A method for extracting high-purity vitamin B2

Abstract

The application belongs to the technical field of biological extraction, and discloses an extraction method of high-purity vitamin B2, which comprises the following steps: 1) pretreatment of vitamin B2 fermentation liquor; 2) impurity removal; 3) deodorization and decolorization; 4) ultrafiltration membrane filtration; 5) centrifugal separation; 6) multistage dilution purification; and 7) drying and packaging. The yield of vitamin B2 extracted by the method and the content of the product are high, and the purity of the product reaches more than 98 %.

Description

Technical Field

[0001] This invention belongs to the field of vitamin B2 preparation technology, specifically providing a method for extracting high-purity vitamin B2. Background Technology

[0002] Currently, vitamin B2 extraction methods mainly include chemical synthesis, fermentation, and natural product extraction. While chemical synthesis offers high efficiency, it may introduce harmful substances, affecting product safety. Fermentation, though relatively safe, is complex and costly. Traditional natural product extraction methods, such as solvent extraction, suffer from solvent residues and low extraction rates. Therefore, a new technology is urgently needed that can ensure product quality and safety while improving extraction efficiency and reducing costs.

[0003] As people's awareness of health and nutrition continues to increase, vitamin B2 (riboflavin), as one of the essential B vitamins for the human body, is becoming increasingly important. Vitamin B2 participates in biological oxidation and energy metabolism in the body and is crucial for maintaining the normal function of the skin, eyes, and nervous system. However, traditional vitamin B2 extraction methods suffer from problems such as low efficiency, high cost, and significant environmental impact, making it difficult to meet the growing market demand.

[0004] CN2022112036352 discloses a method for extracting vitamin B2 from a vitamin B2 extraction mother liquor. The specific steps include passing the vitamin B2 extraction mother liquor through a tubular ceramic membrane for pre-clarification, obtaining a clear liquid and a concentrated liquid; further passing the clear liquid through an ultrafiltration membrane to obtain a clarified vitamin B2-containing mother liquor; evaporating and concentrating the obtained vitamin B2-containing mother liquor to precipitate vitamin B2; allowing it to stand and settle to obtain a concentrated vitamin B2-containing liquid; filtering the obtained concentrated vitamin B2 to obtain crude vitamin B2, which is then purified by acid dissolution to obtain the finished riboflavin product. However, the above method has the following problems: the product obtained from concentration and filtration contains a large number of small molecule proteins, polysaccharides, and flavonoids, affecting purity; and the bacteria and culture medium in the fermentation broth can darken the product's color. This invention aims to provide a new, efficient, environmentally friendly, and cost-controllable method for vitamin B2 extraction to improve the production efficiency and purity of vitamin B2. The above-mentioned method, which involves processes such as standing, settling, and filtering the mother liquor, cannot completely filter out impurities and decolorize it, and still suffers from technical problems of low efficiency and poor purity. Summary of the Invention

[0005] The purpose of this invention is to provide a method for extracting high-purity vitamin B2. The method described in this invention can significantly improve the extraction efficiency and purity of vitamin B2.

[0006] The present invention is achieved through the following technical solution.

[0007] A method for extracting high-purity vitamin B2 includes step 1) pretreatment of vitamin B2 fermentation broth, step 2) impurity removal, step 3) deodorization and decolorization, step 4) ultrafiltration membrane filtration, step 5) centrifugation, step 6) multi-stage dilution and purification, and step 7) drying and packaging.

