Method for producing inositol by fermentation

Abstract

The invention belongs to the technical field of microbial fermentation, particularly relates to a method for producing inositol by fermentation, and solves the problems of high acetic acid content, low inositol yield, low conversion rate and low production cost in the process of producing inositol by fermentation by taking an inositol producing strain as an initial strain and controlling the glycerol supplementing flow rate in stages according to the characteristics of a thallus fermentation period and keeping a dissolved oxygen set value constant. And the unit consumption of glycerol is high. The concentration of inositol in the fermentation liquor is 118g / L or above, the conversion rate is 96% or above, the accumulation amount of acetic acid is less than 2g / L, and the unit consumption of glycerol is 0.6 or below.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a method for producing inositol by fermentation. Background Art

[0002] As a precursor of inositol phosphates and lipids, inositol participates in many life activities of the body, such as growth regulation, biofilm formation, osmotic tolerance, signal transduction, etc. It is a water-soluble vitamin containing a six-carbon ring. It has been widely used as an effective ingredient in the national standard in many industries such as medicine, food, and cosmetics. In the food industry, inositol is mainly used as a nutrient and food fortification additive to give food nutritional and health functions. In the feed industry, inositol is used as a biological promoter to prevent animal diseases and promote growth. In the pharmaceutical industry, inositol shows an insulin-like effect, which can reduce postprandial blood sugar and has been proven to treat diseases such as diabetes and fatty liver. In the cosmetics industry, inositol is a raw material for making comprehensive vitamins, which has the effects of promoting cell growth, preventing aging, and anti-oxidation. In recent years, thanks to the continuous development of the downstream market, the demand for inositol has increased year by year, but it is still in short supply.

[0003] The production methods of inositol mainly include hydrolysis, chemical synthesis, enzyme conversion, and biological fermentation. Among them, the hydrolysis method is to hydrolyze inositol hexaphosphate under acid or alkaline conditions to obtain inositol. The disadvantage is that acid / alkali and phosphorus-rich wastewater will be produced, which is easy to cause environmental pollution. The chemical synthesis method synthesizes inositol, which has complex processes, high costs, and serious pollution. Although the enzyme conversion method is pollution-free, the preparation and purification cost of the enzyme is high and the yield is low. In order to develop an inositol production method with less pollution, low cost, and high yield, in recent years, engineered bacteria for producing inositol have been constructed, and the production of inositol by biological fermentation has been increasingly widely used. How to balance the distribution of carbon sources in bacterial growth and inositol production has become the main problem of fermentation production of inositol. Tao et al. used glycerol as a carbon source for cell growth and glucose as a carbon source for inositol production to develop an inositol production bacterium that coordinates the utilization of carbon sources, and its fermentation yield can reach up to 106g / L. The patent application with publication number CN116286923A also constructed an inositol-producing bacterium with synergistic carbon source utilization (deposit number CCTCC NO: M20221796), and the inositol production can reach 53 g / L.

