A method for preparing citric acid

Abstract

The present invention relates to the technical field of microbial fermentation, and particularly relates to a method for preparing citric acid, comprising the following steps: S1, dispersing Aspergillus niger spores in the Aspergillus niger bran koji 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, and stirring and reacting to form immobilized bacteria particles; S3, adding a fermentation medium into a fermenter, adjusting the pH of the fermentation medium to 4.5 - 5.0, adding the prepared immobilized bacteria particles into the fermenter for fermentation, with a fermentation temperature of 30 - 32 °C, a dissolved oxygen content of 20 - 30%, and a fermentation period of 38 - 52 h to obtain citric acid. The method for preparing citric acid provided by the present invention significantly enhances the adsorption capacity of the carrier for Aspergillus niger spores, reduces the cell loss rate during fermentation, shortens the fermentation time, and the citric acid conversion rate reaches over 99%.

Description

Technical Field

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

[0002] Citric acid is an important organic acid and is widely used in the fields of food, medicine, chemical industry, etc. At present, fermentation method is mainly used in industry to produce citric acid, including surface fermentation and deep liquid fermentation. Among them, deep liquid fermentation has become the main method for citric acid production due to its advantages such as 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 through free cell fermentation. However, when using free cell fermentation, Aspergillus niger is prone to form mycelial pellets 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 in the fermenter. Moreover, re-inoculation is required after the fermentation is completed, increasing the production cost.

[0003] Compared with traditional free cell fermentation, immobilized fermentation has received more and more attention and application in the production of biochemical products due to its obvious advantages. The existing patent CN201210311914.0 discloses a method for producing citric acid using immobilized Aspergillus niger. Calcium alginate is used as a carrier to entrap and immobilize Aspergillus niger cells for the production of citric acid. When using immobilized cells to produce citric acid, the optimal sucrose concentration is 120 g / L, the optimal temperature is 30 °C, the optimal inoculation amount is 10%, and the optimal rotation speed is 200 r / min. The influence of immobilized cells on the production of citric acid is more obvious than that of free cells. However, in the above-mentioned scheme, due to the weak affinity between the surface of calcium alginate and Aspergillus niger, the cells are prone to loss during the immobilization process, and the carrier is extremely easy to break during the fermentation process, with insufficient mechanical strength, making it difficult to achieve multi-batch cyclic fermentation. In addition, using calcium alginate as the immobilization carrier, its pore structure is not ideal enough, affecting the mass transfer efficiency during the fermentation process and resulting in a low citric acid yield. 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 in the production of citric acid by immobilized Aspergillus niger.

[0005] The technical solution of the present invention is realized as follows: The present invention provides a method for preparing citric acid, which includes the following steps:

[0006] S1. Disperse the Aspergillus niger spores in the bran koji of Aspergillus niger in sterile water to obtain an Aspergillus niger spore suspension;

[0007] S2. Mix the Aspergillus niger spore suspension with modified calcium alginate, add a cross-linking agent solution, and stir and react to form immobilized bacteria particles;

[0008] S3. Add the fermentation medium into the fermenter, adjust the pH of the fermentation medium to 4.5 - 5.0, add the prepared immobilized bacteria particles into the fermenter for fermentation, with the fermentation temperature being 30 - 32°C, the dissolved oxygen content being 20 - 30%, and the fermentation cycle being 38 - 52 h to obtain citric acid.

[0009] The method for preparing citric acid provided by the present invention uses modified calcium alginate as the immobilization carrier, significantly enhancing the adsorption capacity of the carrier for Aspergillus niger spores. Moreover, the modified carrier has excellent mechanical strength and structural stability, greatly reducing the loss rate of bacteria during the fermentation process. In terms of fermentation performance, by precisely 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 mass transfer efficiency of Aspergillus niger are effectively improved, shortening the fermentation time and increasing the conversion rate of citric acid. In addition, the present invention also has significant environmental protection advantages. The immobilization carrier can be reused, reducing waste generation, and there are few pollutant emissions during the fermentation process, showing good industrial application prospects.

[0010] Based on the above technical solutions, 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.

[0011] Based on the above technical solutions, preferably, in step S2, the preparation method of the modified calcium alginate includes:

[0012] S21. Dissolve chitosan in water, add glycidyltrimethylammonium chloride, adjust the pH to 8 - 10, and react at a temperature of 40 - 60°C to obtain quaternized chitosan;

[0013] S22. Dissolve the quaternized chitosan in an acetic acid aqueous solution, stir well until completely dissolved to obtain a chitosan solution; dissolve calcium alginate in deionized water, heat and stir, and after it is completely dissolved, cool it to room temperature; under stirring, slowly drip the chitosan solution into the calcium alginate solution, stir for 1 - 2 h, and adjust the pH to 4.5 - 5.0 to obtain the modified calcium alginate.

