Method for preparing arctigenin through dynamic regulation and control fermentation

Abstract

The invention discloses a method for preparing arctigenin through dynamic regulation and control fermentation, and belongs to the technical field of natural active ingredient extraction. In order to solve the technical problem that the extraction efficiency of arctigenin is poor in the prior art, the invention provides a method for preparing arctigenin through dynamic regulation and control fermentation. According to the method, the arctigenin is prepared by dynamically regulating and controlling fermentation through a multi-path replaceable process, and the method comprises the following steps: dynamically regulating and controlling fermentation in a time-sharing manner, and dynamically regulating and controlling the feeding speed, the stirring speed and the fermentation temperature; the biomass of the thalli is improved by selecting a carbon source adaptive to the metabolic characteristics of the strains; dynamic time-sharing fermentation regulation and control is combined with dynamic regulation and control of material supplementation, stirring rate and fermentation temperature, so that the yield of arctigenin is increased. According to the method for preparing arctigenin through dynamic regulation and control fermentation, the conversion efficiency of arctigenin is improved to the maximum extent, the waste rate is reduced, and energy consumption and production cost are effectively saved.

Description

Technical Field

[0001] The invention belongs to the technical field of natural active ingredient extraction, and in particular relates to a method for preparing arctiin by dynamically regulating fermentation. Background Art

[0002] Arctigenin ((-)-Arctigenin), the molecular formula is , with a molecular weight of 372.41. Burdock seeds (Actium lappa) are the dried, mature fruit of the herbaceous plant Arctium lappa, a member of the Asteraceae family. They are widely documented in ancient Chinese medical texts for their efficacy in dispelling wind-heat, clearing the lungs and clearing rashes, and reducing swelling and detoxifying. Clinically, they are primarily used to treat acute sore throats, wind-heat colds, throat-related coughs, and rashes. The active ingredients in burdock seeds that exert their medicinal effects are the lignin compounds arctiin and arctigenin. Arctiin must first be metabolized into arctigenin before it can be absorbed by the body. Therefore, arctigenin is an active ingredient that can be directly absorbed by the body.

[0003] The prior art discloses a method for extracting arctiin using water as a solvent and then performing enzymatic hydrolysis with burdock's own hydrolase to extract arctiin. However, due to the poor water solubility of arctiin, the solution concentration during enzymatic hydrolysis is low, resulting in high industrial production costs and unsuitable for the extraction of arctiin. Summary of the Invention

[0004] The present invention aims to solve the technical problem of poor extraction efficiency of arctiin in the prior art and provides a method for preparing arctiin by dynamically controlling fermentation.

[0005] One of the purposes of the present invention is to provide a method for preparing arctigenin by dynamic controlled fermentation, the method comprising the following steps: Using burdock powder as a substrate, the activated bacteria were dynamically regulated and fermented to obtain a fermentation liquid containing arctigenin; The dynamic control of fermentation includes: dynamic time-sharing control of fermentation, dynamic control of feeding rate, stirring rate and fermentation temperature; The time-sharing fermentation control steps include: supplementing a sugar source at 0-12 hours of fermentation; adding 0.08% magnesium sulfate and 0.05% calcium chloride at 12-24 hours of fermentation, and supplementing ferrous sulfate according to the real-time enzyme activity; and gradually increasing the concentration of the phosphate buffer at 24-36 hours of fermentation to obtain a fermentation broth; The step of dynamically controlling the feeding rate is as follows: when the pH value of the fermentation broth is lower than 5.8, the feeding rate is increased from the initial 2 mL / min to ≤15 mL / min; when the pH value of the fermentation broth is higher than 6.2, the feeding rate is reduced to ≥2 mL / min; The step of dynamically controlling the stirring rate is as follows: when the dissolved oxygen value is lower than 30% during the fermentation process, adjusting the stirring rate from the initial 200 rpm to 600 rpm; The step of dynamically controlling the fermentation temperature is as follows: when the arctiin concentration in the fermentation liquid is detected by online HPLC to be cumulatively increased by 5 mg / g, the fermentation temperature is reduced by 1° C. and the fermentation cycle is extended by 10%.

[0006] In a preferred embodiment of the present invention, the bacterial species is any one of Lactobacillus, Bifidobacterium or Bacillus.

