °C for mutagenesis for 20 min, centrifuged at 3300 rpm for 10 min, and the supernatant was discarded. It was washed three times with physiological saline, and the strain was spread on a separation medium with glucose as the sole carbon source and placed in an incubator at 37 °C for 48 h. Filter paper was made into 5-mm-sized pieces with a hole puncher, soaked in a solution prepared by equal-volume mixing of 0.5% creatine and 5% 1-naphthol. Finally, the pieces of paper were tightly covered on the single colonies in the separation plate and left standing at 37 °C for 1 h. Strains with red spots were selected as the primary screening strains.
[0009] The primary screening strains were subjected to shake-flask fermentation experiments, and the acetoin yield was detected by gas chromatography. Finally, a strain that could efficiently metabolize glucose to produce acetoin was screened out and named B.velezensis AHHY-1A.
[0010] Furthermore, the seed culture parameters:
[0011] Seed medium: It includes carbon source compounds, nitrogen source compounds, and inorganic salts, and the initial pH is controlled at 6.5 - 7.0;
[0012] Culture conditions: The temperature is 25 - 40 °C, the shaker speed is 100 - 300 rpm, and the culture time is 12 - 16 h.
[0013] Furthermore, the carbon source compounds in the seed medium include at least one or more of yeast powder, glucose, and beef extract.
[0014] Furthermore, the shake-flask fermentation parameters:
[0015] Fermentation medium: It includes carbon source compounds, nitrogen source compounds, inorganic salts, and trace elements, and the initial pH is controlled at 6.0 - 7.0;
[0016] Culture conditions: The temperature is 25 - 40 °C, the shaker speed is 100 - 300 rpm, and the fermentation time is 48 h.
[0017] Furthermore, the carbon source compounds in the fermentation medium include at least glucose, and the concentration of the glucose is 10 - 100 g / L.
[0018] Furthermore, the carbon source compounds in the fermentation medium also include other carbon sources, and the other carbon sources include one or more combinations of sugars (except glucose), alcohols, and organic acids, and the concentration of the other carbon sources is 5 - 30 g / L.
[0019] Preferably, the other carbon sources include one or more combinations of glycerol, mannitol, sorbitol, yeast powder, and wort.
[0020] Furthermore, the nitrogen source compounds include one or more combinations of nitrates, ammonium salts, yeast powder, soybean powder, tryptone, and corn steep liquor, and the concentration of the nitrogen source compounds is 5 - 30 g / L.
[0021] Preferably, the nitrogen source compounds include one or more combinations of yeast powder, ammonium sulfate, ammonium nitrate, and tryptone.
[0022] Furthermore, the inorganic salts include one or more of carbonates, chlorides, nitrates, phosphates, and sulfates; the metal part of the inorganic salts includes one or more of calcium, iron, potassium, cobalt, copper, magnesium, sodium, or zinc; and the concentration of the inorganic salts is 5 - 20 g / L.
[0023] Preferably, the inorganic salts include one or more combinations of manganese chloride, magnesium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, calcium chloride, ferrous sulfate, potassium chloride, and sodium chloride.
[0024] Furthermore, during the shake - flask fermentation process, glucose is added to the fermentation system once or multiple times; preferably, glucose is added during the fermentation cycle of 24 - 48 h; preferably, glucose is added until the glucose concentration in the fermentation system is 10 - 60 g / L.
[0025] It should be further noted that the method for measuring the acetoin yield is as follows:
[0026] Preparation of the fermentation broth sample and acetoin standard: Centrifuge the fermentation broth, and take the supernatant and dilute it 10 times for standby. Using acetoin as the standard, prepare acetoin standard solutions with different concentrations. Extract the diluted supernatant and the acetoin standard solutions with the extractant ethyl acetate respectively. The upper organic phase is filtered through a 0.45 - μm microporous membrane and used for gas chromatography detection. The standard curve has a good linear relationship between 0 - 3.0 g / L.
[0027] Gas chromatography detection conditions: The chromatographic column is Column BGB - 174 (30 m×0.25 mm I.D. 0.25 μm df (P / N: 27430 - 025)), and the detection conditions are: SPL1 temperature: 220°C; FID temperature: 240°C; Program temperature rise: The initial temperature is 100°C and is held for 2 min, then it rises to 130°C at a rate of 2.00°C / min, and then it rises to 180°C at a rate of 15°C / min; Carrier gas: N2, using the split mode (split ratio: 1:5).