[0014] Specifically, first, the quaternization modification of chitosan is carried out to improve the selective adsorption capacity of the carrier for Aspergillus niger, and Aspergillus niger spores can be quickly immobilized at the initial stage of fermentation; then the combination 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.

[0015] Based on the above technical solutions, preferably, the mass ratio of chitosan to glycidyltrimethylammonium chloride is 1:1 - 3, and the reaction time is 2 - 4 h.

[0016] Based on the above technical solutions, 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%.

[0017] Based on the above technical solutions, 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 stirring reaction is carried out for 2 - 4 h.

[0018] Based on the above technical solutions, preferably, in step S2, the cross - linker is calcium chloride, and the addition amount of calcium chloride is 5 - 10% of the mass of the modified calcium alginate solution.

[0019] Specifically, calcium chloride is used as the cross - linker. Through the ionic cross - linking of calcium ions with the carboxyl groups on the molecular chain of the modified calcium alginate, a three - dimensional network structure is formed between the molecular chains of the modified calcium alginate. At the same time, the presence of quaternized chitosan enhances the stability of the cross - linked network, forming a dual immobilization effect of electrostatic adsorption and physical entrapment. The cross - linked structure provides a suitable micro - environment for Aspergillus niger.

[0020] Based on the above technical solutions, 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 hydrolysate, and the bran hydrolysate is used to make the volume of the fermentation medium up to 1 L.

[0021] More preferably, the preparation method of the bran hydrolysate includes: weighing 15 g / L of bran, adding 800 mL of water, boiling for 1 h, filtering to obtain the filtrate, and then making the volume up to 1 L with water.

[0022] Based on the above technical solutions, preferably, in step S3, the addition amount of the immobilized bacteria particles is 10 - 20 g per liter of the fermentation medium.

[0023] Based on the above technical solutions, preferably, step S3 specifically includes: after the first batch of fermentation is completed, the fermentation broth is discharged, the immobilized bacteria particles are retained in the fermenter, fresh fermentation medium is supplemented, and then the next batch of fermentation is carried out.

[0024] The present invention provides a citric acid prepared by the preparation method described in any one of the above.

[0025] The preparation method of the citric acid of the present invention has the following beneficial effects compared with the prior art:

[0026] (1) The citric acid preparation method provided by the present invention uses modified calcium alginate as an immobilization carrier, significantly enhancing the adsorption capacity of the carrier for Aspergillus niger spores. Moreover, the modified carrier has excellent mechanical strength and structural stability, reducing the cell loss rate during fermentation. At the same time, the porous structure of the immobilization carrier effectively improves the metabolic activity and mass transfer efficiency of Aspergillus niger, shortening the fermentation time, and the citric acid conversion rate reaches over 99%. This method has simple process operation, and the immobilized bacteria particles can be reused 5 - 8 batches, significantly reducing the production cost.

[0027] (2) The present invention quaternizes and modifies chitosan, introducing stable positive charge quaternary ammonium groups on the molecular chain, enabling the modified carrier to have a strong electrostatic adsorption effect with the cell wall of Aspergillus niger, promoting the rapid immobilization of Aspergillus niger spores at the initial stage of fermentation. At the same time, the combination of quaternized chitosan and calcium alginate not only provides good mechanical strength but also forms a porous network structure. This porous structure can not only provide sufficient space for the growth of Aspergillus niger but also ensure the rapid transfer of substrates such as glucose and products such as citric acid. Detailed implementation mode

[0028] Next, in combination with the implementation modes of the present invention, the technical solutions in the implementation modes of the present invention will be clearly and completely described. Obviously, the described implementation modes are only a part of the implementation modes of the present invention, rather than all of them. Based on the implementation modes in the present invention, all other implementation modes obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0029] In the present invention, there is no special limitation on the specific type of Aspergillus niger, and it can be a common Aspergillus niger strain used for fermenting citric acid in the art.

[0030] Example 1

[0031] This example provides a method for preparing citric acid, including the following steps:

[0032] S1. Disperse the Aspergillus niger spores in the Aspergillus niger bran koji in sterile water to make an Aspergillus niger spore suspension with a spore concentration of 5×10 8 spores / mL.

[0033] S2. Mix 10 mL of the Aspergillus niger spore suspension with 30 g of modified calcium alginate to obtain a mixed solution. Take 3.0 g of calcium chloride and prepare it into a calcium chloride solution with deionized water so that the concentration of the calcium chloride solution is 2.5%. Drop the mixed solution into the calcium chloride solution, stir and react at 35°C for 4 h, and obtain immobilized bacteria particles through filtration, washing, and drying.

