A method for fermentative production of inositol

Abstract

The present application belongs to the field of microbial fermentation technology, and particularly relates to a method for fermentatively producing inositol, which takes inositol production bacteria as a starting strain, controls the glycerol supplement flow rate in stages according to the characteristics of the bacterial fermentation cycle, and sets a constant dissolved oxygen value, thereby solving the problems of high by-product acetic acid, low inositol yield, low conversion rate and high glycerol unit consumption in the process of fermentatively producing inositol. In the present application, the inositol concentration in the fermentation liquor is above 118 g / L, the conversion rate is above 96%, the acetic acid accumulation is less than 2 g / L, and the glycerol unit consumption is below 0.6.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a method for fermenting and producing inositol. Background Technology

[0002] Inositol, as a precursor to inositol phosphate and lipids, participates in various vital activities such as growth regulation, biomembrane formation, osmotic tolerance, and signal transduction. It is a water-soluble vitamin containing a six-carbon ring and is widely used as an active ingredient in the pharmaceutical, food, and cosmetic industries according to national standards. In the food industry, inositol is mainly used as a nutrient and food fortification additive, giving food nutritional and health benefits. In the feed industry, inositol acts as a bio-promoter, preventing animal diseases and promoting growth. In the pharmaceutical industry, inositol exhibits insulin-like effects, lowering postprandial blood glucose and has been proven to treat diseases such as diabetes and fatty liver. In the cosmetics industry, inositol is a raw material for manufacturing multivitamins, promoting cell growth, preventing aging, and providing antioxidant effects. In recent years, thanks to the continuous development of downstream markets, the demand for inositol has been increasing year by year, but supply still falls short of demand.

[0003] The main methods for producing inositol include hydrolysis, chemical synthesis, enzymatic conversion, and bio-fermentation. Hydrolysis, which involves hydrolyzing inositol hexaphosphate under acidic or alkaline conditions, produces acid / alkali and phosphorus-rich wastewater, easily causing environmental pollution. Chemical synthesis is complex, costly, and polluting. While enzymatic conversion is pollution-free, enzyme preparation and purification are costly and yields are low. To develop less polluting, lower-cost, and higher-yield inositol production methods, engineered bacteria for inositol production have been constructed in recent years. Bio-fermentation for inositol production is becoming increasingly widely used. However, balancing the distribution of carbon sources between cell growth and inositol production is a major challenge in fermentation-based inositol production. Tao et al. developed an inositol-producing bacterium that coordinates carbon source utilization, using glycerol as the carbon source for cell growth and glucose as the carbon source for inositol production, achieving a fermentation yield of up to 10⁶ g / L. Patent application CN116286923A also constructed an inositol-producing bacterium that utilizes carbon sources in synergy (accession number CCTCC NO: M20221796), with an inositol yield of up to 53 g / L.

[0004] The principle of inositol production by fermentation of the aforementioned inositol-producing bacteria is to utilize glycerol to provide energy for various life activities of the bacteria, while glucose serves as a substrate, yielding inositol through a two-step enzymatic reaction. The inositol fermentation process mainly consists of three stages: the early stage of bacterial growth, the middle stage of enzyme induction, and the late stage of inositol conversion. Tao et al.'s feeding method involved controlling dissolved oxygen at 30-50%, feeding a mixed carbon source (sugar to glycerol mass ratio 1:1), and adding glucose in batches during conversion. This control mode is prone to excessive glycerol supplementation, leading to a large accumulation of the byproduct acetic acid, and even glycerol accumulation, resulting in stagnation of inositol accumulation. Patent CN116286923A uses a feeding method that links dissolved oxygen to feeding; when DO exceeds 35%, a mixed carbon source (sugar to glycerol mass ratio 5:1) is added. This instantaneous addition of large amounts of glycerol causes a rapid decrease in dissolved oxygen, easily leading to anaerobic metabolism and the production of the byproduct acetic acid. Studies have shown that excessive acetic acid accumulation can affect the activity of various enzymes in *E. coli*, causing various metabolic activities within the bacteria to cease. For example, a decrease in the activity of NADH-dependent enzymes in the metabolic pathway will prevent the NADH produced during glycerol decomposition from being properly oxidized, ultimately leading to its re-oxidation back into glycerol, forming a closed-loop cycle. This weakens or even eliminates the cell's ability to utilize glycerol. More importantly, glycerol is a high-cost component, representing one of the main costs in inositol fermentation production. Therefore, reducing glycerol consumption is a primary way to lower inositol production costs. However, insufficient glycerol feeding results in a slow inositol accumulation rate, a long fermentation cycle, and low efficiency. Therefore, optimizing the inositol fermentation process is crucial for increasing inositol yield, improving sugar alcohol conversion, and reducing production costs. Summary of the Invention

