Method for preparing magnesium citrate through fermentation

Abstract

The invention discloses a method for preparing magnesium citrate through fermentation, and belongs to the technical field of fermentation engineering. Aspergillus niger, Aspergillus tubingensis and Aspergillus awamori are mixed in proportion to prepare a mixed seed solution as a composite leavening agent, the composite leavening agent is added into a composite fermentation culture medium containing a shepherd's purse extract, and finally magnesium citrate is prepared. Compared with a method adopting a single strain for fermentation and a method adopting a citric acid fermentation culture medium without the shepherd's purse extract for fermentation, the method has a better effect. According to the method, the synthesis potential of existing strains is fully explored, different growth requirements of various bacteria are controlled and met in the same reactor, all the strains play a synergistic effect, the fermentation period of citric acid is shortened, the yield and conversion rate of citric acid are increased, and the method has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation engineering, and in particular to a method for preparing magnesium citrate by fermentation. Background Art

[0002] Magnesium citrate (Trimagnesium dicitrate), also known as trimagnesium dicitrate, is a white or slightly yellow powder. It is non-toxic and non-corrosive. The magnesium it contains is easily absorbed by the human body, so it is an important source of magnesium nutritional supplements. The common medical use of magnesium citrate is as a saline laxative, which patients take before surgery to help empty the intestines. In addition, it is also used to treat constipation or prevent kidney stones.

[0003] Magnesium citrate is usually prepared from citric acid and magnesium-containing compounds, and the important raw material citric acid is mostly synthesized by fermentation. The patent "CN109207373B A microbial strain with high citric acid production and a method for fermenting starch sugar to produce citric acid" discloses a strain that can use starch sugar to ferment citric acid. The strain is used to ferment sugar raw materials to produce citric acid, and the citric acid yield is improved compared with before the transformation. However, the new strain of Aspergillus niger is constructed by genetic engineering, which poses a biosafety risk. The paper "Research on the process of producing citric acid by mixed fermentation of rice straw" uses a variety of bacteria for mixed fermentation to produce citric acid. After 140h of fermentation, the final citric acid yield is 79 g / L, which is still low and the fermentation cycle is still long. Therefore, selecting suitable strains for combination and providing an appropriate culture environment for production to shorten the citric acid fermentation cycle, improve the conversion rate and yield are key issues that need to be solved in the preparation of magnesium citrate by fermentation. Summary of the invention

[0004] In view of the above prior art, the object of the present invention is to provide a method for preparing magnesium citrate by fermentation.

[0005] To achieve the above object, the present invention adopts the following technical solution: The first aspect of the present invention provides a method for preparing magnesium citrate by fermentation, comprising the following steps: (1) inoculating Aspergillus niger, Aspergillus tubingensis and Aspergillus awamori into a composite fermentation medium and fermenting to obtain a citric acid fermentation broth; (2) purifying the citric acid fermentation broth to obtain citric acid; reacting the citric acid with a magnesium compound to obtain magnesium citrate after purification; The composite fermentation medium comprises the following components in parts by weight: 1-10 parts of shepherd's purse extract, 100-120 parts of starch saccharification liquid, 2-10 parts of (NH4)2SO4, 2-5 parts of NH4Cl, 2-5 parts of KH2PO4, 0.1-1 parts of VB1, 2-10 parts of Tween 80, and 100-150 parts of trace element mixed liquid.

[0006] Furthermore, the magnesium compound includes magnesium carbonate.

[0007] Furthermore, the shepherd's purse extract is prepared by the following method: Fresh shepherd's purse leaves were washed and dried in an oven at 70°C, crushed to 400 mesh and sieved to obtain shepherd's purse leaf powder; the shepherd's purse leaf powder was extracted in 75% v / v ethanol at room temperature with a solid-liquid ratio of 1:2, each extraction time was 12 h, and the extraction was performed 3 times in total; the extracts obtained each time were combined, concentrated under reduced pressure at 50°C, and then dried in an oven at 70°C to obtain shepherd's purse extract.

