Preparation method of citric acid

Abstract

The present invention relates to the technical field of microbial fermentation, particularly to a citric acid preparation method, which comprises: S1, dispersing aspergillus niger spores in aspergillus niger mouldy bran in sterile water to obtain an aspergillus niger spore suspension; s2, mixing the aspergillus niger spore suspension with the modified calcium alginate, adding a cross-linking agent solution, and stirring for reaction to form immobilized bacteria particles; s3, adding the fermentation culture medium into a fermentation tank, adjusting the pH value of the fermentation culture medium to 4.5-5.0, adding the prepared immobilized bacterium particles into the fermentation tank, and fermenting at the fermentation temperature of 30-32 DEG C, the dissolved oxygen content of 20-30% and the fermentation period of 38-52 hours to obtain the citric acid. According to the citric acid preparation method provided by the invention, the adsorption capacity of the carrier to aspergillus niger spores is remarkably enhanced, the loss rate of thalli in the fermentation process is reduced, the fermentation time is shortened, and the conversion rate of the citric acid reaches 99% or above.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and in particular to a method for preparing citric acid. Background Art

[0002] Citric acid is an important organic acid, which is widely used in the fields of food, medicine, chemical industry, etc. At present, the industry mainly uses fermentation to produce citric acid, including surface fermentation and deep liquid fermentation. Among them, deep liquid fermentation has become the main method of citric acid production due to its advantages of simple process and high production efficiency. In the deep fermentation process, Aspergillus niger is mainly used as the production strain, and the biosynthesis of citric acid is carried out by free cell fermentation. However, when free cell fermentation is used, Aspergillus niger is prone to form mycelium during the fermentation process, which not only affects the mass transfer efficiency of nutrients and oxygen, but also causes problems such as entanglement of the stirring blades of the fermentation tank, and re-inoculation is required after the fermentation is completed, which increases the production cost.

[0003] Compared with traditional free cell fermentation, immobilized fermentation has gained more and more attention and application in the production of biochemical products because of its obvious advantages. Existing patent CN201210311914.0 discloses a method for producing citric acid using immobilized Aspergillus niger, wherein calcium alginate is used as a carrier to embed immobilized Aspergillus niger cells for producing citric acid. When using immobilized cells to produce citric acid, the optimum sucrose concentration is 120g / L, the optimum temperature is 30°C, the optimum inoculation amount is 10%, and the optimum speed is 200r / min. The effect of immobilized cells on the production of citric acid is more obvious than that of free cells. However, in the above scheme, due to the weak affinity between the surface of calcium alginate and Aspergillus niger, the bacteria are easily lost during the immobilization process, and the carrier is very easy to break during the fermentation process, and the mechanical strength is insufficient, making it difficult to achieve multi-batch cyclic fermentation; in addition, using calcium alginate as an immobilized carrier, its pore structure is not ideal, which affects the mass transfer efficiency during the fermentation process, resulting in a low yield of citric acid. Summary of the invention

[0004] In view of this, the present invention proposes a method for preparing citric acid to solve the problem of low fermentation efficiency of citric acid produced by existing immobilized Aspergillus niger.

[0005] The technical solution of the present invention is achieved as follows: The present invention provides a method for preparing citric acid, comprising the following steps: S1. dispersing the Aspergillus niger spores in the Aspergillus niger bran in sterile water to obtain an Aspergillus niger spore suspension; S2, mixing the Aspergillus niger spore suspension with modified calcium alginate, adding a cross-linking agent solution, and stirring to react to form immobilized bacterial particles; S3. Add the fermentation medium into the fermentation tank, adjust the pH of the fermentation medium to 4.5-5.0, add the prepared immobilized bacteria particles into the fermentation tank for fermentation, the fermentation temperature is 30-32° C., the dissolved oxygen content is 20-30%, the fermentation period is 38-52 hours, and citric acid is obtained.