[0005] To address the shortcomings of the existing technology, this invention provides a method for fermenting and producing inositol. This method is based on the principle of inositol-producing bacteria fermenting and producing inositol. According to the characteristics of the bacterial fermentation cycle using glycerol as the growth carbon source and glucose as the conversion carbon source, the method improves the production process, controls the glycerol replenishment rate in stages, and keeps the dissolved oxygen setpoint constant. This solves the problems of high acetic acid byproduct, low inositol yield, low conversion rate, and high glycerol consumption in the fermentation and production of inositol.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for producing inositol by fermentation includes the following steps:

[0008] Dissolved oxygen was controlled at 30%-50% during fermentation; the feeding method was as follows:

[0009] During the enzyme induction stage, a mixed carbon source consisting of glucose and glycerol is added, with the glycerol flow rate controlled at 1-2 g / L / h. This enzyme induction stage occurs after the addition of the inducer, which is present at the OD of the fermentation broth. 550 Join when it reaches 15;

[0010] During the rapid conversion phase, a mixed carbon source consisting of glucose and glycerol is added, with the glycerol flow rate controlled at 1-2 g / L / h; the OD of the fermentation broth is then adjusted. 550 When it reaches 35, it enters the rapid conversion phase;

[0011] During the low-rate conversion phase, a single carbon source consisting of glycerol is added, with the glycerol flow rate controlled at 0.5-1.5 g / L / h; the low-rate conversion phase begins when the inositol production rate drops below 2 g / L / h.

[0012] In some implementation schemes, dissolved oxygen is controlled at 35%.

[0013] In some implementation schemes, during the enzyme induction phase, the glycerol supplementation flow rate is controlled at 1.25 g / L / h;

[0014] In some implementation schemes, during the rapid conversion phase, the glycerol supplementation flow rate is controlled at 1.8 g / L / h;

[0015] In some implementation schemes, during the low-rate conversion phase, the glycerol supplementation flow rate is controlled at 1.25 g / L / h.

[0016] In some implementations, the aeration rate during the fermentation process is fixed at 0.5-1 vvm, preferably 1 vvm;

[0017] In some implementation schemes, the fermentation speed is controlled in conjunction with dissolved oxygen, with the speed range being 300–700 rpm.

[0018] In some implementations, the fermentation temperature is 30-37°C and the pH is 6.8-7.0 during the fermentation process.

[0019] In some embodiments, during the fermentation process: the pre-induction growth stage, i.e., the stage from the start of fermentation to the addition of the inducer, is controlled at a temperature of 37°C and a pH of 6.8; the enzyme induction stage is controlled at a temperature of 30°C and a pH of 6.8; and the conversion stage is controlled at a temperature of 37°C and a pH of 7.0.

[0020] In some embodiments, the inducer is a conventionally chosen substance that can be selected by those skilled in the art, such as arabinose, preferably added at an amount of 1-5 g / L.

[0021] In some implementations, the pH is controlled during the fermentation process by adding 12.5% ​​ammonia water (mass fraction).

[0022] In some embodiments, during fermentation, the initial fermentation medium comprises the following components: glycerol 5-10 g / L, glucose 5-10 g / L, potassium dihydrogen phosphate 2-8 g / L, magnesium sulfate heptahydrate 1-4 g / L, ammonium sulfate 1-4 g / L, yeast extract 0.1-1 g / L, citric acid 1-4 g / L, vitamin B1 10-40 mg / L, antifoaming agent PPE 0.5-1 mL / L, FeCl3 2-5 mg / L, CoCl·6H2O 1-4 mg / L, CuCl2·2H2O 1-4 mg / L, NaMnO4·2H2O 1-4 mg / L, and water as the solvent.