[0008] Furthermore, the preparation method of starch saccharification liquid is as follows: after the starch raw material is crushed, the slurry is adjusted, and amylase is added to liquefy at 100° C.-120° C. for 40-60 minutes to obtain starch liquefied liquid, the starch liquefied liquid is cooled to 50-60° C., saccharifying enzyme is added, and the starch saccharification liquid is kept warm for 30-40 minutes to obtain starch saccharification liquid; The starchy raw material includes corn.

[0009] Furthermore, the trace element mixed solution includes: 0.6g / L aminoacetic acid, 3.0g / L MgSO4·7H2O, 3.0g / L FeSO4·7H2O, 0.15g / L CoSO4, 0.15g / L CaCl2·2H2O, 0.15g / L ZnSO4·7H2O, and 0.25g / L CuSO4·5H2O.

[0010] Furthermore, in step (1), the Aspergillus niger seed liquid, the Aspergillus tubingensis seed liquid and the Aspergillus awamori seed liquid are mixed in a volume ratio of (0.1-5):(0.1-5):(0.1-5) to obtain a mixed seed liquid, and the mixed seed liquid is inoculated into a composite fermentation medium for fermentation culture.

[0011] Furthermore, the number of viable bacteria in the Aspergillus niger seed solution, Aspergillus tubingensis seed solution or Aspergillus awamori seed solution is ≥ 1×10 6 cfu / mL.

[0012] Furthermore, the inoculation amount of the mixed seed liquid is 15%-20% of the volume of the composite fermentation medium.

[0013] Furthermore, in step (1), the fermentation conditions are as follows: the fermentation temperature is 20-40° C., the dissolved oxygen content is 10-20%, and the fermentation is stopped when the reducing sugar in the composite fermentation medium is ≤4 g / L.

[0014] Furthermore, in step (2), the purification operation is as follows: the citric acid fermentation broth is subjected to membrane sterilization, electrodialysis, decolorization, ion exchange and concentrated crystallization to obtain citric acid.

[0015] Beneficial effects of the present invention: The present invention mixes Aspergillus niger, Aspergillus tubingensis and Aspergillus awamori in proportion to prepare mixed seed liquid as a composite fermentation agent, adds the composite fermentation agent to a composite fermentation medium containing shepherd's purse extract, and finally prepares output magnesium citrate. Aspergillus niger, Aspergillus tubingensis and Aspergillus awamori adopted in the present invention all adopt aerobic fermentation, and there is no antagonism between each strain. When it is used for fermentation production, the acidity of citric acid reaches 30.1%, the conversion rate of citric acid is 98.9%, the fermentation cycle is 47h, and the output is 242g / L, which has better effect than the method of fermenting with a single strain and using a citric acid fermentation medium fermented without adding shepherd's purse extract. The present invention fully taps the synthetic potential of existing strains, controls and satisfies the different growth requirements of multiple bacteria in the same reactor, and each strain plays a synergistic role, shortens the fermentation cycle of citric acid, improves the output and conversion rate of citric acid, and has broad application prospects. DETAILED DESCRIPTION

[0016] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0017] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0018] As described in the background technology, in the prior art, the Aspergillus niger treated with mutagenesis and genetic modification is often used as a fermentation strain to prepare citric acid, but the strain after genetic modification and mutagenesis treatment has a biosafety risk. Meanwhile, although there is an attempt to use different strains to prepare citric acid by fermentation in the prior art, there is still a problem of long fermentation cycle and low citric acid production. In addition, during the fermentation process, microorganisms have a certain dependence on culture medium conditions. In addition to the substances necessary for the survival of microorganisms, the addition of beneficial substances has a significant impact on microbial production speed, fermentation efficiency, etc. Therefore, it is reasonable to select strains and explore suitable culture conditions, further improve the output of citric acid, improve citric acid conversion rate and shorten the fermentation cycle for magnesium citrate manufacturers.

[0019] The experimental materials used in the examples of the present invention without specific description are all conventional experimental materials in the field and can be purchased through commercial channels. Amylase was purchased from Jiangsu Caiwei Biotechnology Co., Ltd.; saccharifying enzyme was purchased from Shandong Fengtai Biotechnology Co., Ltd. The strains used in the present invention: Aspergillus niger ( Aspergillus niger ), deposited in the China Industrial Microbiological Culture Collection Center in Beijing, China, strain number CICC 41178; Aspergillus tubingensis ( Aspergillus tubingensis ), deposited in the China Industrial Microbiological Culture Collection Center in Beijing, China, strain number CICC41176; Aspergillus awamori ( Aspergillus awamori ), deposited in the China Industrial Microbiological Culture Collection Center in Beijing, China, with the strain number CICC 40838. The above strains were purchased through public channels.