[0008] The specific steps are as follows: Step 1) Pretreatment of vitamin B2 fermentation broth: Add 0.5-2 times the volume fraction of purified water to the vitamin B2 fermentation broth for dilution, heat to 45-60℃, and then pass it through a 50-200nm ceramic membrane. The membrane temperature is controlled at 45℃-60℃ to remove bacteria and large particulate solid impurities, and obtain the pretreated broth. Step 2) Protein removal: Add alkaline solution to the pretreatment solution to adjust the pH to 10-10.5, control the temperature at 30℃-35℃, then centrifuge and filter to remove alkaline proteins. Adjust the pH to 7.5 with acidic solution, add 0.1% by weight of protein hydrolase, and then use the Sevage method to centrifuge again to remove small molecule proteins, and obtain the treatment solution. Step 3) Deodorization and decolorization: Add 0.1-0.2% by weight of adsorbent to the treatment solution. The adsorbent by weight includes: 90 parts activated carbon particles, 5 parts diatomaceous earth and 5 parts adsorption fiber. Stir and adsorb at room temperature for 10-20 minutes, then centrifuge and filter to remove the residue. The slag-removed solution is passed through a resin column a; the adsorbent in the resin column includes, by mass fraction: 80-90 styrene macroporous weakly basic anion exchange resin microspheres and 10-20 adsorption fibers, with a particle size of 0.5-0.1 mm. The mixture is homogeneous, and the flow rate is less than twice the volume of the resin column until the transmittance reaches 55% or more. If the transmittance is less than 55%, the column pass is repeated. The adsorbed solution is passed through resin column b, which uses D-101 non-polar macroporous adsorption resin with a particle size of 0.5-0.1 mm. The flow rate is less than twice the volume of the resin column until the transmittance reaches more than 60%. If the transmittance is less than 60%, the column is passed through again to obtain the initial clear solution. Step 4) Ultrafiltration membrane filtration: Pass the initial clarified liquid obtained in step 3) through an ultrafiltration membrane with a diameter of 20-50 nm, and control the membrane temperature at 60℃-65℃ to remove solid impurities and obtain a clarified liquid. Step 5) Centrifugation: Evaporate and concentrate the clarified liquid obtained in Step 4), then cool it to 1℃-5℃, stir and crystallize for 4-6 hours, centrifuge, collect the crystals, add hydrochloric acid to the remaining solution, adjust the pH to 2.5-3, precipitate the crystals, place them in a centrifuge for centrifugation, mix the crystals twice to obtain the wet product of vitamin B2. Step 6) Multi-stage dilution and purification: Dilute the wet vitamin B2 sample from step 5) with water, centrifuge again to remove some impurities and water, repeat this process 1-5 times to obtain high-purity vitamin B2 crystals; Step 7) Drying and Packaging: Vacuum dry the centrifuged vitamin B2 wet crystals to obtain the finished vitamin B2 product.

[0009] Furthermore, in step 2), the alkaline solution is saturated limewater or sodium hydroxide solution, and the acidic solution is hydrochloric acid.

[0010] Furthermore, in step 3), after resin column a is saturated, it is washed with 0.5% sodium chloride solution with twice the volume of resin column a, then washed with 9% hydrochloric acid solution with 3-5 times the volume of resin column a, and finally rinsed with water until the pH value is neutral to achieve regeneration.

[0011] Furthermore, in step 3), after resin column b is saturated, it is washed with 65% ethanol solution with twice the volume of resin column b, then washed with 5% hydrochloric acid solution with 3-5 times the volume of resin column b, and finally rinsed with water until the pH value is neutral to achieve regeneration.

[0012] Furthermore, in step 3), the styrene macroporous weakly basic anion exchange resin microspheres are formed by aqueous polymerization of styrene and divinylbenzene. During the polymerization process, a dispersant and a pore-forming agent are added. After polymerization, tetraethylenepentamine is added for curing to obtain styrene macroporous weakly basic anion exchange resin microspheres.

[0013] The beneficial effects achieved by this invention mainly include, but are not limited to, the following aspects: This method can significantly improve the extraction efficiency and purity of vitamin B2, with an extraction yield >83% and product purity ≥98%. This method does not add any exogenous materials such as oxidants throughout the entire process, thus reducing wastewater treatment costs and environmental pollution; This method simplifies the extraction process and reduces production costs; This method is applicable to the extraction of vitamin B2 from various microbial fermentation broths and has broad application prospects. Detailed Implementation

[0014] To enable those skilled in the art to better understand the technical solutions in this application, the technical solutions will be fully described below in conjunction with specific embodiments of this application. Example

[0015] A method for extracting high-purity vitamin B2 includes step 1) pretreatment of vitamin B2 fermentation broth, step 2) impurity removal, step 3) deodorization and decolorization, step 4) ultrafiltration membrane filtration, step 5) centrifugation, step 6) multi-stage dilution and purification, and step 7) drying and packaging.