[0023] In some implementations, during the pre-induction growth phase, if the initial fermentation medium is deficient in glycerol, supplemental feeding can be used to induce OD fermentation by the inositol-producing bacteria. 550 The concentration can reach 15, and the feed flow rate can be controlled at 1-2 g / L / h; preferably, the feed is added after the glycerol in the initial fermentation medium is depleted.

[0024] In some implementations, a mixed carbon source is supplemented during the pre-induction growth stage, the enzyme-inducing stage, and the rapid conversion stage. Glycerol serves as the carbon source for cell growth, and the amount of glycerol added determines the cell growth rate, cell concentration, and cell activity. Glucose serves as the substrate for conversion to inositol, and the amount of glucose determines the product concentration. The mixed carbon source consists of glucose and glycerol in a mass ratio of 2-5:1, preferably 3:1. More preferably, the glucose concentration is 450-550 g / L, and the glycerol concentration is 100-180 g / L. Even more preferably, the glucose concentration is 450 g / L, and the glycerol concentration is 150 g / L.

[0025] During the low-speed conversion stage, when the sugar concentration is high or it is considered that no more sugar needs to be added in the next tank, glycerol, a single carbon source, is added. After the glycerol is depleted, the tank is removed. The single carbon source is glycerol with a concentration of 250-500 g / L.

[0026] In some implementations, feeding is performed when the dissolved oxygen content in the fermentation broth increases by 10%-30% from the set dissolved oxygen value; the set dissolved oxygen value is 30%-50%, preferably 35%.

[0027] In some implementations, the feeding method is intermittent feeding, wherein the intermittent feeding cycle is 1-4 hours; that is, when the dissolved oxygen in the fermentation broth increases by 10%-30% from the set dissolved oxygen value, feeding is carried out for 1-4 hours and then feeding is stopped. When the dissolved oxygen in the fermentation broth increases by another 10%-30% from the set dissolved oxygen value, feeding is carried out again for 1-4 hours at the set flow rate for the current stage, and so on.

[0028] The characteristics of the aforementioned stages are: from the start of fermentation to the depletion of initial glycerol, and the OD of the fermentation broth. 550The dissolved oxygen level reached 15, and the period of dissolved oxygen recovery was the early growth induction stage; the OD of the fermentation broth... 550 Once the temperature reaches 15°C, the period from when the inducing agent is added and the temperature is lowered until the induction process ends is the enzyme production induction phase. The amount of glycerol added during this phase determines the cell growth rate and the amount of acetic acid produced as a byproduct. After induction, the temperature is raised to enter the rapid conversion phase. In the later stages of fermentation, cell activity decreases, entering the low-speed conversion phase. During this phase, the amount of glycerol added determines the inositol accumulation rate and the acetic acid accumulation rate. During the low-speed conversion phase, feeding is stopped when the residual sugar content in the fermentation broth falls below 0.5 g / L. When the glycerol is depleted, the fermentation can be removed from the tank.

[0029] The principle of this invention

[0030] The fermentation process of inositol is mainly divided into three stages: the early stage of cell growth, the middle stage of enzyme induction, and the late stage of inositol conversion. This invention, based on the characteristics of the cell fermentation cycle, controls the glycerol feed rate in stages to maintain constant dissolved oxygen, keeping the cells in a stable state and promoting stable fermentation. The coordinated control of feed and dissolved oxygen, preferably with intermittent feeding stops, helps in identifying overfeeding, adjusting the feed rate in a timely manner, and consuming excess feed. Those skilled in the art will understand that, based on the fermentation principle of inositol production using glycerol as the growth carbon source and glucose as the conversion carbon source, this improved fermentation process is also applicable to other inositol-producing bacteria besides the inositol-producing bacteria with accession number CCTCC NO: M20221796 specifically used in this invention. For example, CN113667686A discloses recombinant Escherichia coli strains MI01, MI02, MI03, MI04, and MI05; CN112646760B discloses engineered strains TEJ-11, TEJ-12, TEJ-21, TEJ-22, TEJ-23, TEJ-24, TEJ-25, TEJ-26, and TEJ-31.

