A method for separating and purifying taurine from a microbial fermentation broth

Abstract

The application discloses a method for separating and purifying taurine from microbial fermentation liquor, which comprises the following steps: in the first step, supernatant after fermentation of corynebacterium glutamicum engineering bacteria in a fermentation tank is collected, and after solid-liquid separation, ultrafiltration and activated carbon decolorization treatment are sequentially performed, pretreated liquid is obtained; in the second step, after the PH value of the pretreated liquid is adjusted to 3.0-5.0, the pretreated liquid is loaded to a strong acid cation exchange resin column, the strong acid cation exchange resin column is filled with 732 type hydrogen type strong acid cation exchange resin with a sulfonic acid group as an active group, then deionized water and ammonia water are sequentially used for elution, and eluate is collected in sections; in the third step, taurine crystal is obtained through negative pressure concentration, crystallization and vacuum drying of the eluate collected in the second step and containing taurine. Through the above method, taurine in the fermentation liquor can be efficiently separated through ion exchange chromatography, the pollution of macromolecules in the fermentation liquor to the resin is reduced, and the service life of the resin column is improved.

Description

Technical Field

[0001] This invention relates to the field of taurine preparation, and in particular to a method for separating and purifying taurine from microbial fermentation broth. Background Technology

[0002] Taurine (2-aminoethanesulfonic acid) is a sulfur-containing amino acid widely found in animal tissues. It possesses various physiological functions, including regulating cell osmotic pressure, protecting nerve cells, and promoting lipid metabolism, and is widely used in pharmaceuticals, health foods, functional beverages, and pet food. Currently, taurine production methods mainly include chemical synthesis, natural extraction, and microbial fermentation. Chemical synthesis (such as the ethylene oxide method and the ethanolamine method) is the main industrial production method, but it suffers from problems such as harsh reaction conditions, numerous byproducts, and severe environmental pollution. Natural extraction methods extract taurine from marine organisms such as shellfish and fish, but the raw material sources are limited, extraction costs are high, and large-scale production is difficult. Microbial fermentation has become a research hotspot due to its environmental friendliness and renewable raw materials; however, the fermentation broth has a complex composition, containing impurities such as bacterial cells, high concentrations of inorganic salts, residual culture medium, and pigments. How to efficiently separate and purify taurine from the fermentation broth is a key bottleneck restricting the industrialization of this technology. In existing technologies, ion exchange chromatography has been used to purify taurine from crude extracts of natural products. However, for fermentation broth systems, directly applying traditional ion exchange processes to the fermentation broth can lead to resin contamination by large molecules such as proteins and polysaccharides, reducing resin lifespan. Furthermore, traditional processes do not optimize chromatographic parameters for the characteristics of the fermentation broth, making it difficult to meet industrial requirements in terms of product purity and yield. Therefore, there is a need to develop a highly efficient method for the separation and purification of taurine suitable for microbial fermentation broth systems, which has significant future industrial application value. Summary of the Invention

[0003] The main technical problem solved by this invention is to provide a method for separating and purifying taurine from microbial fermentation broth, which is applicable to ion exchange chromatography and taurine purification in fermentation broth systems, and reduces the contamination of resin columns by macromolecules.

[0004] To solve the above-mentioned technical problems, one technical solution adopted by the present invention is: to provide a method for separating and purifying taurine from microbial fermentation broth, wherein the method for separating and purifying taurine from microbial fermentation broth includes the following steps: Step 1: Pretreatment. The supernatant from the fermentation of engineered Corynebacterium glutamicum in the fermenter is collected and subjected to solid-liquid separation, ultrafiltration, and activated carbon decolorization in sequence to obtain the pretreated liquid. Step 2: Ion exchange chromatography separation. After adjusting the pH of the pretreatment solution to between 3.0 and 5.0, the sample is loaded onto a strong acid cation exchange resin column. The strong acid cation exchange resin column is filled with 732 type hydrogen form strong acid cation exchange resin with sulfonic acid groups as active groups. Then, it is eluted with deionized water and ammonia water in sequence, and the eluent is collected in segments. Step 3: Collecting the taurine-containing eluent collected in Step 2 involves concentrating under negative pressure, crystallizing, and vacuum drying to obtain taurine crystals.

[0005] In a preferred embodiment of the present invention, the solid-liquid separation in step one is carried out by high-speed centrifugation, with a centrifugation speed of 4000~1000 rpm.

[0006] In a preferred embodiment of the present invention, the ultrafiltration in step one uses an ultrafiltration membrane with a molecular weight cutoff of 3000-10000 Da.