[0007] In a preferred embodiment of the present invention, when the bacterial species is Lactobacillus, the preparation method of activated Lactobacillus is: inoculating Lactobacillus into MRS culture medium containing 15% burdock seed powder, pre-inducing in an aerobic environment at 37°C for 2 hours to obtain activated Lactobacillus.

[0008] In a preferred embodiment of the present invention, when the bacterial species is bifidobacterium, the preparation method of activated bifidobacterium is: activating bifidobacteria in stages under an anaerobic environment, initially proliferating with 1% lactose for the first 6 hours, and then inoculating the bifidobacteria into MRS medium containing 15% burdock seed powder and 0.2% cysteine, and pre-inducing in an aerobic environment at 37°C for 2 hours to obtain activated bifidobacteria.

[0009] In a preferred embodiment of the present invention, the composition of the MRS medium is: 10 g peptone, 10 g beef powder, 5 g yeast powder, 20 g glucose, 0.1 g magnesium sulfate, 5 g sodium acetate, 2 g sodium citrate, 2 g dipotassium hydrogen phosphate, 0.05 g manganese sulfate, 1 g Tween 80, pH = 6.2.

[0010] In a preferred embodiment of the present invention, when the bacterial species is Bacillus, the preparation method of activated Bacillus is as follows: after the Bacillus is heat-treated at 80°C to activate the spores, the Bacillus spores are inoculated into LB medium containing 15% burdock powder, and shake-cultured until the bacterial density OD 600 =0.6, and activated Bacillus was obtained.

[0011] In a preferred embodiment of the present invention, the composition of the LB medium is: 10 g tryptone, 5 g yeast extract, 10 g NaCl, pH=7.0.

[0012] In a preferred embodiment of the present invention, the method of supplementing the sugar source is continuous feeding or fractional addition; the continuous feeding refers to the continuous feeding of 10% glucose during the fermentation process of 0-12 hours; the fractional addition refers to the addition of 2% glucose every 2 hours during the fermentation process of 0-12 hours, and the fractional addition is performed until the total sugar concentration is 50 g / L.

[0013] In a preferred embodiment of the present invention, the step of dynamically supplementing ferrous sulfate is: when the real-time enzyme activity test result is lower than 50 U / mL, dynamically supplementing 0.1% ferrous sulfate.

[0014] In a preferred embodiment of the present invention, the step of gradually increasing the concentration of phosphate buffer is: increasing the concentration of phosphate buffer by 0.005 mol / L every 6 hours, and the concentration of phosphate buffer is gradually increased from an initial 0.01 mol / L to 0.03 mol / L.

[0015] The beneficial effects of the present invention are as follows: the present invention provides a method for preparing arctiin by dynamically controlling fermentation, wherein burdock powder is used as a substrate, and activated bacteria are subjected to dynamic controlled fermentation to obtain a fermentation liquid containing arctiin; the dynamic controlled fermentation comprises: dynamic time-sharing controlled fermentation, and dynamic control of feeding, stirring rate and fermentation temperature.

[0016] The present invention realizes dynamic control of fermentation and preparation of arctiin through a multi-path replaceable process; when Lactobacillus is selected as the bacterial species, the polysaccharides and phenolic acids in the burdock powder during the lactobacillus activation process can induce the bacteria to secrete β-glucosidase precursors in advance, laying the foundation for the subsequent enzyme activation stage; when Bifidobacterium is selected as the bacterial species, the reducing property of cysteine ​​is used to maintain the metabolic activity of the bacteria during the activation of the bifidobacterium, forming a redox synergistic effect with the subsequently added ferrous sulfate, thereby reducing the loss of enzyme activity; when Bacillus is selected as the bacterial species, the germination of Bacillus spores activated by high temperature during the Bacillus activation process can efficiently utilize the burdock precursors, and combined with the subsequent phosphate gradient adjustment strategy, the synthesis efficiency of arctiin is significantly improved.