[0031] Beneficial technical effects

[0032] Based on the characteristics of the bacterial fermentation cycle, this invention controls the carbon source composition and glycerol supplementation rate in stages, thereby rationally allocating carbon sources for strain growth and inositol production. This achieves full utilization of carbon sources, reduces the generation of acetic acid as a byproduct during fermentation, increases inositol yield and sugar alcohol conversion rate, and reduces glycerol consumption per unit. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments.

[0034] Unless otherwise specified, the methods used in the following examples are conventional methods, and the reagents used are commercially available reagents.

[0035] The inositol-producing bacteria with accession number CCTCC NO: M20221796 in the examples has been disclosed in application number "202211518107.6" entitled "Screening of ribosome binding sequences and their application in the construction of recombinant inositol bacteria".

[0036] Example 1

[0037] Fermentation medium: glycerol 10g / L, glucose 10g / L, potassium dihydrogen phosphate 7.5g / L, magnesium sulfate heptahydrate 3g / L, ammonium sulfate 3.5g / L, yeast extract 0.8g / L, citric acid 3g / L, vitamin B1 40mg / L, antifoaming agent PPE 1mL / L, FeCl3 5mg / L, CoCl·6H2O 4mg / L, CuCl2·2H2O 2mg / L, NaMnO4·2H2O 3.5mg / L, solvent: water.

[0038] The feed consisted of: a mixed carbon source (including 540 g / L glucose and 180 g / L glycerol, with a glucose to glycerol mass ratio of 3:1); a single carbon source (500 g / L glycerol); pH adjustment with 12.5% ​​ammonia (mass fraction); and an inducer arabinose with a final concentration of 2 g / L. The sterilization conditions were 115°C for 30 min.

[0039] 1. Preparation of seed solution:

[0040] Cryopreserved glycerol-producing inositol bacteria (preservation number: CCTCC NO: M20221796) were streaked onto LB agar plates containing kanamycin resistance (final concentration 50 mg / L) and incubated at 37°C for 12-16 h. Then, 5-6 single colonies were picked and inoculated into LB liquid medium containing kanamycin resistance (final concentration 50 mg / L) and cultured at 37°C and 220 rpm until OD500 was reached. 550 2-3, to obtain seed liquid.

[0041] 2. Inoculate the seed culture at a rate of 5% into a 5L fermenter containing 2L of fermentation medium. Incubate at 37℃, pH 6.8, aeration ratio of 1 vvm, initial fermentation speed of 400 rpm, and dissolved oxygen setpoint of 35%. Fermentation speed and dissolved oxygen are controlled in a tandem (i.e., if dissolved oxygen is below the setpoint, the speed increases; if dissolved oxygen is above the setpoint, the speed decreases). The fermentation speed range is 300–700 rpm. After approximately 12 hours of fermentation, the glycerol in the initial medium is depleted, and the dissolved oxygen recovers to 50%. Samples are taken to analyze the bacterial concentration. The absorbance (OD) at 550 nm is measured using spectrophotometry. 550The OD was 18. After cooling to 30℃, arabinose, the inducer, was added to induce enzyme production. The mixed carbon source was added at a flow rate of 1.25 g / L / h glycerol. The feeding and dissolved oxygen were controlled in tandem. That is, the feeding would stop automatically after 1 hour and the dissolved oxygen would rise to 50%. Then the feeding would be added again at the same flow rate of 1.25 g / L / h for another hour. This process was repeated. The fermentation temperature was 30℃ and the pH was 6.8. After culturing at 30℃ for 10 hours (i.e., 22 hours), the OD value was measured at 35. The temperature was then increased to 37℃ and the pH to 7.0, entering the rapid conversion stage, i.e., fermentation from 22 to 66 hours. During this stage, the glycerol feed rate was 1.8 g / L / h, and the feed and dissolved oxygen were controlled in tandem. After 66 hours of fermentation, based on the detected inositol production rate (less than 2 g / L / h), it was determined that the low-speed conversion stage had begun. Glycerol was added as the single carbon source, and the glycerol feed rate was reduced to 1.5 g / L / h, with feed and dissolved oxygen controlled in tandem. After 82 hours of fermentation, the mixture was transferred to a tank and the fermentation broth was collected.