[0007] In a preferred embodiment of the present invention, the activated carbon decolorization in step one uses powdered activated carbon, with an addition amount of 0.1-1.0% w / v, a decolorization temperature of 40-60℃, and a decolorization time of 30-60 min.

[0008] In a preferred embodiment of the present invention, the flow rate of sample loading in step two is 0.5-2.0 times column volume / hour, and the resin column is equilibrated with deionized water for 2-3 times column volume before sample loading.

[0009] In a preferred embodiment of the present invention, the washing in step two uses deionized water, the washing volume is 2-3 times the column volume, and the washing flow rate is 1.0-2.0 times the column volume / hour.

[0010] In a preferred embodiment of the present invention, in step two, the elution uses an ammonia solution with a concentration of 0.5-2.0 mol / L, the elution flow rate is 0.5-1.5 times the column volume / hour, and the elution volume is 3-5 times the column volume.

[0011] In a preferred embodiment of the present invention, the concentration process in step three is carried out under negative pressure, with a temperature of 50-70°C and a vacuum degree of -0.08 to -0.095 MPa, until the taurine concentration is 50-200 g / L.

[0012] In a preferred embodiment of the present invention, the crystallization in step three is carried out by ethanol precipitation; the ethanol precipitation method involves adding 1 to 3 times the volume of anhydrous ethanol to the concentrate and allowing it to stand at 4 to 10°C for 12 to 24 hours to crystallize.

[0013] In a preferred embodiment of the present invention, the ion exchange resin in step two is regenerated after use using 2-5% dilute sulfuric acid or dilute hydrochloric acid.

[0014] The beneficial effects of this invention are as follows: The comprehensive pretreatment process effectively removes impurities such as bacteria, proteins, polysaccharides, and pigments from the fermentation broth, significantly reducing the contamination of the resin during ion exchange and thus significantly extending the resin's lifespan. During the elution process, the pH value is optimized for the characteristics of the fermentation broth, and deionized water and ammonia are selected for elution, effectively improving the adsorption and elution rates of taurine. The actual purity of taurine obtained through the above method in a single crystallization can reach over 95%. Based on this, the purity can reach over 99% after recrystallization, directly meeting the requirements for pharmaceutical and food grades. Furthermore, the ethanol and ammonia used in the entire process can be absorbed and recycled in subsequent processes, significantly reducing the pollution from chemical waste generated in traditional production routes and improving environmental compatibility. Detailed Implementation

[0015] The preferred embodiments of the present invention will now be described in detail so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0016] The embodiments of the present invention include: Example 1 The engineered strain of Corynebacterium glutamicum was used for fermentation. After fermentation, HPLC analysis showed that the taurine content in the fermentation broth was 2.7 g / L, and the pH value was 7.1. Based on this fermentation broth, the taurine was separated and purified according to the following steps: Step 1 Preprocessing Fermentation broth from *Corynebacterium glutamicum* engineered bacteria in a 5L fermenter was collected. First, the broth was centrifuged at 6000 rpm for 15 min to remove over 99% of the bacterial cells. The supernatant was then collected and passed through an ultrafiltration membrane with a molecular weight cutoff of 5000 Da to remove over 80% of the protein molecules. The permeate was collected, and powdered activated carbon was added at a ratio of 0.5% w / v. The mixture was stirred at 50°C for 45 min, and then filtered through a 0.45 μm filter membrane to remove the activated carbon adsorbing pigments, resulting in a clear pretreated solution. Finally, the final pretreated solution was obtained. Step 2: Ion exchange chromatography separation First, a 732 type hydrogen-form strong acid cation exchange resin with sulfonic acid groups was wet-packed into a glass column (Φ2.5 cm × 25 cm), with a resin bed volume of approximately 120 mL. After packing, the resin was equilibrated with 3 column volumes of deionized water. Then, the pH of the pretreatment solution was adjusted to 4.0 using 1 mol / L dilute hydrochloric acid. The pretreated solution was then loaded at a flow rate of 1 column volume per hour, for a total loading volume of 4 column volumes. During loading, the eluent was collected to detect taurine breakthrough. After loading, the sample was washed with 3 column volumes of deionized water at a flow rate of 1.5 column volumes per hour. After washing, the sample was eluted with a total of 3 column volumes of 1.0 mol / L ammonia solution at a flow rate of 1 column volume per hour. The eluent was collected, and each 0.5 column volume was used as a fraction. The position of taurine was detected using thin-layer chromatography (TLC), and the taurine-containing fractions were combined. Step 3: Collect the taurine-containing eluent collected in Step 2 and concentrate it under reduced pressure at 60°C and -0.09 until the taurine concentration is close to 100 g / L. Then, add 2 times the volume of anhydrous ethanol to the concentrate, stir well, and let it stand at 4°C for 24 hours to crystallize. Finally, filter and collect the taurine crystals, and wash the crystal surface with a small amount of anhydrous ethanol. After washing, place them in a vacuum drying oven and dry them at 60°C to constant weight.