[0004] The principle of the above-mentioned inositol production bacteria fermentation to produce inositol is to use glycerol to provide energy for various life activities of the bacteria, and glucose is used as a substrate to obtain inositol through a two-step enzyme catalytic reaction. The fermentation process of inositol is mainly divided into three stages, the early bacterial growth stage, the mid-term induction enzyme production stage, and the late inositol conversion stage. The feeding method of Tao et al. is to control the dissolved oxygen by 30-50%, flow and add a mixed carbon source (the mass ratio of sugar and glycerol is 1:1), and during the conversion period, glucose is added in batches at one time. This control mode is prone to excessive glycerol supplementation, resulting in a large amount of accumulation of byproduct acetic acid, and even glycerol accumulation, and inositol accumulation stagnation phenomenon. The feeding method of patent CN116286923A is that dissolved oxygen is associated with feeding. When DO is greater than 35%, a mixed carbon source (the mass ratio of sugar and glycerol is 5:1) is added. Glycerol is added instantaneously, and the dissolved oxygen decreases quickly, and it is easy to anaerobic metabolism to produce byproduct acetic acid. Studies have shown that more acetic acid accumulation will affect the activity of various enzymes in Escherichia coli and stop various metabolic activities in the bacteria. For example, the enzyme activity of NADH-dependent related enzymes in the metabolic pathway is reduced, which will cause the NADH produced in the glycerol decomposition process to not be normally oxidized, and will eventually be oxidized to glycerol again, forming a closed loop, and the ability of the bacterial body to utilize glycerol is weakened or even lost. More importantly, glycerol cost is high, which is one of the main costs of fermentation production of inositol, so reducing glycerol unit consumption has also become the main way to reduce the production cost of inositol. However, too little glycerol feed will cause slow inositol accumulation rate, long fermentation cycle, and low efficiency. Therefore, how to optimize the process of fermentation production of inositol becomes a major problem of increasing inositol yield, improving sugar alcohol conversion rate, and reducing production costs. Summary of the invention

[0005] In view of the deficiencies of the above-mentioned prior art, the present invention provides a method for producing inositol by fermentation. The method is based on the principle of producing inositol by fermentation of the above-mentioned inositol-producing bacteria, according to the characteristics of the bacterial fermentation cycle using glycerol as a growth carbon source and glucose as a conversion carbon source, by improving the production process, controlling the glycerol supply flow rate in stages, and maintaining a constant dissolved oxygen set value, thereby solving the problems of high byproduct acetic acid, low inositol yield, low conversion rate, and high glycerol unit consumption in the process of fermentation production of inositol.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

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

[0008] During the fermentation process, the dissolved oxygen is controlled at 30%-50%; the feeding method is:

[0009] During the induction enzyme production stage, a mixed carbon source consisting of glucose and glycerol was added, and the glycerol addition flow rate was controlled to be 1-2 g / L / h. The induction enzyme production stage is the stage after the inducer is added. The inducer is at a temperature of 100 °C in the fermentation liquid OD 550 Join when you reach 15;

[0010] In the rapid conversion stage, a mixed carbon source consisting of glucose and glycerol was added, and the glycerol flow rate was controlled at 1-2 g / L / h. 550 When it reaches 35, it enters the rapid transformation stage;

[0011] In the low-speed conversion stage, a single carbon source consisting of glycerol is added, and the glycerol addition flow rate is controlled to be 0.5-1.5 g / L / h; wherein, when the inositol production rate drops below 2 g / L / h, the low-speed conversion stage is entered.

[0012] In some embodiments, the dissolved oxygen is controlled at 35%.

[0013] In some embodiments, during the enzyme production induction stage, the glycerol supplement flow rate is controlled to be 1.25 g / L / h;

[0014] In some embodiments, during the rapid conversion stage, the glycerol supplement flow rate is controlled to be 1.8 g / L / h;

[0015] In some embodiments, during the low-speed conversion stage, the glycerol feed flow rate is controlled to be 1.25 g / L / h.

[0016] In some embodiments, during the fermentation process, the fixed ventilation rate is 0.5-1 vvm, preferably 1 vvm;

[0017] In some embodiments, during the fermentation process, the rotation speed is controlled in conjunction with dissolved oxygen, and the rotation speed ranges from 300 to 700 rpm.

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

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

[0020] In some embodiments, the inducer can be routinely selected by a person skilled in the art, such as arabinose, and the preferred addition amount is 1-5 g / L.

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

[0022] In some embodiments, during the fermentation process, the initial fermentation medium includes 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 powder 0.1-1 g / L, citric acid 1-4 g / L, vitamin B1 10-40 mg / L, defoamer PPE 0.5-1 mL / L, FeCl3 2-5 mg / L, CoCl·6H2O1-4 mg / L, CuCl2·2H2O1-4 mg / L, NaMnO4·2H2O 1-4 mg / L, and the solvent is water.