[0017] The present invention provides a dynamic time-sharing controlled fermentation, wherein sugar source supplementation is performed at 0-12 h of fermentation, and by selecting a carbon source adapted to the metabolic characteristics of the bacterial strain, the bacterial biomass is increased by 60% (compared with the treatment group with a single carbon source, the arctiin yield is increased by 20%); 0.08% magnesium sulfate and 0.05% calcium chloride are added at 12-24 h of fermentation to activate glycosidase activity, and ferrous sulfate is added according to the real-time enzyme activity (compared with the treatment group without ferrous sulfate supplementation, the arctiin yield is increased by 29%). / Directly activates enzyme activity, Enzyme inactivation is delayed by stabilizing the redox potential. During fermentation, the osmotic pressure is increased by gradually increasing the phosphate buffer concentration between 24 and 36 hours (compared to the group treated with a constant phosphate buffer concentration, arctiin production increased by 39%), thereby initiating stress metabolic pathways in the bacteria to synthesize aglycones, thereby improving arctiin synthesis efficiency. This invention combines dynamic time-sharing fermentation control with dynamic control of feed addition, stirring rate, and fermentation temperature, ultimately stabilizing arctiin production to 32.72 mg / g, an 8.7-fold increase compared to the traditional static arctiin production process (3.75 mg / g).

[0018] It can be seen that the method for preparing arctiin by dynamically controlling fermentation provided by the present invention maximizes the conversion efficiency of arctiin, reduces the waste rate, and effectively saves energy consumption and production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a comparison chart of the arctigenin yields obtained in Examples 1-3; Figure 2 This is a comparison chart of the arctigenin yields obtained in Example 1, Comparative Examples 1 and 4; Figure 3 This is a comparison chart of the arctigenin yields obtained in Example 1, Comparative Examples 2 and 4; Figure 4 This is a comparison chart of the arctigenin yields obtained in Example 1, Comparative Examples 3 and 4. DETAILED DESCRIPTION

[0020] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant persons can modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0021] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments. The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained from commercial channels by those skilled in the art.

[0022] Example 1: Using burdock powder as a substrate, the activated bacteria are subjected to dynamic controlled fermentation to obtain a fermentation liquid containing arctigenin; the dynamic controlled fermentation includes: dynamic time-sharing controlled fermentation, dynamic control of feeding rate, stirring rate and fermentation temperature; The strain is Lactobacillus, and the preparation method of activated Lactobacillus is as follows: inoculating Lactobacillus into MRS medium (10 g of peptone, 10 g of beef powder, 5 g of yeast powder, 20 g of glucose, 0.1 g of magnesium sulfate, 5 g of sodium acetate, 2 g of sodium citrate, 2 g of dipotassium hydrogen phosphate, 0.05 g of manganese sulfate, 1 g of Tween 80, pH=6.2) containing 15% burdock fruit powder, and pre-inducing in an aerobic environment at 37°C for 2 hours to obtain activated Lactobacillus; (1) The time-sharing fermentation steps are as follows: 10% glucose was continuously added during the fermentation period of 0-12 h; Add 0.08% magnesium sulfate and 0.05% calcium chloride during fermentation for 12-24 hours, and supplement ferrous sulfate according to the real-time enzyme activity. When the real-time enzyme activity test result is lower than 50 U / mL, dynamically supplement 0.1% ferrous sulfate; During the fermentation period of 24-36 h, the concentration of the phosphate buffer solution was increased gradually, with the concentration increasing by 0.005 mol / L every 6 h, from an initial concentration of 0.01 mol / L to 0.03 mol / L, to obtain a fermentation broth; (2) The steps for dynamically controlling the feeding rate are as follows: when the pH value of the fermentation broth is lower than 5.8, the feeding rate is increased from the initial 2 mL / min to ≤15 mL / min; when the pH value of the fermentation broth is higher than 6.2, the feeding rate is reduced to ≥2 mL / min; (3) Dynamically control the stirring rate as follows: when the dissolved oxygen value during the fermentation process is lower than 30%, adjust the stirring rate from the initial 200 rpm to 600 rpm; (4) The steps for dynamically controlling the fermentation temperature are as follows: when the concentration of arctigenin in the fermentation broth increases by 5 mg / g as detected by online HPLC, the fermentation temperature is reduced by 1°C and the fermentation cycle is extended by 10%.

[0023] The yield of arctigenin prepared in this example was 32.72 mg / g.