[0042] In this invention, the volume of the glycerol feed flow rate is the initial constant volume.

[0043] In this context, L in the flow rate refers to the rate at which glycerol is added per liter of initial fermentation medium at a rate of 1-2 g glycerol / h, or 1-2 g glycerol / L fermentation medium / h.

[0044] The specific implementation of the feed-feeding and dissolved oxygen co-control is as follows: On the fermentation control panel, select the dissolved oxygen and feed-feeding co-control option, set the feed-feeding interval to 1 hour, and start feed-feeding when the dissolved oxygen level reaches 50%. This invention's feed-feeding and dissolved oxygen co-control method can automatically determine the relationship between the amount of glycerol added and the amount consumed by the cells. If the amount of glycerol added is equal to the amount consumed by the cells, feed-feeding is stopped, the dissolved oxygen level immediately rises, and feed-feeding resumes. If the amount of glycerol added is greater than the amount consumed by the cells, glycerol residue remains, and after stopping feed-feeding, the dissolved oxygen level will not rise until the glycerol is completely consumed, the dissolved oxygen level rises, and the feed-feeding mode is automatically restarted. The feed-feeding interval can be further optimized to adjust the feed flow rate.

[0045] Example 2

[0046] The difference between this embodiment and Embodiment 1 is that the glycerol addition flow rate is 1.25 g / L / h throughout the fermentation process.

[0047] Example 3

[0048] The difference between this example and Example 1 is as follows: the glucose concentration in the mixed carbon source is 450 g / L, and the sugar to glycerol mass ratio is 2.5:1; the glycerol concentration of the single carbon source is 320 g / L; during the fermentation period of 12-21 h (the enzyme induction stage), the glycerol flow rate is 1.5 g / L / h; during the fermentation period of 22-50 h, the rapid conversion stage begins, during which the glycerol flow rate is 1.8 g / L / h during the fermentation period of 22-40 h; during the fermentation period of 41-60 h, the glycerol flow rate is 1.25 g / L / h; and during the fermentation period of 60-84 h (the low-speed conversion stage), the single carbon source glycerol is added at a flow rate of 0.9 g / L / h.

[0049] Example 4

[0050] The difference between this embodiment and Example 1 is that the glucose concentration in the mixed carbon source is 500 g / L, the glycerol concentration is 100 g / L, and the glucose to glycerol mass ratio is 5:1; the glycerol replenishment flow rate during fermentation is 1.25 g / L / h throughout the entire process.

[0051] Example 5

[0052] The difference between this embodiment and Embodiment 1 is that the glucose concentration in the mixed carbon source is 450 g / L, the glycerol concentration is 150 g / L, and the glucose to glycerol mass ratio is 3:1; while the glycerol concentration in the single carbon source is 250 g / L.

[0053] Example 6

[0054] The difference between this embodiment and Example 1 is that the fermentation medium formula is: glycerol 5g / L, glucose 5g / L, potassium dihydrogen phosphate 2.5g / L, magnesium sulfate heptahydrate 2g / L, ammonium sulfate 1g / L, yeast extract 0.2g / L, citric acid 1g / L, vitamin B1 20mg / L, antifoaming agent PPE 0.5mL / L, FeCl3 2mg / L, CoCl·6H2O 1.5mg / L, CuCl2·2H2O 1mg / L, NaMnO4·2H2O 2mg / L, and water as the solvent. The glycerol replenishment flow rate during fermentation is 1.25g / L / h throughout the entire process.

[0055] Comparative Example 1

[0056] The difference between this comparative example and Example 1 is as follows: the glycerol concentration in the mixed carbon source is 270 g / L, and the glucose to glycerol mass ratio is 2:1; during fermentation for 12-21 h, i.e. the enzyme induction stage, the glycerol flow rate is 1.25 g / L / h; during fermentation for 22-58 h, i.e. the rapid conversion stage, the glycerol flow rate is 2.7 g / L / h; and during fermentation for 59-82 h, i.e. the slow conversion stage, glycerol is supplemented with a single carbon source at a flow rate of 0.6 g / L / h.