[0023] In some embodiments, during the pre-induction growth phase, when the initial fermentation medium is insufficient in glycerol, the inositol-producing bacteria can be fed to increase the fermentation OD to 1. 550 It can reach 15, and the feed rate is controlled at 1-2 g / L / h; preferably, the feed is added after the glycerol in the initial fermentation medium is consumed.

[0024] In some embodiments, a mixed carbon source is supplemented in the pre-induction growth stage, the induction enzyme production stage and the rapid conversion stage, wherein glycerol is the carbon source for bacterial growth, and the amount of glycerol added determines the growth rate, bacterial concentration and bacterial activity of the bacteria; glucose is the substrate converted into inositol, and the amount of glucose determines the product concentration; wherein the mixed carbon source is composed of glucose and glycerol in a mass ratio of 2-5:1, preferably glucose and glycerol in a mass ratio of 3:1; more preferably, the concentration of glucose is 450-550 g / L, and the concentration of glycerol is 100-180 g / L; still preferably, the concentration of glucose is 450 g / L, and the concentration of glycerol is 150 g / L;

[0025] In the low-speed conversion stage, the sugar concentration is high or it is considered that no sugar needs to be supplemented after the tank is removed, so a single carbon source, glycerol, is supplemented and the tank is removed after the glycerol is consumed; wherein, the single carbon source is glycerol with a concentration of 250-500g / L.

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

[0027] In some embodiments, 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% based on the dissolved oxygen set value, feeding is performed for 1-4 hours, and the feeding is stopped. When the dissolved oxygen in the fermentation broth increases by another 10%-30% based on the dissolved oxygen set value, feeding is continued for 1-4 hours at the set flow rate for the current stage, and this cycle is repeated.

[0028] The characteristics of the stage are: from the beginning of fermentation to the end of the initial glycerol consumption, the fermentation liquid OD 550Reaching 15, the period of dissolved oxygen recovery is the early growth stage of induction; the fermentation liquid OD 550 After reaching 15, the temperature starts to drop and the inducer is added. The period from the end of induction to the induction stage is the induction enzyme production stage. The amount of glycerol added determines the growth rate of the bacteria and the amount of byproduct acetic acid produced. After the induction is completed, the temperature rises to enter the rapid conversion stage. In the late fermentation stage, the activity of the bacteria decreases and enters the low-speed conversion stage. The amount of glycerol added during the conversion determines the accumulation rate of inositol and the accumulation rate of byproduct acetic acid. In the low-speed conversion stage, when the residual sugar content in the fermentation liquid is lower than 0.5g / L, stop feeding; when the glycerol is consumed, the tank can be released.

[0029] Principle of the Invention

[0030] The fermentation process of inositol is mainly divided into three stages, the early bacterial growth stage, the mid-term induction enzyme production stage, and the late inositol conversion stage. According to the characteristics of the bacterial fermentation cycle, the present invention controls the glycerol supplement flow rate in stages, with constant dissolved oxygen, so that the bacterial body is in a stable state, which is conducive to the stable fermentation. The feed supplement and dissolved oxygen are controlled together, and it is preferred to stop the feed supplement intermittently, which helps to judge the excessive feed supplement situation, change the feed supplement flow rate in time, and consume the excess feed supplement amount. It can be understood by ordinary technicians in the field that based on the fermentation principle of inositol production bacteria using glycerol as a growth carbon source and glucose as a conversion carbon source, in addition to the inositol production bacteria with a preservation number of CCTCC NO: M20221796 specifically used in the present invention, other inositol production bacteria are also suitable for the improved fermentation process of the present invention. For example, the recombinant Escherichia coli bacteria MI01, MI02, MI03, MI04 and MI05 disclosed in CN113667686A; the engineered bacteria TEJ-11, TEJ-12, TEJ-21, TEJ-22, TEJ-23, TEJ-24, TEJ-25, TEJ-26 and TEJ-31 disclosed in CN112646760B.