[0024] Example 2: Using burdock powder as a substrate, the activated bacteria are subjected to dynamic controlled fermentation to obtain a fermentation liquid containing arctigenin; the dynamic controlled fermentation includes: dynamic time-sharing controlled fermentation, dynamic control of feeding rate, stirring rate and fermentation temperature; The strain is bifidobacterium, and the preparation method of activated bifidobacterium is as follows: the bifidobacterium is activated in stages under an anaerobic environment, and initial proliferation is carried out by 1% lactose for the first 6 hours, and then the bifidobacterium is inoculated into MRS medium (10 g of peptone, 10 g of beef powder, 5 g of yeast powder, 20 g of glucose, 0.1 g of magnesium sulfate, 5 g of sodium acetate, 2 g of sodium citrate, 2 g of potassium dihydrogen phosphate, 0.05 g of manganese sulfate, 1 g of Tween 80, pH=6.2) containing 15% burdock seed powder and 0.2% cysteine, and pre-induced in an aerobic environment at 37°C for 2 hours to a bacterial density OD 600 =0.6, to obtain activated bifidobacteria; (1) The time-sharing fermentation steps are as follows: 10% glucose was continuously added during the fermentation period of 0-12 h; Add 0.08% magnesium sulfate and 0.05% calcium chloride during fermentation for 12-24 hours, and supplement ferrous sulfate according to the real-time enzyme activity. When the real-time enzyme activity test result is lower than 50 U / mL, dynamically supplement 0.1% ferrous sulfate; During the fermentation period of 24-36 h, the concentration of the phosphate buffer solution was increased gradually, with the concentration of the phosphate buffer solution increasing by 0.005 mol / L every 6 h, from 0.01 mol / L to 0.03 mol / L, to obtain a fermentation broth; (2) The steps for dynamically controlling the feeding rate are as follows: when the pH value of the fermentation broth is lower than 5.8, the feeding rate is increased from the initial 2 mL / min to ≤15 mL / min; when the pH value of the fermentation broth is higher than 6.2, the feeding rate is reduced to ≥2 mL / min; (3) Dynamically control the stirring rate as follows: when the dissolved oxygen value during the fermentation process is lower than 30%, adjust the stirring rate from the initial 200 rpm to 600 rpm; (4) The steps for dynamically controlling the fermentation temperature are as follows: when the concentration of arctigenin in the fermentation broth increases by 5 mg / g as detected by online HPLC, the fermentation temperature is reduced by 1°C and the fermentation cycle is extended by 10%.

[0025] The yield of arctigenin prepared in this example is 30.63 mg / g.

[0026] Example 3: Using burdock powder as a substrate, the activated bacteria are subjected to dynamic controlled fermentation to obtain a fermentation liquid containing arctigenin; the dynamic controlled fermentation includes: dynamic time-sharing controlled fermentation, dynamic control of feeding rate, stirring rate and fermentation temperature; The strain is Bacillus, and the preparation method of activated Bacillus is as follows: after activating spores by heat treatment at 80°C, the Bacillus spores are inoculated into LB medium (10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, pH=7.0) containing 15% burdock powder, and cultured by shaking until the bacterial density OD 600 =0.6, to obtain activated Bacillus; (1) The time-sharing fermentation steps are as follows: 10% glucose was continuously added during the fermentation period of 0-12 h; Add 0.08% magnesium sulfate and 0.05% calcium chloride during fermentation for 12-24 hours, and supplement ferrous sulfate according to the real-time enzyme activity. When the real-time enzyme activity test result is lower than 50 U / mL, dynamically supplement 0.1% ferrous sulfate; During the fermentation period of 24-36 h, the concentration of the phosphate buffer solution was increased gradually, with the concentration of the phosphate buffer solution increasing by 0.005 mol / L every 6 h, from 0.01 mol / L to 0.03 mol / L, to obtain a fermentation broth; (2) The steps for dynamically controlling the feeding rate are as follows: when the pH value of the fermentation broth is lower than 5.8, the feeding rate is increased from the initial 2 mL / min to ≤15 mL / min; when the pH value of the fermentation broth is higher than 6.2, the feeding rate is reduced to ≥2 mL / min; (3) Dynamically control the stirring rate as follows: when the dissolved oxygen value during the fermentation process is lower than 30%, adjust the stirring rate from the initial 200 rpm to 600 rpm; (4) The steps for dynamically controlling the fermentation temperature are as follows: when the concentration of arctigenin in the fermentation broth increases by 5 mg / g as detected by online HPLC, the fermentation temperature is reduced by 1°C and the fermentation cycle is extended by 10%.