[0057] Comparative Example 2

[0058] The difference between this comparative example and Example 1 is as follows: the glucose concentration in the mixed carbon source is 450 g / L, the glycerol concentration is 150 g / L, and the mass ratio of sugar to glycerol is 3:1; the glycerol concentration of the single carbon source is 312.5 g / L; after dissolved oxygen rebound (i.e., when dissolved oxygen is higher than 35%), the rotation speed is kept constant at 450 rpm, and the feeding control method is dissolved oxygen start feeding mode. Specifically, when the dissolved oxygen is higher than 35%, glycerol is fed at a feeding rate of 3.6 g / L / h, and when the dissolved oxygen is lower than 35%, feeding is stopped. The dissolved oxygen fluctuation range is between 3% and 50%.

[0059] Effect Example

[0060] The contents of inositol, acetic acid, glycerol and glucose in fermentation broth were determined by high performance liquid chromatography (HPLC). The method was as follows: the sample was diluted 100 times with pure water, filtered through a 0.22 μm aqueous filter membrane, and then injected into the HPLC instrument for detection.

[0061] The high-performance liquid chromatography (HPLC) detection conditions are as follows: HPLC instrument (equipped with a differential detector), column: Aminex HPX-87H Column (300x7.8mm); mobile phase: 18mM sulfuric acid aqueous solution; flow rate: 0.5ml / min; column oven temperature: 35℃; detector temperature: 35℃.

[0062] The conversion rate is calculated as follows:

[0063] Conversion rate = Inositol concentration * fermentation tank volume / total sugar consumed * 100%

[0064] The fermentation effect data of the above embodiments and comparative examples are shown in Table 1:

[0065] Table 1. Fermentation results of the examples and comparative examples.

[0066] Group Fermentation cycle h Fermentation volume (L) Production g / L Conversion rate % Acetic acid g / L Glycerin consumption Example 1 82 3 128 98 1.9 0.54 Example 2 82 3 123 97 0 0.50 Example 3 84 3 125 97 1.3 0.52 Example 4 82 3 124 97 0 0.52 Example 5 84 3 125 98 1.6 0.52 Example 6 84 3 119 98 0 0.55 Comparative Example 1 82 2.8 88 88 5.4 0.87 Comparative Example 2 82 3 68 92 6.8 0.75

[0067] Among them, glycerol consumption per unit: the mass of glycerol required to produce 1g of product. The higher the consumption per unit, the higher the cost, and the higher the cost of glycerol.

[0068] As shown in Table 1, in the embodiments of the present invention, when the feed flow rate is controlled in stages and the dissolved oxygen is kept constant (i.e., the dissolved oxygen setpoint fluctuates very little, 35±1%), and the feed and dissolved oxygen are controlled in conjunction, the yield in the lower tank is consistently above 118 g / L, the conversion rate is above 96%, the acetic acid accumulation is less than 2 g / L, and the glycerol consumption is below 0.6 g / L. Therefore, this control mode can reduce the formation of the byproduct acetic acid, increase the yield of inositol, improve the conversion rate, reduce the glycerol consumption, and the fermentation effect varies little between different batches, indicating good fermentation stability.

[0069] In Comparative Example 1, during the rapid conversion stage of fermentation, the glycerol feed flow rate was too high, leading to glycerol accumulation and consequently, excessive acetic acid accumulation, which affected the fermentation results. Compared to Example 1, Comparative Example 1 showed 2.84 times higher acetic acid byproduct; 1.45 times lower inositol yield; 1.11 times lower conversion rate; and 1.6 times higher glycerol consumption per unit area.

[0070] In Comparative Example 2, glycerol supplementation was initiated after glycerol was depleted and dissolved oxygen recovered to 35%. Supplementation ceased when dissolved oxygen fell below 35%. This rapid, large-scale glycerol supplementation caused a sharp drop in dissolved oxygen, reaching as low as 3%. Under hypoxic conditions, anaerobic metabolism in E. coli led to a high content of the byproduct acetic acid, resulting in poor fermentation outcomes. Compared to Example 1, Comparative Example 2 showed 3.58 times higher acetic acid content, 1.88 times lower inositol yield, 1.06 times lower conversion rate, and 1.39 times higher glycerol consumption per unit area.