[0006] The citric acid preparation method provided by the present invention adopts modified calcium alginate as an immobilized carrier, which significantly enhances the carrier's adsorption capacity for Aspergillus niger spores, and the modified carrier has excellent mechanical strength and structural stability, which greatly reduces the bacterial cell loss rate during the fermentation process. In terms of fermentation performance, by accurately controlling the fermentation conditions (pH 4.5-5.0, temperature 30-32°C, dissolved oxygen content 20-30%) and the porous structure design of the modified carrier, the metabolic activity and material transfer efficiency of Aspergillus niger are effectively improved, the fermentation time is shortened, and the citric acid conversion rate is improved. In addition, the present invention also has significant environmental advantages. The immobilized carrier can be reused to reduce waste generation, and the fermentation process has less pollutant emissions, which has good industrial application prospects.

[0007] Based on the above technical solution, preferably, in step S1, the content of Aspergillus niger spores in the Aspergillus niger spore suspension is 3×10 8 -7×10 8 spores / mL.

[0008] On the basis of the above technical solution, preferably, in step S2, the preparation method of modified calcium alginate comprises: S21, dissolving chitosan in water, adding glycidyl trimethyl ammonium chloride, adjusting the pH to 8-10, and the reaction temperature to 40-60° C., and reacting to obtain quaternized chitosan; S22. Dissolve quaternized chitosan in an aqueous acetic acid solution, stir thoroughly until completely dissolved, and obtain a chitosan solution; dissolve calcium alginate in deionized water, heat and stir, and cool to room temperature after it is completely dissolved; while stirring, slowly drop the chitosan solution into the calcium alginate solution, stir for 1 to 2 hours, and adjust the pH to 4.5-5.0 to obtain modified calcium alginate.

[0009] Specifically, firstly, chitosan is quaternized to improve the selective adsorption capacity of the carrier for Aspergillus niger, so that Aspergillus niger spores can be quickly fixed in the early stage of fermentation; then, the composite of quaternized chitosan and calcium alginate not only provides good mechanical strength, but also forms a porous network structure suitable for the growth of Aspergillus niger.

[0010] On the basis of the above technical solution, preferably, the mass ratio of chitosan to glycidyltrimethylammonium chloride is 1:1-3, and the reaction time is 2-4h.

[0011] On the basis of the above technical solution, preferably, the mass percentage of the chitosan solution is 1.0-3.0%, and the mass percentage of the calcium alginate solution is 2.0-4.0%.

[0012] On the basis of the above technical scheme, preferably, in step S2, the volume mass ratio of the Aspergillus niger spore suspension to the modified calcium alginate is 10 mL:2.5 g-3.5 g, and the reaction is stirred for 2-4 hours.

[0013] On the basis of the above technical solution, preferably, in step S2, the cross-linking agent is calcium chloride, and the amount of calcium chloride added is 5-10% of the mass of the modified calcium alginate solution.

[0014] Specifically, calcium chloride is used as a cross-linking agent, and calcium ions are ionically cross-linked with the carboxyl groups on the modified calcium alginate molecular chains to promote the formation of a three-dimensional network structure between the modified calcium alginate molecular chains. At the same time, the presence of quaternary ammonium chitosan enhances the stability of the cross-linked network, forming a dual fixation effect of electrostatic adsorption and physical embedding. The cross-linked structure provides a suitable microenvironment for Aspergillus niger.

[0015] On the basis of the above technical scheme, preferably, in step S3, the composition of the fermentation medium includes: glucose 100-120 g / L, (NH4)2SO4 2-5 g / L, KH2PO4 1-2 g / L, MgSO4·7H2O 0.5 g / L, FeSO4·7H2O 0.05 g / L and bran hydrolyzate, and the fermentation medium is fixed to 1 L with the bran hydrolyzate.