[0027] The yield of arctigenin prepared in this example was 31.26 mg / g.

[0028] Comparative Example 1: The difference between this comparative example and Example 1 is that: (1) a single carbon source is selected in the time-sharing controlled fermentation step. The other steps and parameters are the same as those in Example 1, referred to as single glycogen fermentation.

[0029] The yield of arctigenin prepared in this comparative example was 27.27 mg / g.

[0030] Comparative Example 2: The difference between this comparative example and Example 1 is that: (1) ferrous sulfate was not supplemented in the time-sharing controlled fermentation step. The other steps and parameters were the same as those in Example 1, and in short: ferrous sulfate was not supplemented.

[0031] The yield of arctigenin prepared in this comparative example was 25.36 mg / g.

[0032] Comparative Example 3: The difference between this comparative example and Example 1 is that: (1) the phosphate buffer concentration was not increased gradually in the time-sharing fermentation step. The other steps and parameters were the same as those in Example 1, and the following is abbreviated as: the phosphate buffer concentration was not increased gradually.

[0033] The yield of arctigenin prepared in this comparative example was 23.54 mg / g.

[0034] Comparative Example 4: In this comparative example, a heating reflux apparatus was used to add methanol solution to burdock fruit powder at a material-liquid ratio of 1:10 (g / mL), and the mixture was heated and refluxed for 1.5 h. After the reflux period, the extract was separated by suction filtration using a Büchner funnel and the volume was re-adjusted to 20 mL to obtain a crude extract of arctiin; this is referred to as the raw material group.

[0035] The yield of arctiin in the crude extract of arctiin prepared in this comparative example was 3.75 mg / g.

[0036] Effect experiment: (1) Detection of bacterial biomass The OD values ​​of the bacterial biomass obtained by single glycogen fermentation in Example 1 and Comparative Example 1 were calculated. 600 The results showed that the biomass of the bacteria obtained by single glycogen fermentation was OD 600 =0.9, the bacterial biomass obtained by the method provided by the present invention is OD 600 =1.44; It can be seen that compared with the bacterial biomass obtained by single glycogen fermentation, the method provided by the present invention increases the bacterial biomass by 60%. The present invention is more conducive to the increase of bacterial biomass by selecting a carbon source that adapts to the metabolic characteristics of the bacterial species.

[0037] (2) Detection of arctigenin production The production of arctiin in the fermentation broth containing arctiin obtained in Examples 1-3 was detected by HPLC. Figure 1 As shown, the dynamic controlled fermentation using Lactobacillus, Bifidobacterium or Bacillus can produce arctiin with a higher yield.

[0038] The production of arctiin in the fermentation broth containing arctiin obtained in Example 1 and Comparative Example 1 was detected by HPLC. Figure 2 As shown, compared with the fermentation liquid containing arctiin prepared by single glycogen fermentation (Comparative Example 1), the method provided by the present invention increases the yield of arctiin by 20%.

[0039] The production of arctiin in the fermentation broth containing arctiin obtained in Example 1 and Comparative Example 2 was detected by HPLC. Figure 3 As shown, compared with the fermentation broth containing arctiin prepared without supplementation of ferrous sulfate (Comparative Example 2), the method provided by the present invention increases the yield of arctiin by 29%.

[0040] The production of arctiin in the fermentation broths containing arctiin prepared in Example 1 and Comparative Example 3 was detected by HPLC. Figure 4 As shown, compared with the fermentation broth containing arctiin prepared without gradient increase of phosphate buffer concentration (Comparative Example 3), the method provided by the present invention increases the yield of arctiin by 39%.

[0041] The production of arctiin in the fermentation broths containing arctiin prepared in Example 1 and Comparative Examples 1-4 was detected by HPLC. Figure 2-4 As shown, the yield of arctiin obtained by the traditional static process (Comparative Example 4: raw material group) was only 3.75 mg / g, while the yield of arctiin obtained by Example 1 was 32.72 mg / g. This shows that compared with the traditional static process, the method provided by the present invention increased the yield of arctiin by 8.7 times.