[0031] Beneficial technical effects

[0032] According to the characteristics of the bacterial fermentation cycle, the present invention controls the carbon source composition and the glycerol supplementation flow rate in stages, so that the carbon source for strain growth and inositol production is rationally distributed, the carbon source is fully utilized, the generation of byproduct acetic acid in the fermentation process is reduced, the inositol yield and the sugar alcohol conversion rate are increased, and the glycerol unit consumption is reduced. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments.

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

[0035] The inositol-producing bacteria with the accession number of CCTCC NO: M20221796 in the embodiment has been disclosed in the application number “202211518107.6” and the application name “Screening of ribosome binding sequences and their application in the construction of inositol recombinant 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 powder 0.8g / L, citric acid 3g / L, vitamin B1 40mg / L, defoamer PPE 1mL / L, FeCl3 5mg / L, CoCl·6H2O 4mg / L, CuCl2·2H2O 2mg / L, NaMnO4·2H2O 3.5mg / L, solvent is water.

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

[0039] 1. Preparation of seed solution:

[0040] The frozen glycerol tubular inositol producing bacteria (preservation number: CCTCC NO: M20221796) were streaked on LB solid plates containing kanamycin resistance (final concentration of 50 mg / L), cultured in an incubator at 37°C for 12-16 h, and then 5-6 single colonies were picked and inoculated into LB liquid medium containing kanamycin resistance (final concentration of 50 mg / L), and cultured at 37°C and 220 rpm until the OD 550 2-3, obtain seed solution.

[0041] 2. Inoculate the seed liquid with 5% inoculation amount into a 5L fermentation tank containing 2L fermentation medium, and cultivate it at a temperature of 37°C, pH of 6.8, ventilation ratio of 1vvm, initial speed of 400rmp, dissolved oxygen setting value of 35%, speed and dissolved oxygen joint control (i.e., if the dissolved oxygen is lower than the setting value, the speed increases, and if the dissolved oxygen is higher than the setting value, the speed decreases), and the speed range is 300-700rpm. After fermentation for about 12h, the glycerol in the initial culture medium is consumed, and the dissolved oxygen rises to 50%. Take samples to test the bacterial concentration, and use spectrophotometry to detect the absorbance value (OD 550), OD was 18, and the inducer arabinose was added after the temperature dropped to 30°C to enter the induction enzyme production stage. The mixed carbon source was added at a glycerol flow rate of 1.25g / L / h. The feeding was linked to the dissolved oxygen, that is, the feeding stopped automatically after 1 hour, and when the dissolved oxygen returned to 50%, the same flow rate of 1.25g / L / h was used to feed for another 1 hour. This process was repeated. The fermentation temperature was 30°C and the pH was 6.8. After culturing at 30°C for 10 hours, i.e. 22 hours later, sampling was performed to detect that the OD reached 35, the temperature was raised to 37°C, the pH was raised to 7.0, and the rapid conversion stage began, i.e., the fermentation lasted for 22-66 hours. During this stage, the glycerol feed rate was 1.8 g / L / h, and the feed and dissolved oxygen were controlled in conjunction. After 66 hours of fermentation, according to the detected inositol production rate (less than 2 g / L / h), it was determined that the fermentation entered a slow conversion stage, glycerol was fed as a single carbon source, and the glycerol feed rate was reduced to 1.5 g / L / h, and the feed and dissolved oxygen were controlled in conjunction. The fermentation was tanked after 82 hours, and the fermentation liquid was collected.

[0042] The volume of the glycerol feed flow rate in the present invention is the initial constant volume.

[0043] The L in the flow rate refers to the glycerol replenishment rate of 1-2 g glycerol / h per liter of initial fermentation medium, that is, 1-2 g glycerol / L fermentation medium / h.