[0016] The specific steps are as follows: Step 1) Pretreatment of vitamin B2 fermentation broth: Add 2 times the volume fraction of purified water to the vitamin B2 fermentation broth for dilution, heat to 60℃, and then pass it through a 200nm ceramic membrane. The membrane temperature is controlled at 60℃ to remove the cells and large particulate solid impurities, and obtain the pretreated broth. Step 2) Impurity removal: Add alkaline solution to the pretreatment solution to adjust the pH to 10.5, control the temperature at 35℃, then centrifuge and filter to remove alkaline proteins. Adjust the pH to 7.5 with acidic solution, add 0.1% by weight of protein hydrolase, and then use the Sevage method to centrifuge again to remove small molecule proteins, and obtain the treatment solution. The alkaline solution is a saturated lime water solution, and the acidic solution is hydrochloric acid; Step 3) Deodorization and decolorization: Add 0.2% by weight of adsorbent to the treatment solution. The adsorbent by weight includes: 90 parts activated carbon particles, 5 parts diatomaceous earth and 5 parts adsorption fiber. Stir and adsorb at room temperature for 10 minutes, then centrifuge and filter to remove the residue. The solution after slag removal is passed through resin column a; the adsorbent in the resin column includes, by mass fraction: 80 styrene macroporous weakly basic anion exchange resin microspheres and 20 adsorption fibers, with a particle size of 0.5 mm. They are mixed evenly, and the flow rate is less than 2 times the volume of the resin column until the transmittance reaches more than 55%. If the transmittance is less than 55%, the column is repeated. The adsorbed solution is passed through resin column b, which uses D-101 non-polar macroporous adsorption resin with a particle size of 1 mm. The flow rate is less than twice the volume of the resin column until the transmittance reaches more than 60%. If the transmittance is less than 60%, the column is passed through again to obtain the initial clear solution. Step 4) Ultrafiltration membrane filtration: Pass the initial clarified liquid obtained in step 3) through a 20nm ultrafiltration membrane, with the membrane temperature controlled at 60℃-65℃, to remove solid impurities and obtain a clarified liquid; Step 5) Centrifugation: The clarified liquid obtained in Step 4) is evaporated and concentrated, then cooled to 1°C, stirred and crystallized for 6 hours, centrifuged, and the crystals are collected. Hydrochloric acid is added to the remaining solution, the pH is adjusted to 2.5, and the crystal precipitate is precipitated. The precipitate is then centrifuged and separated. The crystals are mixed twice to obtain a wet product of vitamin B2. Step 6) Multi-stage dilution and purification: Dilute the wet vitamin B2 sample from step 5) with water, centrifuge again to remove some impurities and water, repeat this process once to obtain high-purity vitamin B2 crystals; Step 7) Drying and Packaging: Vacuum dry the centrifuged vitamin B2 wet crystals to obtain the finished vitamin B2 product.

[0017] After resin column a is saturated, it is washed with 0.5% sodium chloride solution with twice the volume of resin column a, then washed with 9% hydrochloric acid solution with five times the volume of resin column a, and finally rinsed with water until the pH value is neutral to achieve regeneration.

[0018] After resin column b is saturated, wash it with 65% ethanol solution with twice the volume of resin column b, then wash it with 5% hydrochloric acid solution with 3-5 times the volume of resin column b, and finally rinse it with water until the pH value is neutral to achieve regeneration. Example

[0019] A method for extracting high-purity vitamin B2 includes step 1) pretreatment of vitamin B2 fermentation broth, step 2) impurity removal, step 3) deodorization and decolorization, step 4) ultrafiltration membrane filtration, step 5) centrifugation, step 6) multi-stage dilution and purification, and step 7) drying and packaging.