[0016] More preferably, the preparation method of the bran hydrolyzate comprises: weighing 15 g / L of bran, adding 800 mL of water, boiling for 1 hour, filtering to obtain the filtrate, and then making up the volume to 1 L with water.

[0017] Based on the above technical solution, preferably, in step S3, the amount of immobilized bacterial particles added is 10-20 g per liter of fermentation medium.

[0018] On the basis of the above technical solution, preferably, step S3 specifically includes: after the first batch of fermentation is completed, the fermentation liquid is discharged, the immobilized bacterial particles are retained in the fermentation tank, fresh fermentation medium is added, and then the next batch of fermentation is carried out.

[0019] The present invention provides citric acid prepared by any one of the above preparation methods.

[0020] The preparation method of citric acid of the present invention has the following beneficial effects compared with the prior art: (1) The citric acid preparation method provided by the present invention adopts modified calcium alginate as an immobilized carrier, which significantly enhances the carrier's adsorption capacity for Aspergillus niger spores, and the modified carrier has excellent mechanical strength and structural stability, which reduces the bacterial cell loss rate during the fermentation process; at the same time, the porous structure of the immobilized carrier effectively improves the metabolic activity and material transfer efficiency of Aspergillus niger, shortens the fermentation time, and the citric acid conversion rate reaches more than 99%. The method is simple to operate, and the immobilized bacterial particles can be reused for 5-8 batches, which significantly reduces the production cost; (2) The present invention introduces a stable positively charged quaternary ammonium group into the molecular chain by quaternizing chitosan, so that the modified carrier can produce a strong electrostatic adsorption effect with the cell wall of Aspergillus niger, thereby promoting the rapid fixation of Aspergillus niger spores in the early stage of fermentation; at the same time, the composite of quaternized chitosan and calcium alginate not only provides good mechanical strength, but also forms a porous network structure, which can provide sufficient space for the growth of Aspergillus niger and ensure the rapid transfer of substrates such as glucose and products such as citric acid. DETAILED DESCRIPTION

[0021] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] In the present invention, there is no particular limitation on the specific type of Aspergillus niger, and the Aspergillus niger species commonly used in the art for fermentation to produce citric acid may be used.

[0023] Example 1 This embodiment provides a method for preparing citric acid, comprising the following steps: S1. Disperse the Aspergillus niger spores in the Aspergillus niger bran in sterile water to make a spore concentration of 5×10 8 A suspension of Aspergillus niger spores with spores / mL; S2, 10mL of Aspergillus niger spore suspension was mixed with 30g of modified calcium alginate to obtain a mixed solution, 3.0g of calcium chloride was prepared into a calcium chloride solution with deionized water to make the concentration of the calcium chloride solution be 2.5%, the mixed solution was added dropwise to the calcium chloride solution, stirred and reacted at 35°C for 4h, filtered, washed and dried to obtain immobilized bacterial particles; S3, add 1L fermentation medium into the fermentation tank, wherein the components of the fermentation medium are glucose 110 g / L, (NH4)2SO4 3.5 g / L, KH2PO4 1.5 g / L, MgSO4·7H2O 0.5 g / L, FeSO4·7H2O 0.05 g / L and bran hydrolyzate (weigh 15g / L bran, add 800 mL water, boil for 1h, filter to obtain the filtrate, and then adjust the volume to 1L with water), and adjust the volume of the fermentation medium to 1L with bran hydrolyzate. Adjust the pH of the fermentation medium to 4.5-5.0, add 15g of the prepared immobilized bacteria particles into the fermentation tank for fermentation, the fermentation temperature is 30℃, the stirring speed can be initially set to 150 rpm, the dissolved oxygen content is 25%, and the fermentation time is 45h to obtain citric acid.