[0044] The specific implementation method of the joint control of feeding and dissolved oxygen is: select the joint control of dissolved oxygen and feeding on the fermentation control panel, set the feeding interval to 1h, and start feeding when the dissolved oxygen is 50%. The joint control method of feeding and dissolved oxygen of the present invention can automatically judge the relationship between the amount of glycerol added and the consumption of the bacteria. If the amount of glycerol added is equivalent to the consumption of the bacteria, the feeding will be stopped, the dissolved oxygen will rise immediately, and the feeding will continue; if the amount of glycerol added is greater than the consumption of the bacteria, glycerol will remain, and the dissolved oxygen will not rise after the feeding is stopped until the glycerol is consumed, the dissolved oxygen rises, and the feeding mode is automatically turned on. Through the feeding interval, the feeding flow rate can be further optimized to adjust.

[0045] Example 2

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

[0047] Example 3

[0048] The difference between this example and Example 1 is that the glucose concentration in the mixed carbon source is 450 g / L, and the mass ratio of sugar to glycerol is 2.5:1; the single carbon source glycerol concentration is 320 g / L; fermentation is 12-21 hours, i.e., the induction enzyme production stage, and the glycerol flow rate is 1.5 g / L / h; fermentation is 22-50 hours, and enters the rapid conversion stage, wherein fermentation is 22-40 hours, and the glycerol flow rate is 1.8 g / L / h; fermentation is 41-60 hours, and the glycerol flow rate is 1.25 g / L / h; fermentation is 60-84 hours, i.e., the low-speed conversion stage, and single carbon source glycerol is supplemented at a glycerol flow rate of 0.9 g / L / h.

[0049] Example 4

[0050] The difference between this embodiment and embodiment 1 is that the glucose concentration in the mixed carbon source is 500 g / L, the glycerol concentration is 100 g / L, and the mass ratio of glucose to glycerol is 5:1; the glycerol supplement flow rate during the fermentation process is 1.25 g / L / h throughout the 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 mass ratio of glucose to glycerol is 3:1; the glycerol concentration of the single carbon source is 250 g / L.

[0053] Example 6

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

[0055] Comparative Example 1

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

[0057] Comparative Example 2

[0058] The difference between this comparative example and Example 1 is that: 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 the dissolved oxygen rebounds (that is, when the dissolved oxygen is higher than 35%), the rotation speed is constant at 450 rpm, and the feeding control mode is the dissolved oxygen start feeding mode, specifically, when the dissolved oxygen is higher than 35%, the glycerol is fed at a rate of 3.6 g / L / h, and when the dissolved oxygen is lower than 35%, the feeding is stopped, and the dissolved oxygen fluctuation range is between 3% and 50%.

[0059] Effect Example

[0060] High performance liquid chromatography (HPLC) was used to detect the contents of inositol, acetic acid, glycerol and glucose in the fermentation broth. The detection method was as follows: the sample to be tested was diluted 100 times with pure water, filtered using a 0.22 μm aqueous filter membrane, and then sampled and detected using a high performance liquid chromatograph.

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

[0062] The conversion rate is calculated as follows:

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

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

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

[0066] Group Fermentation cycle h Fermentation volume L Yield g / L Conversion Rate % Acetic acid g / L Glycerol 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 unit consumption: the mass of glycerol required to produce 1g of product. The higher the unit consumption, the more expensive the cost, and the higher the glycerol cost.

[0068] As shown in Table 1, in the embodiment of the present invention, when the feed flow rate is controlled in stages, the dissolved oxygen is constant (i.e., the dissolved oxygen set value fluctuates very little, 35±1%), and the feed and dissolved oxygen are controlled in combination for inositol fermentation, the lower tank output is all 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. Therefore, this control mode can reduce the generation of byproduct acetic acid, increase inositol production, increase conversion rate, reduce glycerol unit consumption, and the fermentation effect difference of different batches is small, indicating good fermentation stability.