[0020] The specific steps are as follows: Step 1) Pretreatment of vitamin B2 fermentation broth: Add 2 times the volume fraction of purified water to the vitamin B2 fermentation broth for dilution, heat to 60℃, and then pass it through a 200nm ceramic membrane. The membrane temperature is controlled at 60℃ to remove the cells and large particulate solid impurities, and obtain the pretreated broth. Step 2) Impurity removal: Add alkaline solution to the pretreatment solution to adjust the pH to 10.5, control the temperature at 35℃, then centrifuge and filter to remove alkaline proteins. Adjust the pH to 7.5 with acidic solution, add 0.1% by weight of protein hydrolase, and then use the Sevage method to centrifuge again to remove small molecule proteins, and obtain the treatment solution. The alkaline solution is a saturated lime water solution, and the acidic solution is hydrochloric acid; Step 3) Deodorization and decolorization: Add 0.2% by weight of adsorbent to the treatment solution. The adsorbent by weight includes: 90 parts activated carbon particles, 5 parts diatomaceous earth and 5 parts adsorption fiber. Stir and adsorb at room temperature for 20 minutes, then centrifuge and filter to remove the residue. The solution after slag removal is passed through resin column a; the adsorbent in the resin column includes, by mass fraction: 90 styrene macroporous weakly basic anion exchange resin microspheres and 10 adsorption fibers with a particle size of 0.5 mm. The mixture is homogeneous, and the flow rate is less than twice the volume of the resin column until the transmittance reaches 55% or more. If the transmittance is less than 55%, the column is passed through again. The adsorbed solution is passed through resin column b, which uses D-101 non-polar macroporous adsorption resin with a particle size of 0.5 mm. The flow rate is less than twice the volume of the resin column until the transmittance reaches more than 60%. If the transmittance is less than 60%, the column is passed through again to obtain the initial clear solution. Step 4) Ultrafiltration membrane filtration: Pass the initial clarified liquid obtained in step 3) through a 50nm ultrafiltration membrane, with the membrane temperature controlled at 60℃, to remove solid impurities and obtain a clarified liquid; Step 5) Centrifugation: The clarified liquid obtained in Step 4) is evaporated and concentrated, then cooled to 1°C, stirred and crystallized for 6 hours, centrifuged, and the crystals are collected. Hydrochloric acid is added to the remaining solution, the pH is adjusted to 2.5, and the crystal precipitate is precipitated. The precipitate is then centrifuged and separated. The crystals are mixed twice to obtain a wet product of vitamin B2. Step 6) Multi-stage dilution and purification: Dilute the wet vitamin B2 sample from step 5) with water, centrifuge again to remove some impurities and water, repeat this process once to obtain high-purity vitamin B2 crystals; Step 7) Drying and packaging: The centrifuged vitamin B2 wet crystals are vacuum dried at 80°C for 8 hours to obtain the finished vitamin B2 product. Example

[0021] The styrene macroporous weakly basic anion exchange resin microspheres are formed by aqueous polymerization of styrene and divinylbenzene. During the polymerization process, a dispersant and a pore-forming agent are added. After polymerization, tetraethylenepentamine is added for curing to obtain styrene macroporous weakly basic anion exchange resin microspheres. Example

[0022] For the experimental comparison, the experimental group was Example 2, and the control group was the fermentation broth centrifuged filtration, plate and frame filtration and 100nm ceramic membrane filtration, with other conditions unchanged, as shown in Table 1 below.

[0023] Table 1 Fermentation broth quantity Product yield Product purity Flavonoid content Protein content Color experimental group 10L 21.2% 98.6% 0.13% 0.55% pale yellow, bright control group 10L 20.5% 96.5% 0.55% 1.79% Brown, dull Conclusion: As can be clearly seen from Table 1, the product yield in Example 2 increased by 0.7% and the purity increased by 2.1%. Among them, the flavonoids and proteins were significantly reduced, and the color was more vivid. Example

[0024] For the experimental comparison, the experimental group was Example 2, and the control group was formed by replacing the resin in Example 2 with phenolic resin, while other conditions remained unchanged, as shown in Table 2 below.

[0025] Table 2 Fermentation broth quantity Product yield Product purity Flavonoid content Protein content Color experimental group 10L 21.2% 98.6% 0.13% 0.55% pale yellow, bright control group 10L 20.9% 97.7% 0.44% 1.15% Yellowish-brown, dull Conclusion: As can be clearly seen from Table 2, the product yield increased by 0.3% and the purity increased by 0.9% in Example 2. However, the flavonoids and proteins were still significantly reduced, and the color was more vivid.

[0026] Finally, it should be noted that the above examples are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A method for extracting high-purity vitamin B2, comprising: Step 1) Pretreatment of vitamin B2 fermentation broth; Step 2) Impurity removal; Step 3) Deodorization and decolorization; Step 4) Ultrafiltration membrane filtration; Step 5) Centrifugation; Step 6) Multi-stage dilution and purification; Step 7) Drying and packaging.