[0024] Wherein, the preparation method of modified calcium alginate comprises: S21, dissolving 10g chitosan in water, adding 20g glycidyl trimethylammonium chloride, adjusting the pH to 8-10, the reaction temperature to 50°C, the reaction time to 3h, to obtain quaternized chitosan; S22. Dissolve the quaternized chitosan in 200 mL of 2% acetic acid aqueous solution, stir thoroughly until completely dissolved, and obtain a chitosan solution with a mass percentage of 2.0%; dissolve calcium alginate in 300 mL of deionized water, heat and stir, and cool to room temperature after it is completely dissolved to obtain a calcium alginate solution with a mass percentage of 3.0%; while stirring, slowly add the chitosan solution dropwise to the calcium alginate solution, stir for 1.5 hours, adjust the pH to 4.5-5.0, and obtain modified calcium alginate.

[0025] Example 2 S1. Disperse the Aspergillus niger spores in the Aspergillus niger bran in sterile water to make a spore concentration of 3×10 8 A suspension of Aspergillus niger spores with spores / mL; S2, 10mL of Aspergillus niger spore suspension was mixed with 25g of modified calcium alginate to obtain a mixed solution, 1.25g of calcium chloride was prepared into a calcium chloride solution with deionized water to make the concentration of the calcium chloride solution 2%, and the mixed solution was added dropwise to the calcium chloride solution, stirred and reacted at 30°C for 6h, filtered, washed and dried to obtain immobilized bacterial particles; S3. Add 1L of fermentation medium into the fermentation tank, wherein the components of the fermentation medium are glucose 100 g / L, (NH4)2SO4 2 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.5 g / L, FeSO4·7H2O 0.05 g / L and bran hydrolyzate (weigh 15g / L of bran, add 800 mL of water, boil for 1h, filter to obtain the filtrate, and then adjust the volume to 1L with water), and adjust the volume of the fermentation medium to 1L with bran hydrolyzate. Adjust the pH of the fermentation medium to 4.5-5.0, add 10g of the prepared immobilized bacteria particles into the fermentation tank for fermentation, the fermentation temperature is 30℃, the stirring speed can be initially set to 100 rpm, the dissolved oxygen content is 20%, and the fermentation time is 52h to obtain citric acid.

[0026] Wherein, the preparation method of modified calcium alginate comprises: S21, dissolving 10g of chitosan in water, adding 10g of epoxypropyltrimethylammonium chloride, adjusting the pH to 8-10, the reaction temperature to 40°C, the reaction time to 6h, to obtain quaternized chitosan; S22. Dissolve the quaternized chitosan in 100 mL of 2% acetic acid aqueous solution, stir thoroughly until completely dissolved, and obtain a chitosan solution with a mass percentage of 1.0%; dissolve calcium alginate in 200 mL of deionized water, heat and stir, and cool to room temperature after it is completely dissolved to obtain a calcium alginate solution with a mass percentage of 2.0%; while stirring, slowly add the chitosan solution dropwise to the calcium alginate solution, stir for 1 hour, adjust the pH to 4.5-5.0, and obtain modified calcium alginate.

[0027] Example 3 S1. Disperse the Aspergillus niger spores in the Aspergillus niger bran in sterile water to make a spore concentration of 7×10 8 A suspension of Aspergillus niger spores with spores / mL; S2, 10mL of Aspergillus niger spore suspension was mixed with 35g of modified calcium alginate to obtain a mixed solution, 3.5g of calcium chloride was prepared into a calcium chloride solution with deionized water to make the concentration of the calcium chloride solution 3%, and the mixed solution was added dropwise to the calcium chloride solution, stirred and reacted at 40°C for 2h, filtered, washed and dried to obtain immobilized bacterial particles; S3, add 1L fermentation medium into the fermentation tank, wherein the components of the fermentation medium are glucose 120 g / L, (NH4)2SO4 5 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.5 g / L, FeSO4·7H2O 0.05 g / L and bran hydrolyzate (weigh 15g / L bran, add 800 mL water, boil for 1h, filter to obtain the filtrate, and then adjust the volume to 1L with water), and adjust the volume of the fermentation medium to 1L with bran hydrolyzate. Adjust the pH of the fermentation medium to 4.5-5.0, add 20g of the prepared immobilized bacteria particles into the fermentation tank for fermentation, the fermentation temperature is 32°C, the stirring speed can be initially set to 200 rpm, the dissolved oxygen content is 30%, and the fermentation time is 38h to obtain citric acid.