[0020] Example 1 (1) Preparation of starch saccharification liquid The corn was crushed to a particle size of 70 mesh, water was added to adjust the slurry so that the concentration of the corn after adjustment was 25wt%, amylase was added, and liquefied at 110°C for 50 minutes to obtain a starchy liquefied liquid, the starchy liquefied liquid was cooled to 55°C, saccharifying enzyme was added, and the temperature was kept for saccharification for 35 minutes to obtain a starchy saccharified liquid, wherein the amount of amylase added was 2% of the mass of the corn, and the amount of saccharifying enzyme added was 3% of the mass of the corn.

[0021] (2) Preparation of Shepherd's Purse Extract Fresh shepherd's purse leaves were washed and dried in an oven at 70°C, crushed to 400 mesh and sieved to obtain shepherd's purse leaf powder. The shepherd's purse leaf powder was extracted in 75% v / v ethanol at room temperature with a solid-liquid ratio of 1:2, each extraction time was 12 hours, and the extraction was performed 3 times in total; the extracts obtained each time were combined, concentrated under reduced pressure at 50°C, and then dried in an oven at 70°C to obtain shepherd's purse extract.

[0022] (3) Preparation of composite fermentation medium Mix 1.5 g shepherd's purse extract, 110 g starch saccharification liquid, 5 g (NH4)2SO4, 3.5 g NH4Cl, 3.5 g KH2PO4, 0.5 g VB1, 5.5 g Tween 80, and 120 mL trace element mixed solution, add pure water to make the volume to 1 L, and obtain a composite fermentation medium.

[0023] Among them, the trace element mixed solution includes: 0.6g / L aminoacetic acid, 3.0g / L MgSO4·7H2O, 3.0g / L FeSO4·7H2O, 0.15g / L CoSO4, 0.15g / L CaCl2·2H2O, 0.15g / L ZnSO4·7H2O, and 0.25g / L CuSO4·5H2O.

[0024] A citric acid fermentation medium was also prepared. The difference between the citric acid fermentation medium and the composite fermentation medium was that the citric acid fermentation medium did not contain shepherd's purse extract.

[0025] Example 2: Preparation of magnesium citrate (1) Preparation of composite fermentation agent The viable count was 6×10 7 cfu / mL of Aspergillus niger seed liquid, Aspergillus tubingensis seed liquid and Aspergillus awamori seed liquid were uniformly mixed in a volume ratio of 1:1:1 to obtain a mixed seed liquid, which was used as a subsequent composite fermentation agent.

[0026] (2) Preparation of citric acid fermentation broth The composite fermentation agent was inoculated into the composite fermentation medium prepared in Example 1. Before addition, the volume of the composite fermentation agent was 18% of the volume of the composite fermentation medium. After mixing evenly, fermentation was carried out at 28°C. The initial ventilation volume of the fermentation was 0.15v / (v·min), the initial stirring speed was 250r / min, the initial tank pressure was 0.15MPa, and the initial dissolved oxygen content was 100%. As the fermentation proceeded, when the dissolved oxygen content was ≤10%, the ventilation volume and stirring speed were adjusted to control the dissolved oxygen content to 10%-20%. When the reducing sugar in the composite fermentation medium was 4g / L, the fermentation was stopped to obtain a citric acid fermentation liquid.

[0027] (3) Fermentation broth treatment and product acquisition The obtained citric acid fermentation liquid is subjected to membrane sterilization, electrodialysis, decolorization, ion exchange and concentrated crystallization treatment to obtain citric acid. The citric acid is dissolved and mixed with magnesium carbonate powder, and magnesium citrate is obtained after purification.

[0028] Example 3: Preparation of magnesium citrate (1) Preparation of composite fermentation agent The viable count was 6×10 7 cfu / mL of Aspergillus niger seed liquid, Aspergillus tubingensis seed liquid and Aspergillus awamori seed liquid were uniformly mixed in a volume ratio of 1:2:1 to obtain a mixed seed liquid, which was used as a subsequent composite fermentation agent.