[0017] The taurine crystals obtained after the above treatment weighed 6.53g. Based on the total amount of taurine in 5L of fermentation broth (2.7L×5 =13.5g), the yield was 48.4%, and the purity was 96.2% as determined by high performance liquid chromatography (HPLC).

[0018] Based on the above, recrystallization was carried out. The recrystallization method was to redissolve the taurine obtained from the first crystallization in 5 times the volume of crystals in 60°C warm water, then add 0.2% w / v activated carbon, stir at 50°C for 30 min, filter, cool the filtrate to 4°C for crystallization, wash, and then place it in a vacuum drying oven and dry at 60°C to constant weight. The mass of taurine after recrystallization was 5.68 g, the total yield was 42.1%, and the purity was as high as 98.9% as determined by high performance liquid chromatography (HPLC).

[0019] Following the method of Example 1, Examples 2 and 3, and Comparative Examples 1 and 2, were obtained by adjusting only the following parameters: The above results show that when the pH value, flow rate, and elution concentration of the sample deviate from the control range of the process parameters of this invention, either too high or too low, it will lead to a significant decrease in product purity or yield.

[0020] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for separating and purifying taurine from microbial fermentation broth, characterized in that, The method for separating and purifying taurine from microbial fermentation broth includes the following steps: Step 1: Pretreatment. The supernatant from the fermentation of engineered Corynebacterium glutamicum in the fermenter is collected and subjected to solid-liquid separation, ultrafiltration, and activated carbon decolorization in sequence to obtain the pretreated liquid. Step 2: Ion exchange chromatography separation. After adjusting the pH of the pretreatment solution to between 3.0 and 5.0, the sample is loaded onto a strongly acidic cation exchange resin column. The column is filled with 732 type hydrogen-form strongly acidic cation exchange resin with sulfonic acid groups as the active groups. Then, the sample is eluted sequentially with deionized water and ammonia water, and the eluent is collected in fractions. Step 3: Collecting the taurine-containing eluent collected in Step 2 involves concentrating under negative pressure, crystallizing, and vacuum drying to obtain taurine crystals.

2. The method for separating and purifying taurine from microbial fermentation broth according to claim 1, characterized in that, In step one, the solid-liquid separation is performed by high-speed centrifugation at a speed of 4000-1000 rpm.

3. The method for separating and purifying taurine from microbial fermentation broth according to claim 1, characterized in that, The ultrafiltration in step one uses an ultrafiltration membrane with a molecular weight cutoff of 3000-10000 Da.

4. The method for separating and purifying taurine from microbial fermentation broth according to claim 1, characterized in that, In step one, the activated carbon decolorization uses powdered activated carbon, with an addition amount of 0.1-1.0% w / v, a decolorization temperature of 40-60℃, and a decolorization time of 30-60 min.

5. The method for separating and purifying taurine from microbial fermentation broth according to claim 1, characterized in that, In step two, the sample loading flow rate is 0.5-2.0 times the column volume / hour, and the resin column is equilibrated with deionized water for 2-3 times the column volume before loading.

6. The method for separating and purifying taurine from microbial fermentation broth according to claim 1, characterized in that, In step two, the washing process uses deionized water, with a washing volume of 2-3 times the column volume and a washing flow rate of 1.0-2.0 times the column volume per hour.

7. The method for separating and purifying taurine from microbial fermentation broth according to claim 1, characterized in that, In step two, the elution uses an ammonia solution with a concentration of 0.5-2.0 mol / L, an elution flow rate of 0.5-1.5 column volumes / hour, and an elution volume of 3-5 column volumes.

8. The method for separating and purifying taurine from microbial fermentation broth according to claim 1, characterized in that, In step three, the concentration process is carried out under negative pressure, at a temperature of 50-70℃ and a vacuum degree of -0.08 to -0.095 MPa, until the taurine concentration is 50-200 g / L.

9. The method for separating and purifying taurine from microbial fermentation broth according to claim 1, characterized in that, The ethanol precipitation method used in step three involves adding 1 to 3 times the volume of anhydrous ethanol to the concentrate and allowing it to stand at 4 to 10°C for 12 to 24 hours to crystallize.

10. The method for separating and purifying taurine from microbial fermentation broth according to claim 1, characterized in that, The ion exchange resin in step two is regenerated after use using 2-5% dilute sulfuric acid or dilute hydrochloric acid.

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