[0034] 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.

[0035] Wherein, the preparation method of modified calcium alginate comprises:

[0036] 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;

[0037] 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.

[0038] Example 2

[0039] 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;

[0040] 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;

[0041] 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.

[0042] Wherein, the preparation method of modified calcium alginate comprises:

[0043] 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;

[0044] 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.

[0045] Example 3

[0046] 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;

[0047] 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;

[0048] 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.

[0049] Wherein, the preparation method of modified calcium alginate comprises:

[0050] 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;

[0051] 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.

[0052] Comparative Example 1

[0053] This comparative example provides a method for preparing citric acid, comprising the following steps:

[0054] 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;

[0055] 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;

[0056] S3. Add 1 L of the fermentation medium into the fermenter. The components of the fermentation medium are glucose at 110 g / L, (NH4)2SO4 at 3.5 g / L, KH2PO4 at 1.5 g / L, MgSO4·7H2O at 0.5 g / L, FeSO4·7H2O at 0.05 g / L, and bran hydrolysate (weigh 15 g / L of bran, add 800 mL of water, boil for 1 h, filter to obtain the filtrate, and make up the volume to 1 L with water). Use the bran hydrolysate to make up the volume of the fermentation medium to 1 L. Adjust the pH of the fermentation medium to 4.5 - 5.0, add 15 g of the prepared immobilized bacteria particles into the fermenter for fermentation. The fermentation temperature is 30 - 32 °C, the stirring speed can be initially set at 150 rpm, the dissolved oxygen content is 25%, and the fermentation time is 45 h to obtain citric acid.

[0057] Comparative Example 2

[0058] This comparative example provides a method for preparing citric acid, including the following steps:

[0059] S1. Disperse the Aspergillus niger spores in the bran koji of Aspergillus niger in sterile water to make an Aspergillus niger spore suspension with a spore concentration of 4×10 8 spores / mL.

[0060] S2. Mix 10 mL of the Aspergillus niger spore suspension with 30 g of modified calcium alginate to obtain a mixture. Take 3.0 g of calcium chloride and prepare it into a calcium chloride solution with deionized water so that the concentration of the calcium chloride solution is 2.5%. Drop the mixture into the calcium chloride solution, stir and react at 35 °C for 4 h, and after filtration, washing, and drying, obtain the immobilized bacteria particles.

[0061] S3. Add 1 L of the fermentation medium into the fermenter. The components of the fermentation medium are glucose at 110 g / L, (NH4)2SO4 at 3.5 g / L, KH2PO4 at 1.5 g / L, MgSO4·7H2O at 0.5 g / L, FeSO4·7H2O at 0.05 g / L, and bran hydrolysate (weigh 15 g / L of bran, add 800 mL of water, boil for 1 h, filter to obtain the filtrate, and make up the volume to 1 L with water). Use the bran hydrolysate to make up the volume of the fermentation medium to 1 L. Adjust the pH of the fermentation medium to 4.5 - 5.0, add 15 g of the prepared immobilized bacteria particles into the fermenter for fermentation. The fermentation temperature is 30 °C, the stirring speed can be initially set at 150 rpm, the dissolved oxygen content is 25%, and the fermentation time is 45 h to obtain citric acid.

[0062] Among them, the preparation method of the modified calcium alginate includes:

[0063] Chitosan was dissolved in 200 mL of 2% aqueous acetic acid solution and stirred thoroughly until completely dissolved to obtain a chitosan solution with a mass percentage of 2.0%. Calcium alginate was dissolved in 300 mL of deionized water, heated and stirred. After it was completely dissolved, the temperature was lowered to room temperature to obtain a calcium alginate solution with a mass percentage of 3.0%. Under stirring, the chitosan solution was slowly added dropwise to the calcium alginate solution, stirred for 1.5 h, and the pH was adjusted to 4.5 - 5.0 to obtain modified calcium alginate.

[0064] Comparative Example 3

[0065] This comparative example provides a preparation method of citric acid, including the following steps:

[0066] S1. Aspergillus niger spores in Aspergillus niger bran koji were dispersed in sterile water to prepare an Aspergillus niger spore suspension with a spore concentration of 4×10 8 spores / mL.

[0067] S2. 10 mL of the Aspergillus niger spore suspension was mixed with 30 g of modified calcium alginate to obtain a mixed solution, which was stirred and reacted at 35°C for 4 h, and then filtered, washed, and dried to obtain immobilized bacteria particles.