[0029] (2) Preparation of citric acid fermentation broth The composite fermentation agent was inoculated into the composite fermentation medium prepared in Example 1. Before addition, the volume of the composite fermentation agent was 18% of the volume of the composite fermentation medium. After mixing evenly, fermentation was carried out at 28°C. The initial ventilation volume of the fermentation was 0.15v / (v·min), the initial stirring speed was 250r / min, the initial tank pressure was 0.15MPa, and the initial dissolved oxygen content was 100%. As the fermentation proceeded, when the dissolved oxygen content was ≤10%, the ventilation volume and stirring speed were adjusted to control the dissolved oxygen content to 10%-20%. When the reducing sugar in the composite fermentation medium was 4g / L, the fermentation was stopped to obtain a citric acid fermentation liquid.

[0030] (3) Fermentation broth treatment and product acquisition The obtained citric acid fermentation liquid is subjected to membrane sterilization, electrodialysis, decolorization, ion exchange and concentrated crystallization treatment to obtain citric acid. The citric acid is dissolved and mixed with magnesium carbonate powder, and magnesium citrate is obtained after purification.

[0031] Example 4: Preparation of magnesium citrate (1) Preparation of composite fermentation agent The viable count was 6×10 7 cfu / mL of Aspergillus niger seed liquid, Aspergillus tubingensis seed liquid and Aspergillus awamori seed liquid were uniformly mixed in a volume ratio of 1:1:2 to obtain a mixed seed liquid, which was used as a subsequent composite fermentation agent.

[0032] (2) Preparation of citric acid fermentation broth The composite fermentation agent was inoculated into the composite fermentation medium prepared in Example 1. Before addition, the volume of the composite fermentation agent was 18% of the volume of the composite fermentation medium. After mixing evenly, fermentation was carried out at 28°C. The initial ventilation volume of the fermentation was 0.15v / (v·min), the initial stirring speed was 250r / min, the initial tank pressure was 0.15MPa, and the initial dissolved oxygen content was 100%. As the fermentation proceeded, when the dissolved oxygen content was ≤10%, the ventilation volume and stirring speed were adjusted to control the dissolved oxygen content to 10%-20%. When the reducing sugar in the composite fermentation medium was 4g / L, the fermentation was stopped to obtain a citric acid fermentation liquid.

[0033] (3) Fermentation broth treatment and product acquisition The obtained citric acid fermentation liquid is subjected to membrane sterilization, electrodialysis, decolorization, ion exchange and concentrated crystallization treatment to obtain citric acid. The citric acid is dissolved and mixed with magnesium carbonate powder, and magnesium citrate is obtained after purification.

[0034] Comparative Example 1 The viable count was 6×10 7 cfu / mL of Aspergillus niger seed liquid was used as the subsequent fermentation agent.

[0035] (1) Preparation of citric acid fermentation broth The starter was inoculated into the composite fermentation medium prepared in Example 1. Before addition, the volume of the starter was 18% of the volume of the composite fermentation medium. After mixing evenly, fermentation was carried out at 28°C. The initial ventilation volume of the fermentation was 0.15v / (v·min), the initial stirring speed was 250r / min, the initial tank pressure was 0.15MPa, and the initial dissolved oxygen content was 100%. As the fermentation proceeded, when the dissolved oxygen content was ≤10%, the ventilation volume and stirring speed were adjusted to control the dissolved oxygen content to 10%-20%. When the reducing sugar in the composite fermentation medium was 4g / L, the fermentation was stopped to obtain a citric acid fermentation liquid.

[0036] (2) Fermentation broth treatment and product acquisition The obtained citric acid fermentation liquid is subjected to membrane sterilization, electrodialysis, decolorization, ion exchange and concentrated crystallization treatment to obtain citric acid. The citric acid is dissolved and mixed with magnesium carbonate powder, and magnesium citrate is obtained after purification.

[0037] Comparative Example 2 The viable count was 6×10 7 cfu / mL of Aspergillus tubingensis seed liquid was used as the subsequent fermentation medium.