2. The method for extracting high-purity vitamin B2 according to claim 1, characterized in that, The specific steps of the extraction method are as follows: Step 1) Pretreatment of vitamin B2 fermentation broth: Add 0.5-2 times the volume fraction of purified water to the vitamin B2 fermentation broth for dilution, heat to 45-60℃, and then pass it through a 50-200nm ceramic membrane. The membrane temperature is controlled at 45℃-60℃ to remove bacteria and large particulate solid impurities, and obtain the pretreated broth. Step 2) Protein removal: Add alkaline solution to the pretreatment solution to adjust the pH to 10-10.5, control the temperature at 30℃-35℃, then centrifuge and filter to remove alkaline proteins. Adjust the pH to 7.5 with acidic solution, add 0.1% by weight of protein hydrolase, and then use the Sevage method to centrifuge again to remove small molecule proteins, and obtain the treatment solution. Step 3) Deodorization and decolorization: Add 0.1-0.2% by weight of adsorbent to the treatment solution. The adsorbent by weight includes: 90 parts activated carbon particles, 5 parts diatomaceous earth and 5 parts adsorption fiber. Stir and adsorb at room temperature for 10-20 minutes, then centrifuge and filter to remove the residue. The slag-removed solution is passed through a resin column a; the adsorbent in the resin column includes, by mass fraction: 80-90 styrene macroporous weakly basic anion exchange resin microspheres and 10-20 adsorption fibers, with a particle size of 0.5-0.1 mm. The mixture is homogeneous, and the flow rate is less than twice the volume of the resin column until the transmittance reaches 55% or more. If the transmittance is less than 55%, the column pass is repeated. The adsorbed solution is passed through resin column b, which uses D-101 non-polar macroporous adsorption resin with a particle size of 0.5-1 mm. The flow rate is less than twice the volume of the resin column until the transmittance reaches more than 60%. If the transmittance is less than 60%, the column is passed through again to obtain the initial clear solution. Step 4) Ultrafiltration membrane filtration: Pass the initial clarified liquid obtained in step 3) through an ultrafiltration membrane with a diameter of 20-50 nm, and control the membrane temperature at 60℃-65℃ to remove solid impurities and obtain a clarified liquid. Step 5) Centrifugation: Evaporate and concentrate the clarified liquid obtained in Step 4), then cool it to 1℃-5℃, stir and crystallize for 4-6 hours, centrifuge, collect the crystals, add hydrochloric acid to the remaining solution, adjust the pH to 2.5-3, precipitate the crystals, place them in a centrifuge for centrifugation, mix the crystals twice to obtain the wet product of vitamin B2. Step 6) Multi-stage dilution and purification: Dilute the wet vitamin B2 sample from step 5) with water, centrifuge again to remove some impurities and water, repeat this process 1-5 times to obtain high-purity vitamin B2 crystals; Step 7) Drying and Packaging: Vacuum dry the centrifuged vitamin B2 wet crystals to obtain the finished vitamin B2 product.

3. The method for extracting high-purity vitamin B2 according to claim 2, characterized in that, In step 2), the alkaline solution is saturated limewater or sodium hydroxide solution, and the acidic solution is hydrochloric acid.

4. The method for extracting high-purity vitamin B2 according to claim 2, characterized in that, In step 3), after resin column a is saturated, it is washed with 0.5% sodium chloride solution with twice the volume of resin column a, then washed with 9% hydrochloric acid solution with 3-5 times the volume of resin column a, and finally rinsed with water until the pH value is neutral to achieve regeneration.

5. The method for extracting high-purity vitamin B2 according to claim 2, characterized in that, In step 3), after resin column b is saturated, it is washed with 65% ethanol solution with twice the volume of resin column b, then washed with 5% hydrochloric acid solution with 3-5 times the volume of resin column b, and finally rinsed with water until the pH value is neutral to achieve regeneration.

6. The method for extracting high-purity vitamin B2 according to claim 2, characterized in that, In step 3), the styrene macroporous weakly basic anion exchange resin microspheres are formed by aqueous polymerization of styrene and divinylbenzene. During the polymerization process, a dispersant and a pore-forming agent are added. After polymerization, tetraethylenepentamine is added for curing to obtain styrene macroporous weakly basic anion exchange resin microspheres.