[0068] S3. 1 L of fermentation medium was added to a fermenter. The components of the fermentation medium were 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 hydrolysate (15 g / L of bran was weighed, 800 mL of water was added, boiled for 1 h, filtered to obtain the filtrate, and the volume was made up to 1 L with water). The fermentation medium was made up to 1 L with the bran hydrolysate. The pH of the fermentation medium was adjusted to 4.5 - 5.0, and the prepared 15 g of immobilized bacteria particles were added to the fermenter for fermentation. The fermentation temperature was 30°C, the stirring speed was initially set at 150 rpm, the dissolved oxygen content was 25%, and the fermentation time was 45 h to obtain citric acid.

[0069] Among them, the preparation method of modified calcium alginate includes:

[0070] S21. 10 g of chitosan was dissolved in water, 20 g of glycidyltrimethylammonium chloride was added, the pH was adjusted to 8 - 10, the reaction temperature was 50°C, and the reaction time was 3 h to obtain quaternized chitosan.

[0071] S22. Dissolve quaternized chitosan in 200 mL of 2% aqueous acetic acid solution, and stir thoroughly until completely dissolved to obtain a chitosan solution with a mass percentage of 2.0%. Dissolve calcium alginate in 300 mL of deionized water, heat and stir, and after it is completely dissolved, cool it to room temperature to obtain a calcium alginate solution with a mass percentage of 3.0%. Under stirring, slowly drip the chitosan solution into the calcium alginate solution, stir for 1.5 h, and adjust the pH to 4.5 - 5.0 to obtain modified calcium alginate.

[0072] Performance detection

[0073] All adopt immobilized continuous fermentation, and the cultivation method of the first batch is the same as that of single-batch fermentation. When the sugar mass concentration in the culture medium is lower than 5 g / L, pour out the culture medium, and then supplement it with a new culture medium for the second batch of fermentation. Detect the acid production amount and fermentation conversion rate of the fermentation in the examples and comparative examples, where the acid production amount of fermentation: the acid production amount of fermentation is represented by the concentration of citric acid in the citric acid fermentation broth, with the unit of g / L. The concentration of the citric acid fermentation broth is simply called acidity, and the acidity parameter is measured by the method referred to in "GB 1886.235-2016 National Food Safety Standard Food Additive Citric Acid"; Fermentation conversion rate: The conversion rate of citric acid (%) = (acid production amount of fermentation × volume of citric acid fermentation broth / weight of total sugar) × 100%. The detection results are shown in Table 1.

[0074] Table 1

[0075]

[0076] As can be seen from Table 1, the preparation method of the present invention can effectively reduce the fermentation cycle for citric acid preparation, which only takes 38 - 52 h, and the fermentation conversion rate reaches more than 99%, with a high acid production amount of fermentation and fermentation conversion rate; and it has good repeatability. During the eight-batch continuous fermentation, the fermentation performance does not decrease significantly, and the entire immobilized fermentation system is stable and efficient.

[0077] The above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing citric acid, characterized in that: It includes the following steps: S1. Dispersing Aspergillus niger spores in bran koji of Aspergillus niger 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 and reacting to form immobilized bacteria particles. The modified calcium alginate is prepared by compounding a quaternized 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. Adding a fermentation medium into a fermenter, adjusting the pH of the fermentation medium to 4.5 - 5.0, adding the prepared immobilized bacteria particles into the fermenter for fermentation, with a fermentation temperature of 30 - 32°C, a dissolved oxygen content of 20 - 30%, and a fermentation period of 38 - 52 h to obtain citric acid; In step S2, the cross-linking agent is calcium chloride, and the addition amount of calcium chloride is 5 - 10% of the mass of the modified calcium alginate solution.

2. The preparation method of 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 preparation method of citric acid according to claim 1, characterized in that: In step S2, the preparation method of the modified calcium alginate includes: S21. Dissolving chitosan in water, adding glycidyltrimethylammonium chloride, adjusting the pH to 8 - 10, and reacting at a reaction temperature of 40 - 60°C to obtain quaternized chitosan; S22. Dissolving the quaternized chitosan in an acetic acid aqueous solution, fully stirring until completely dissolved to obtain a chitosan solution; dissolving calcium alginate in deionized water, heating and stirring, and after it is completely dissolved, cooling it to room temperature; under stirring, slowly dropping the chitosan solution into the calcium alginate solution, stirring for 1 - 2 h, and adjusting the pH to 4.5 - 5.0 to obtain the modified calcium alginate.

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

5. The preparation method of citric acid according to claim 1, characterized in that: 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 stirring and reacting for 2 - 4 h.

6. The preparation method of citric acid according to claim 1, characterized in that: 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 hydrolysate, and the fermentation medium is made up to 1 L with the bran hydrolysate.

7. The preparation method of citric acid according to claim 1, characterized in that: In step S3, the addition amount of the immobilized bacteria particles is 10 - 20 g per liter of the fermentation medium.

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