[0038] (1) Preparation of citric acid fermentation broth The starter was inoculated into the composite fermentation medium prepared in Example 1. Before addition, the volume of the starter was 18% of the volume of the composite fermentation medium. After mixing evenly, fermentation was carried out at 28°C. The initial ventilation volume of the fermentation was 0.15v / (v·min), the initial stirring speed was 250r / min, the initial tank pressure was 0.15MPa, and the initial dissolved oxygen content was 100%. As the fermentation proceeded, when the dissolved oxygen content was ≤10%, the ventilation volume and stirring speed were adjusted to control the dissolved oxygen content to 10%-20%. When the reducing sugar in the composite fermentation medium was 4g / L, the fermentation was stopped to obtain a citric acid fermentation liquid.

[0039] (2) Fermentation broth treatment and product acquisition The obtained citric acid fermentation liquid is subjected to membrane sterilization, electrodialysis, decolorization, ion exchange and concentrated crystallization treatment to obtain citric acid. The citric acid is dissolved and mixed with magnesium carbonate powder, and magnesium citrate is obtained after purification.

[0040] Comparative Example 3 The viable count was 6×10 7 cfu / mL of Aspergillus awamori seed liquid was used as the subsequent fermentation medium.

[0041] (1) Preparation of citric acid fermentation broth The starter was inoculated into the composite fermentation medium prepared in Example 1. Before addition, the volume of the starter was 18% of the volume of the composite fermentation medium. After mixing evenly, fermentation was carried out at 28°C. The initial ventilation volume of the fermentation was 0.15v / (v·min), the initial stirring speed was 250r / min, the initial tank pressure was 0.15MPa, and the initial dissolved oxygen content was 100%. As the fermentation proceeded, when the dissolved oxygen content was ≤10%, the ventilation volume and stirring speed were adjusted to control the dissolved oxygen content to 10%-20%. When the reducing sugar in the composite fermentation medium was 4g / L, the fermentation was stopped to obtain a citric acid fermentation liquid.

[0042] (2) Fermentation broth treatment and product acquisition The obtained citric acid fermentation liquid is subjected to membrane sterilization, electrodialysis, decolorization, ion exchange and concentrated crystallization treatment to obtain citric acid. The citric acid is dissolved and mixed with magnesium carbonate powder, and magnesium citrate is obtained after purification.

[0043] Comparative Example 4 The difference from Example 2 is that in this comparative example 4, the citric acid fermentation medium prepared in Example 1 is used to replace the composite fermentation medium, and the rest of the process is the same.

[0044] Test Example 1 According to "GB 1886.235-2016 Food Safety National Standard Food Additive Citric Acid", the concentration (i.e., acidity) of the citric acid fermentation broth in Example 2-Example 4 and Comparative Example 1-Comparative Example 4 was tested, and the conversion rate of citric acid was calculated; the fermentation cycle and yield of citric acid in Example 2-Example 4 and Comparative Example 1-Comparative Example 4 were statistically analyzed, and the results are shown in Table 1.

[0045] Among them, conversion rate (%) = [(concentration of citric acid fermentation broth × volume of fermentation broth) / weight of total sugar in citric acid fermentation broth] × 100%; the fermentation cycle of citric acid is the time from the start of fermentation to the stop of fermentation; the output of citric acid is the ratio of the mass of citric acid after concentration and crystallization to the volume of citric acid fermentation broth.

[0046] Table 1 Acidity, conversion rate, fermentation cycle and yield of citric acid As can be seen from Table 1, Comparative Examples 1-3 respectively use Aspergillus niger, Aspergillus tubingensis and Aspergillus awamori to ferment and prepare magnesium citrate, wherein the conversion rate of citric acid is 17.6-89.5%, the fermentation cycle is 62-143h, and the output is 19-153g / L. It can be seen that when a single strain is used for fermentation, the output, conversion rate and fermentation cycle of citric acid are not ideal. Its effect is compared with the results of using Aspergillus niger, Aspergillus tubingensis and Aspergillus awamori three strains to be compounded into a composite fermentation agent and then mixed fermentation in Examples 2-4 of the present invention. There is a certain gap; in particular, in Example 1, statistics show that its acidity is 30.1%, the conversion rate of citric acid is 98.9%, the fermentation cycle is 47h, and the output is 242g / L, relative to Comparative Examples 1-3 and Comparative Example 4 using citric acid fermentation medium without adding shepherd's purse extract, all have better effects. It can be seen that the present invention adopts Aspergillus niger, Aspergillus tubingensis and Aspergillus awamori as composite fermentation agents, and synergistically ferments in a composite fermentation medium added with shepherd's purse extract, which can effectively improve the yield and conversion rate of citric acid, shorten the fermentation cycle, and has broad application prospects.