[0028] Advantages of the present invention:
[0029] (1) Provided a Bacillus velezensis strain capable of biosynthesizing acetoin using glucose and other carbon sources as substrates. This strain was named B.velezensis AHHY-1A, which had a high yield in the fermentation production of acetoin and was able to metabolize glucose to produce 20.85 g / L of acetoin; (2) The method for fermenting and producing acetoin using the strain B.velezensis AHHY-1A provided by the present invention had a simple production process, low raw material cost, and few by-products, and was suitable for large-scale industrial production. This method could serve as the basis for further developing the technology of biosynthesizing acetoin by biological methods. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] The present invention will be further described below in conjunction with the accompanying drawings.
[0031] Figure 1 It was the gas chromatography detection result of 2.0 g / L acetoin standard product;
[0032] Figure 2 It was the gas chromatography detection result of the 48 h fermentation broth in Example 1;
[0033] Figure 3 It was the gas chromatography detection result of the 48 h fermentation broth in Example 2. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.
[0035] Example 1
[0036] A method for fermenting and preparing acetoin using Bacillus velezensis (B.velezensis AHHY-1A):
[0037] Step 1: Medium preparation
[0038] Seed medium (g / L): Tryptone 10, yeast extract 5, sodium chloride 10, pH 7.0;
[0039] Fermentation medium (g / L): Glucose 50, yeast extract 8, tryptone 6, potassium dihydrogen phosphate 3, disodium hydrogen phosphate 5, manganese sulfate monohydrate 0.005, magnesium sulfate 0.5, sodium chloride 5, pH 7.0;
[0040] Step 2: Seed preparation
[0041] Add 50 mL of seed medium to a 500 mL Erlenmeyer flask and sterilize it at 121 °C under high temperature and high pressure for 20 min. Take out the glycerol bacteria from the -80 °C refrigerator (the final concentration of the bacterial liquid preservation is 15 - 30%), and after melting, pipette 500 μL into 50 mL of seed medium, and culture it on a shaker at 25 - 40 °C and 100 - 300 rpm for 12 h to obtain the seed liquid;
[0042] Step 3: Shake flask fermentation culture
[0043] Inoculate the seed liquid into 50 mL of fermentation medium at an inoculation amount of 1%, and culture it on a shaker at 25 - 40 °C and 100 - 300 rpm for 48 h. Sample once every 12 h to measure the glucose concentration. When the glucose concentration is lower than 30 g / L, add glucose.
[0044] Step 4: Product detection
[0045] Take the fermentation broth for centrifugation (12000 rpm, 5 min), discard the lower layer of bacteria, retain the supernatant, dilute it 10 times and set aside. Use acetoin as the standard product to prepare acetoin standard solutions with concentrations of 0.5, 1.0, 2.0, and 3.0 g / L (as shown in Figure 1 the gas chromatography detection results of the 2.0 g / L acetoin standard product). Extract the diluted supernatant and acetoin standard solutions with different concentrations with an equal volume of ethyl acetate respectively, collect the upper organic phase, filter it through a 0.45 μm microporous filter membrane, and detect the acetoin yield by high-performance gas chromatography. The measured results are as shown in Figure 2 shown.
[0046] Example 2
[0047] A method for fermenting and preparing acetoin using Bacillus velezensis (B.velezensis AHHY-1A):
[0048] Step 1: Medium preparation
[0049] Seed medium (g / L): Glucose 5, starch 30, yeast powder 2, tryptone 4, sodium chloride 5, pH 7.0;
[0050] Fermentation medium (g / L): Glucose 70, yeast powder 2, beef extract 5, tryptone 10, potassium dihydrogen phosphate 2, disodium hydrogen phosphate 2, potassium chloride 0.5, magnesium sulfate 0.5, ferrous sulfate heptahydrate 0.003, sodium chloride 10, pH 7.0;
[0051] Step 2: Seed preparation
[0052] Add 50 mL of seed medium to a 500 mL Erlenmeyer flask and sterilize it at 121 °C under high temperature and high pressure for 20 min. Take out the glycerol bacteria from the -80 °C refrigerator (the final concentration of the bacterial liquid preservation is 15 - 30%), and after melting, pipette 500 μL into 50 mL of seed medium and culture it on a shaker at 25 - 40 °C and 220 rpm for 16 h to obtain the seed liquid;
[0053] Step 3: Shake flask fermentation culture
[0054] Inoculate the seed liquid into 50 mL of fermentation medium at an inoculation amount of 2% and culture it on a shaker at 25 - 40 °C and 100 - 300 rpm for 48 h. Sample once every 12 h to measure the glucose concentration. When the glucose concentration is lower than 40 g / L, add glucose.