[0071] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for producing inositol by fermentation, characterized in that, Includes the following steps: Dissolved oxygen was controlled at 30%-50% during fermentation; the feeding method was as follows: During the enzyme induction stage, a mixed carbon source consisting of glucose and glycerol is added, and the glycerol flow rate is controlled at 1-2 g / L / h. This enzyme induction stage occurs after the addition of the inducer, which is present at the OD of the fermentation broth. 550 Join when it reaches 15; During the rapid conversion phase, a mixed carbon source consisting of glucose and glycerol is added, and the glycerol flow rate is controlled at 1-2 g / L / h; wherein, when the fermentation broth OD 550 When it reaches 35, it enters the rapid conversion phase; During the low-rate conversion phase, a single carbon source consisting of glycerol is added, and the glycerol flow rate is controlled at 0.5-1.5 g / L / h; the low-rate conversion phase begins when the inositol production rate drops below 2 g / L / h. During the fermentation process, the aeration rate is fixed at 0.5-1 vvm; The mixed carbon source consists of glucose and glycerol in a mass ratio of 2-5:1, and the glucose concentration is 450-540 g / L; Feeding is initiated when the dissolved oxygen level in the fermentation broth increases by 10%-30% from the set dissolved oxygen level; the set dissolved oxygen level is 30%-50%. The feeding method is intermittent feeding, and the intermittent feeding cycle is 1-4 hours.

2. The method according to claim 1, characterized in that, During the enzyme induction stage, a mixed carbon source consisting of glucose and glycerol is added, and the glycerol flow rate is controlled at 1.25 g / L / h. During the rapid conversion phase, a mixed carbon source consisting of glucose and glycerol is added, and the glycerol flow rate is controlled at 1.8 g / L / h. During the low-speed conversion stage, a single carbon source consisting of glycerol is added, and the glycerol flow rate is controlled at 1.25 g / L / h.

3. The method according to claim 1 or 2, characterized in that, During the fermentation process, the aeration rate is fixed at 1 vvm.

4. The method according to claim 1 or 2, characterized in that, The mixed carbon source consists of glucose and glycerol in a mass ratio of 3:

1.

5. The method according to claim 4, characterized in that, The glucose concentration was 450 g / L, and the glycerol concentration was 150 g / L.

6. The method according to claim 1, characterized in that, The dissolved oxygen setting is 35%.

7. The method according to claim 1, characterized in that, During the fermentation process, the fermentation temperature is 30-37℃ and the pH is 6.8-7.

0.

8. The method according to claim 1, characterized in that, The single carbon source is glycerol with a concentration of 250-500 g / L.

9. The method according to claim 1, characterized in that, During fermentation, the initial fermentation medium consists of the following components: glycerol 5-10 g / L, glucose 5-10 g / L, potassium dihydrogen phosphate 2-8 g / L, magnesium sulfate heptahydrate 1-4 g / L, ammonium sulfate 1-4 g / L, yeast extract 0.1-1 g / L, citric acid 1-4 g / L, vitamin B1 10-40 mg / L, antifoaming agent PPE 0.5-1 mL / L, FeCl3 2-5 mg / L, CoCl•6H2O 1-4 mg / L, CuCl2•2H2O 1-4 mg / L, NaMnO4•2H2O 1-4 mg / L, and water as the solvent.

10. The method according to claim 9, characterized in that, If the initial fermentation medium is insufficient in glycerol, replenish the glycerol to the fermentation broth OD as follows. 550 To reach 15: supplement with a mixed carbon source consisting of glucose and glycerol, and control the glycerol flow rate at 1-2 g / L / h.

11. The method according to claim 10, characterized in that, Control the glycerol flow rate to 1.25 g / L / h.

12. The method according to claim 1, characterized in that, During the low-speed conversion stage, feeding is stopped when the residual sugar content in the fermentation broth is below 0.5 g / L.

13. The method according to claim 12, characterized in that, When the glycerin is used up, remove it from the container.