[0042] Any matters not described in detail in this specification are well known to those skilled in the art. Although the present invention has been disclosed above with reference to preferred embodiments, these are not intended to limit the present invention. Anyone skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A method for preparing arctigenin by dynamically controlling fermentation, characterized in that: The method comprises the following steps: Using burdock powder as a substrate, the activated bacteria were dynamically regulated and fermented to obtain a fermentation liquid containing arctigenin; The dynamic control of fermentation includes: dynamic time-sharing control of fermentation, dynamic control of feeding rate, stirring rate and fermentation temperature; The time-sharing fermentation control steps include: supplementing a sugar source at 0-12 hours of fermentation; adding 0.08% magnesium sulfate and 0.05% calcium chloride at 12-24 hours of fermentation, and supplementing ferrous sulfate according to the real-time enzyme activity; and gradually increasing the concentration of the phosphate buffer at 24-36 hours of fermentation to obtain a fermentation broth; The step of dynamically controlling the feeding rate is as follows: when the pH value of the fermentation broth is lower than 5.8, the feeding rate is increased from the initial 2 mL / min to ≤15 mL / min; when the pH value of the fermentation broth is higher than 6.2, the feeding rate is reduced to ≥2 mL / min; The step of dynamically controlling the stirring rate is as follows: when the dissolved oxygen value is lower than 30% during the fermentation process, adjusting the stirring rate from the initial 200 rpm to 600 rpm; The step of dynamically controlling the fermentation temperature is as follows: when the arctiin concentration in the fermentation liquid is detected by online HPLC to be cumulatively increased by 5 mg / g, the fermentation temperature is reduced by 1° C. and the fermentation cycle is extended by 10%.

2. The method according to claim 1, wherein The bacterial species is any one of Lactobacillus, Bifidobacterium or Bacillus.

3. The method according to claim 2, wherein When the bacterial species is Lactobacillus, the preparation method of activated Lactobacillus is: inoculating Lactobacillus into MRS culture medium containing 15% burdock seed powder, pre-inducing in an aerobic environment at 37° C. for 2 hours to obtain activated Lactobacillus.

4. The method according to claim 2, wherein When the bacterial species is bifidobacterium, the preparation method of activated bifidobacterium is as follows: bifidobacterium is activated in stages under an anaerobic environment, initial proliferation is carried out using 1% lactose for the first 6 hours, and then the bifidobacterium is inoculated into an MRS culture medium containing 15% burdock seed powder and 0.2% cysteine, and pre-induced in an aerobic environment at 37°C for 2 hours to obtain activated bifidobacterium.

5. The method according to any one of claims 3 to 4, characterized in that The composition of the MRS culture medium is: 10 g peptone, 10 g beef powder, 5 g yeast powder, 20 g glucose, 0.1 g magnesium sulfate, 5 g sodium acetate, 2 g sodium citrate, 2 g dipotassium hydrogen phosphate, 0.05 g manganese sulfate, 1 g Tween 80, pH=6.

2.

6. The method according to claim 2, characterized in that When the bacterial species is Bacillus, the preparation method of the activated Bacillus is as follows: after the Bacillus is heat-treated at 80°C to activate the spores, the Bacillus spores are inoculated into LB medium containing 15% burdock powder, and shake-cultured until the bacterial density OD 600 =0.6, and activated Bacillus was obtained.

7. The method according to claim 6, wherein The composition of the LB medium is: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, pH=7.

0.

8. The method according to claim 1, wherein The method of supplementing the sugar source is continuous feeding or fractional addition; the continuous feeding refers to the continuous feeding of 10% glucose during the fermentation process of 0-12 hours; the fractional addition refers to the addition of 2% glucose every 2 hours during the fermentation process of 0-12 hours, and the fractional addition is performed until the total sugar concentration is 50 g / L.

9. The method according to claim 1, wherein The step of dynamically supplementing ferrous sulfate is: when the real-time enzyme activity detection result is lower than 50 U / mL, dynamically supplementing 0.1% ferrous sulfate.

10. The method according to claim 1, wherein The step of gradually increasing the concentration of the phosphate buffer is as follows: increasing the concentration of the phosphate buffer by 0.005 mol / L every 6 hours, and the concentration of the phosphate buffer is gradually increased from an initial 0.01 mol / L to 0.03 mol / L.

Images