[0055] Step 4: Product detection: The same as in Example 1, and the measured results are as Figure 3 shown.
[0056] The above specific implementation part has specifically introduced the analysis method involved in the present invention. It should be noted that the above introduction is only to help those skilled in the art better understand the method and idea of the present invention, rather than a limitation on the relevant content. Without departing from the principle of the present invention, those skilled in the art can also make appropriate adjustments or modifications to the present invention, and the above adjustments and modifications should also fall within the protection scope of the present invention.
Claims
1. A method for preparing acetoin by fermenting Bacillus velezensis strains, characterized in that, Using glucose as a raw material and Bacillus velezensis as a starting strain, acetoin is produced through seed culture and shake flask fermentation.
2. The method for fermenting Bacillus velezensis strain to prepare acetoin according to claim 1, wherein Seed culture parameters: Seed culture medium: It includes a carbon source compound, a nitrogen source compound, and inorganic salts, with the initial pH controlled at 6.5 - 7.0; Culture conditions: Temperature is 25 - 40 °C, the shaking speed of the shaker is 100 - 300 rpm, and the culture time is 12 - 16 h.
3. The method for fermenting Bacillus velezensis strain to prepare acetoin according to claim 2, wherein The carbon source compound in the seed culture medium contains at least one or more of yeast powder, glucose, and beef extract.
4. The method for fermenting the Bacillus velezensis strain to prepare acetoin according to claim 1, characterized in that, Shake flask fermentation parameters: Fermentation medium: It includes a carbon source compound, a nitrogen source compound, inorganic salts, and trace elements, with the initial pH controlled at 6.0 - 7.0; Culture conditions: Temperature is 25 - 40 °C, the shaking speed of the shaker is 100 - 300 rpm, and the fermentation time is 48 h.
5. The method for fermenting Bacillus velezensis strain to prepare acetoin according to claim 4, characterized in that, The carbon source compound in the fermentation medium includes at least glucose, and the concentration of the glucose is 10 - 100 g / L.
6. The method for fermenting Bacillus velezensis strain to prepare acetoin according to claim 5, wherein The carbon source compound in the fermentation medium also includes other carbon sources. The other carbon sources include one or more combinations of sugars, alcohols, and organic acids other than glucose, and the concentration of the other carbon sources is 5 - 30 g / L.
7. The method for preparing acetoin by fermenting the Bacillus velezensis strain according to claim 6, characterized in that, The other carbon sources include one or more combinations of glycerol, mannitol, sorbitol, yeast powder, and malt extract.
8. The method for fermenting Bacillus velezensis strain to prepare acetoin according to claim 2 or 4, characterized in that, The nitrogen source compound includes one or more combinations of nitrates, ammonium salts, yeast powder, soybean powder, tryptone, and corn steep liquor, and the concentration of the nitrogen source compound is 5 - 30 g / L; The inorganic salts include one or more of carbonates, chlorides, nitrates, phosphates, and sulfates; the metal part of the inorganic salts includes one or more of calcium, iron, potassium, cobalt, copper, magnesium, sodium, or zinc; the concentration of the inorganic salts is 5 - 20 g / L.
9. The method for fermenting Bacillus velezensis strain to prepare acetoin according to claim 4, characterized in that, During the shake flask fermentation process, glucose is added to the fermentation system one or more times.
10. The method for preparing acetoin by fermenting the Bacillus velezensis strain according to claim 9, characterized in that, Glucose is added during the fermentation period of 24 - 48 h; Glucose is added to the fermentation system until the glucose concentration in the fermentation system is 10 - 60 g / L.