[0028] Wherein, the preparation method of modified calcium alginate comprises: S21, dissolving 10g chitosan in water, adding 30g glycidyl trimethylammonium chloride, adjusting the pH to 8-10, the reaction temperature to 60°C, the reaction time to 2h, to obtain quaternized chitosan; S22. Dissolve quaternized chitosan in 300 mL of 2% acetic acid aqueous solution, stir thoroughly until completely dissolved, and obtain a chitosan solution with a mass percentage of 3.0%; dissolve calcium alginate in 400 mL of deionized water, heat and stir, and cool to room temperature after it is completely dissolved to obtain a calcium alginate solution with a mass percentage of 4.0%; while stirring, slowly add the chitosan solution dropwise to the calcium alginate solution, stir for 2 hours, adjust the pH to 4.5-5.0, and obtain modified calcium alginate.

[0029] Comparative Example 1 This comparative example provides a method for preparing citric acid, comprising the following steps: S1. Disperse the Aspergillus niger spores in the Aspergillus niger bran in sterile water to make a spore concentration of 5×10 8 A suspension of Aspergillus niger spores with spores / mL; S2, 10mL of Aspergillus niger spore suspension was mixed with 30g of calcium alginate to obtain a mixed solution, 3.0g of calcium chloride was prepared into a calcium chloride solution with deionized water to make the concentration of the calcium chloride solution be 2.5%, the mixed solution was added dropwise to the calcium chloride solution, stirred and mixed at 35°C for 4h, filtered, washed and dried to obtain immobilized bacterial particles; S3, add 1L fermentation medium into the fermentation tank, wherein the components of the fermentation medium are glucose 110 g / L, (NH4)2SO4 3.5 g / L, KH2PO4 1.5 g / L, MgSO4·7H2O 0.5 g / L, FeSO4·7H2O 0.05 g / L and bran hydrolyzate (weigh 15g / L bran, add 800 mL water, boil for 1h, filter to obtain the filtrate, and then adjust the volume to 1L with water), and adjust the volume of the fermentation medium to 1L with bran hydrolyzate. Adjust the pH of the fermentation medium to 4.5-5.0, add 15g of the prepared immobilized bacteria particles into the fermentation tank for fermentation, the fermentation temperature is 30-32℃, the stirring speed can be initially set to 150 rpm, the dissolved oxygen content is 25%, and the fermentation time is 45h to obtain citric acid.

[0030] Comparative Example 2 This comparative example provides a method for preparing citric acid, comprising the following steps: S1. Disperse the Aspergillus niger spores in the Aspergillus niger bran in sterile water to make a spore concentration of 4×10 8 A suspension of Aspergillus niger spores with spores / mL; S2, 10mL of Aspergillus niger spore suspension was mixed with 30g of modified calcium alginate to obtain a mixed solution, 3.0g of calcium chloride was prepared into a calcium chloride solution with deionized water to make the concentration of the calcium chloride solution be 2.5%, the mixed solution was added dropwise to the calcium chloride solution, stirred and reacted at 35°C for 4h, filtered, washed and dried to obtain immobilized bacterial particles; S3, add 1L fermentation medium into the fermentation tank, wherein the components of the fermentation medium are glucose 110 g / L, (NH4)2SO4 3.5 g / L, KH2PO4 1.5 g / L, MgSO4·7H2O 0.5 g / L, FeSO4·7H2O 0.05 g / L and bran hydrolyzate (weigh 15g / L bran, add 800 mL water, boil for 1h, filter to obtain the filtrate, and then adjust the volume to 1L with water), and adjust the volume of the fermentation medium to 1L with bran hydrolyzate. Adjust the pH of the fermentation medium to 4.5-5.0, add 15g of the prepared immobilized bacteria particles into the fermentation tank for fermentation, the fermentation temperature is 30℃, the stirring speed can be initially set to 150 rpm, the dissolved oxygen content is 25%, and the fermentation time is 45h to obtain citric acid.