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

[0070] In Comparative Example 2, the glycerol supplementation method is to start supplementing glycerol after the glycerol is consumed and the dissolved oxygen rises to 35%, and stop supplementing glycerol when the dissolved oxygen is lower than 35%. When more glycerol is supplemented instantaneously, the dissolved oxygen decreases rapidly, and the lowest can reach 3%. Under low oxygen conditions, the anaerobic metabolism of Escherichia coli will lead to a high content of byproduct acetic acid, resulting in poor fermentation results. Compared with Example 1, the byproduct acetic acid in Comparative Example 2 is 3.58 times higher; the inositol yield is 1.88 times lower, the conversion rate is 1.06 times lower, and the glycerol unit consumption is 1.39 times higher.

[0071] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is 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 in the protection scope of the present invention.

Claims

1. A method for producing inositol by fermentation, characterized in that: The steps include: During the fermentation process, the dissolved oxygen is controlled at 30%-50%; the feeding method is: In the induction enzyme production stage, a mixed carbon source consisting of glucose and glycerol is added, and the glycerol flow rate is controlled to 1-2g / L / h, preferably 1.25g / L / h; wherein, the induction enzyme production stage is the stage after the addition of the inducer, and the inducer is at the fermentation liquid OD 550 Join when you reach 15; In the rapid conversion stage, a mixed carbon source consisting of glucose and glycerol is added, and the glycerol flow rate is controlled to 1-2 g / L / h, preferably 1.8 g / L / h; wherein, when the fermentation liquid OD 550 When it reaches 35, it enters the rapid transformation stage; In the low-speed conversion stage, a single carbon source consisting of glycerol is added, and the glycerol flow rate is controlled to 0.5-1.5 g / L / h, preferably 1.25 g / L / h; wherein, when the inositol production rate drops below 2 g / L / h, the low-speed conversion stage is entered.

2. The method according to claim 1, characterized in that During the fermentation process, the fixed ventilation volume is 0.5-1 vvm; preferably, the ventilation volume is 1 vvm.

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

0.

4. The method according to claim 1, characterized in that: The mixed carbon source is composed of glucose and glycerol in a mass ratio of 2-5:1, preferably glucose and glycerol in a mass ratio of 3:1; more preferably, the concentration of glucose is 450-550 g / L, and the concentration of glycerol is 100-180 g / L; also preferably, the concentration of glucose is 450 g / L, and the concentration of glycerol is 150 g / L.

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

6. The method according to claim 1, characterized in that During the fermentation process, the initial fermentation medium includes the following components: glycerol 5-10g / L, glucose 5-10g / L, potassium dihydrogen phosphate 2-8g / L, magnesium sulfate heptahydrate 1-4g / L, ammonium sulfate 1-4g / L, yeast powder 0.1-1g / L, citric acid 1-4g / L, vitamin B1 10-40mg / L, defoaming agent PPE 0.5-1mL / L, FeCl3 2-5mg / L, CoCl·6H2O1-4mg / L, CuCl2·2H2O 1-4mg / L, NaMnO4·2H2O 1-4mg / L, and the solvent is water.

7. The method according to claim 6, characterized in that When the initial fermentation medium was insufficient in glycerol, feed was added to the fermentation broth until the OD 550 Reaching 15: adding a mixed carbon source consisting of glucose and glycerol, and controlling the glycerol flow rate to 1-2 g / L / h, preferably 1.25 g / L / h.

8. The method according to claim 1, characterized in that In the low-speed conversion stage, feeding is stopped when the residual sugar content in the fermentation broth is lower than 0.5 g / L; preferably, the tank is drained when the glycerol is consumed.

9. The method according to any one of claims 1 to 8, characterized in that: When the dissolved oxygen content in the fermentation broth increases by 10%-30% based on the set value of dissolved oxygen, feeding is performed; the set value of dissolved oxygen is 30%-50%, preferably 35%.

10. The method according to claim 9, characterized in that The feeding method is intermittent feeding, and the period of the intermittent feeding is 1-4h.