[0047] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing magnesium citrate by fermentation, characterized in that: The following steps are involved: (1) inoculating Aspergillus niger, Aspergillus tubingensis and Aspergillus awamori into a composite fermentation medium and fermenting to obtain a citric acid fermentation broth; (2) purifying the citric acid fermentation broth to obtain citric acid; reacting the citric acid with a magnesium compound to obtain magnesium citrate after purification; The composite fermentation medium comprises the following components in parts by weight: 1-10 parts of shepherd's purse extract, 100-120 parts of starch saccharification liquid, 2-10 parts of (NH4)2SO4, 2-5 parts of NH4Cl, 2-5 parts of KH2PO4, 0.1-1 parts of VB1, 2-10 parts of Tween 80, and 100-150 parts of trace element mixed liquid.

2. The method for preparing citric acid by fermentation according to claim 1, characterized in that: The magnesium compound includes magnesium carbonate.

3. The method for preparing citric acid by fermentation according to claim 1, characterized in that: The shepherd's purse extract is prepared by the following method: Fresh shepherd's purse leaves were washed and dried in an oven at 70°C, crushed to 400 mesh and sieved to obtain shepherd's purse leaf powder; the shepherd's purse leaf powder was extracted in 75% v / v ethanol at room temperature with a solid-liquid ratio of 1:2, each extraction time was 12 h, and the extraction was performed 3 times in total; the extracts obtained each time were combined, concentrated under reduced pressure at 50°C, and then dried in an oven at 70°C to obtain shepherd's purse extract.

4. The method for preparing citric acid by fermentation according to claim 1, characterized in that: The preparation method of starch saccharification liquid is as follows: after the starch raw material is crushed, the slurry is adjusted, and amylase is added to liquefy at 100° C.-120° C. for 40-60 minutes to obtain starch liquefied liquid, and the starch liquefied liquid is cooled to 50-60° C., saccharifying enzyme is added, and the starch saccharification liquid is kept warm for 30-40 minutes to obtain starch saccharification liquid; The starchy raw material includes corn.

5. The method for preparing citric acid by fermentation according to claim 1, characterized in that: The trace element mixed solution includes: 0.6g / L aminoacetic acid, 3.0g / L MgSO4·7H2O, 3.0g / L FeSO4·7H2O, 0.15g / L CoSO4, 0.15g / L CaCl2·2H2O, 0.15g / L ZnSO4·7H2O, and 0.25g / L CuSO4·5H2O.

6. The method for preparing citric acid by fermentation according to claim 1, characterized in that: In step (1), Aspergillus niger seed liquid, Aspergillus tubingensis seed liquid and Aspergillus awamori seed liquid are mixed in a volume ratio of (0.1-5):(0.1-5):(0.1-5) to obtain a mixed seed liquid, and the mixed seed liquid is inoculated into a composite fermentation medium for fermentation culture.

7. The method for preparing citric acid by fermentation according to claim 6, characterized in that: The number of viable bacteria in the Aspergillus niger seed solution, Aspergillus tubingensis seed solution or Aspergillus awamori seed solution is ≥ 1×10 6 cfu / mL.

8. The method for preparing citric acid by fermentation according to claim 6, characterized in that: The inoculation amount of the mixed seed liquid is 15%-20% of the volume of the composite fermentation medium.

9. The method for preparing citric acid by fermentation according to claim 1, characterized in that: In step (1), the fermentation conditions are as follows: the fermentation temperature is 20-40° C., the dissolved oxygen content is 10-20%, and the fermentation is stopped when the reducing sugar in the composite fermentation medium is ≤4 g / L.

10. The method for preparing citric acid by fermentation according to claim 1, characterized in that: In step (2), the purification operation is as follows: the citric acid fermentation broth is subjected to membrane sterilization, electrodialysis, decolorization, ion exchange and concentrated crystallization to obtain citric acid.

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