[0031] Wherein, the preparation method of modified calcium alginate comprises: Dissolve chitosan in 200 mL of 2% acetic acid aqueous solution, stir thoroughly until completely dissolved, and obtain a chitosan solution with a mass percentage of 2.0%; dissolve calcium alginate in 300 mL of deionized water, heat and stir, and cool to room temperature after it is completely dissolved to obtain a calcium alginate solution with a mass percentage of 3.0%; while stirring, slowly add the chitosan solution dropwise to the calcium alginate solution, stir for 1.5 hours, adjust the pH to 4.5-5.0, and obtain modified calcium alginate.

[0032] Comparative Example 3 This comparative example provides a method for preparing citric acid, comprising the following steps: S1. Disperse the Aspergillus niger spores in the Aspergillus niger bran in sterile water to make a spore concentration of 4×10 8 A suspension of Aspergillus niger spores with spores / mL; S2, mixing 10 mL of Aspergillus niger spore suspension with 30 g of modified calcium alginate to obtain a mixed solution, stirring and reacting at 35° C. for 4 h, filtering, washing, and drying to obtain immobilized bacterial particles; S3, add 1L fermentation medium into the fermentation tank, wherein the components of the fermentation medium are glucose 110 g / L, (NH4)2SO4 3.5 g / L, KH2PO4 1.5 g / L, MgSO4·7H2O 0.5 g / L, FeSO4·7H2O 0.05 g / L and bran hydrolyzate (weigh 15g / L bran, add 800 mL water, boil for 1h, filter to obtain the filtrate, and then adjust the volume to 1L with water), and adjust the volume of the fermentation medium to 1L with bran hydrolyzate. Adjust the pH of the fermentation medium to 4.5-5.0, add 15g of the prepared immobilized bacteria particles into the fermentation tank for fermentation, the fermentation temperature is 30℃, the stirring speed can be initially set to 150rpm, the dissolved oxygen content is 25%, and the fermentation time is 45h to obtain citric acid.

[0033] Wherein, the preparation method of modified calcium alginate comprises: S21, dissolving 10g chitosan in water, adding 20g glycidyl trimethylammonium chloride, adjusting the pH to 8-10, the reaction temperature to 50°C, the reaction time to 3h, to obtain quaternized chitosan; S22. Dissolve the quaternized chitosan in 200 mL of 2% acetic acid aqueous solution, stir thoroughly until completely dissolved, and obtain a chitosan solution with a mass percentage of 2.0%; dissolve calcium alginate in 300 mL of deionized water, heat and stir, and cool to room temperature after it is completely dissolved to obtain a calcium alginate solution with a mass percentage of 3.0%; while stirring, slowly add the chitosan solution dropwise to the calcium alginate solution, stir for 1.5 hours, adjust the pH to 4.5-5.0, and obtain modified calcium alginate.

[0034] Performance Testing All of them adopt fixed continuous fermentation, and the cultivation method of the first batch is the same as that of single batch fermentation. When the mass concentration of sugar in the culture medium is lower than 5g / L, the culture medium is poured out, and then a new culture medium is added for the second batch of fermentation. The fermentation acid production and fermentation conversion rate of the embodiment and the comparative example are detected, wherein the fermentation acid production: the fermentation acid production is represented by the concentration of citric acid in the citric acid fermentation broth, the unit is g / L, the concentration of the citric acid fermentation broth is referred to as acidity, and the acidity parameter is measured with reference to the method of "GB 1886.235-2016 Food Safety National Standard Food Additive Citric Acid"; fermentation conversion rate: citric acid conversion rate (%) = (fermentation acid production × volume of citric acid fermentation broth / weight of total sugar) × 100%. The test results are shown in Table 1.

[0035] Table 1

[0036] As can be seen from Table 1, the preparation method of the present invention can effectively reduce the fermentation cycle of citric acid preparation to only 38-52 hours, and the fermentation conversion rate reaches more than 99%, with high fermentation acid production and fermentation conversion rate; and has good repeatability. In eight batches of continuous fermentation, the fermentation performance did not decrease significantly, and the entire immobilized fermentation system was stable and efficient.

[0037] The above description is only a preferred 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 preparing citric acid, characterized in that: The following steps are involved: S1. dispersing the Aspergillus niger spores in the Aspergillus niger bran in sterile water to obtain an Aspergillus niger spore suspension; S2, mixing the Aspergillus niger spore suspension with modified calcium alginate, adding a crosslinking agent solution, stirring and reacting to form immobilized bacterial particles, wherein the modified calcium alginate is prepared by compounding a quaternary ammonium chitosan solution with a mass percentage of 1.0-3.0% and a calcium alginate solution with a mass percentage of 2.0-4.0%; S3. Add the fermentation medium into the fermentation tank, adjust the pH of the fermentation medium to 4.5-5.0, add the prepared immobilized bacteria particles into the fermentation tank for fermentation, the fermentation temperature is 30-32° C., the dissolved oxygen content is 20-30%, the fermentation period is 38-52 hours, and citric acid is obtained.

2. The method for preparing citric acid according to claim 1, wherein: In step S1, the content of Aspergillus niger spores in the Aspergillus niger spore suspension is 3×10 8 -7×10 8 spores / mL.

3. The method for preparing citric acid according to claim 1, wherein: In step S2, the preparation method of modified calcium alginate includes: S21, dissolving chitosan in water, adding glycidyl trimethyl ammonium chloride, adjusting the pH to 8-10, and the reaction temperature to 40-60° C., and reacting to obtain quaternized chitosan; S22. Dissolve quaternized chitosan in an aqueous acetic acid solution, stir thoroughly until completely dissolved, and obtain a chitosan solution; dissolve calcium alginate in deionized water, heat and stir, and cool to room temperature after it is completely dissolved; while stirring, slowly drop the chitosan solution into the calcium alginate solution, stir for 1 to 2 hours, and adjust the pH to 4.5-5.0 to obtain modified calcium alginate.

4. A method for preparing citric acid as claimed in claim 3, characterized in that: The mass ratio of chitosan to glycidyltrimethylammonium chloride is 1:1-3, and the reaction time is 2-4h.

5. The method for preparing citric acid according to claim 1, wherein: In step S2, the volume mass ratio of the Aspergillus niger spore suspension to the modified calcium alginate is 10 mL:2.5 g-3.5 g, and the reaction is stirred for 2-4 hours.

6. The method for preparing citric acid according to claim 1, wherein: In step S2, the cross-linking agent is calcium chloride, and the amount of calcium chloride added is 5-10% of the mass of the modified calcium alginate solution.

7. The method for preparing citric acid according to claim 1, wherein: In step S3, the composition of the fermentation medium includes: glucose 100-120 g / L, (NH4)2SO4 2-5 g / L, KH2PO4 1-2 g / L, MgSO4·7H2O 0.5 g / L, FeSO4·7H2O 0.05 g / L and bran hydrolyzate, and the fermentation medium is fixed to 1L with the bran hydrolyzate.

8. The method for preparing citric acid according to claim 1, wherein: In step S3, the amount of immobilized bacterial particles added is 10-20 g per liter of fermentation medium.

9. A citric acid prepared by the preparation method according to any one of claims 1 to 8.

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