Method for preparing L-cystine by adopting three-phase horizontal spiral centrifuge

Abstract

The invention belongs to the field of amino acid preparation, and discloses a method for preparing L-cystine by adopting a three-phase horizontal spiral centrifuge, which comprises the following steps: (1) sequentially carrying out thallus wall breaking and flocculation operation on an L-cystine precipitate-bacterium-salt mixed solution to obtain a multi-phase mixed system; and (2) separating the multi-phase mixed system obtained in the step (1) through a three-phase horizontal spiral centrifuge to respectively obtain L-cystine precipitate, protein heavy liquid and brine clear liquid, and further purifying the L-cystine precipitate to obtain the L-cystine. According to the method, the separation steps are effectively shortened, the separation operation of a ceramic membrane is reduced, and the separation efficiency of L-cystine is improved.

Description

Technical Field

[0001] This invention belongs to the field of amino acid preparation, specifically the preparation of L-cysteine, and more specifically, a method for preparing L-cysteine ​​using a three-phase horizontal screw centrifuge. Background Technology

[0002] L-Cystine is an essential amino acid for the human body, with the molecular formula C6H12O. 12 N₂O₄S₂, with a molecular weight of 240.3, is a white hexagonal plate-like crystal or a white crystalline powder. It is soluble in dilute acid and alkaline solutions, extremely insoluble in water, and insoluble in ethanol. It exists in small amounts in proteins, primarily in keratin, found in hair, nails, and other similar materials.

[0003] L-cystine is used in medicine as a raw material for the production of antidotes and expectorants. In the food industry, it is used as a dairy additive and a bread ripening accelerator. When heated with sugars in food, it can produce various special aromas and prevent oil oxidation, making it widely used in food processing. In the cosmetics industry, it is a raw material for the production of cold poaching liquid.

[0004] L-cystine is mainly produced by fermentation (e.g., CN 112813012 A, CN 102517352 A). This route first uses microbial fermentation to prepare L-serine and L-cysteine ​​synthases, then pre-concentrates and adds thiol substrates to carry out enzymatic catalytic reactions and oxidative dehydrogenation reactions to obtain crude L-cystine. After a series of filtration, impurity removal, and crystallization steps, the finished L-cystine product is obtained.

[0005] In the L-cysteine ​​purification process, centrifugation is generally used to separate the salt solution from the L-cysteine-bacteria, followed by dissolution to separate the bacterial protein and L-cysteine. This purification process is complex, has a long route, and results in a low overall yield. Especially in the initial centrifugation stage, the mixing of L-cysteine ​​and bacteria leads to high precipitate viscosity and rapid sedimentation, placing high demands on the selection of centrifuge equipment and centrifugation parameters. Summary of the Invention

[0006] This invention provides a method for preparing L-cysteine ​​using a three-phase horizontal screw centrifuge, which effectively shortens the separation steps and improves the separation efficiency of L-cysteine.

[0007] The technical solution of the present invention is as follows: A method for preparing L-cystine using a three-phase horizontal decanter centrifuge includes the following steps: (1) The L-cystine precipitate-bacteria-salt mixed solution was subjected to cell disruption and flocculation operations in sequence to obtain a multiphase mixed system; (2) The multiphase mixture obtained in step (1) is separated by a three-phase horizontal screw centrifuge to obtain L-cystine precipitate, protein heavy liquid and saline solution respectively. The L-cystine precipitate is further purified to obtain L-cystine.

[0008] This invention first subjectes the L-cysteine ​​precipitate-bacteria-salt mixed solution to cell disruption and flocculation in sequence. Then, the L-cysteine, bacteria, and salt solution are simultaneously separated using a horizontal screw centrifuge in a three-phase separation mode, forming three phases: L-cysteine ​​precipitate, protein heavy liquid, and clear brine. The bacterial content in the obtained L-cysteine ​​precipitate is significantly reduced, eliminating the need for subsequent separation using ceramic membranes, greatly shortening the separation steps, improving the separation effect, and resulting in a high yield and purity of L-cysteine.

[0009] In this invention, the L-cysteine ​​precipitation-bacteria-salt mixed solution can be obtained using existing technology, and is a reaction mixture obtained after L-serine fermentation and oxidation. Further, the concentration of L-cysteine ​​in the L-cysteine ​​precipitation-bacteria-salt mixed solution is 5~15wt%. The L-cysteine ​​precipitation-bacteria-salt mixed solution also contains a certain amount of bacteria and salt, and their content is not subject to particularly strict requirements during treatment.

[0010] Preferably, in step (1), the cell wall disruption method is as follows: Add the surfactant, then heat and stir.

[0011] Preferably, the surfactant includes one or more of the Triton series surfactants and the Tween series surfactants.

[0012] Preferably, the amount of surfactant added is 0.1 to 0.5 wt% of the L-cystine precipitate-bacteria-salt mixed solution.

[0013] Preferably, the heating and stirring temperature is 60-80℃.

[0014] As a preferred option, in step (1), the stirring speed and time are judged based on the absence of intact cells under microscopic examination.

[0015] Preferably, the specific method used in step (1) for flocculation is not particularly limited and can be any conventional method in the field, as long as it can completely flocculate the cell / protein after cell wall disruption. This method can be one or more of the following: salting out, isoelectric point precipitation, heating, adding flocculants, or coagulants. For example, flocculation can be performed by adding an equal volume of 2‰ polyacrylamide at pH 4-6, or by adding ammonium sulfate solids to a concentration >8% in the system and adjusting the pH to between 4 and 6.

[0016] Preferably, in step (1), the flocculation operation ends when the absorbance of the supernatant is <0.5 Abs.

[0017] Preferably, in step (2), the separation factor of the three-phase horizontal decanter centrifuge is 1000~2000 xg. Increasing the separation factor can increase the yield of L-cystine after centrifugation by the three-phase horizontal decanter centrifuge. However, if the separation factor is too high, it will lead to an increase in the bacterial ratio and increase the difficulty of subsequent separation. It is further preferred to be 1000~1200 xg.

[0018] Preferably, in step (2), the residence time in the three-phase horizontal screw centrifuge is 2 to 10 minutes, and more preferably 2.5 to 8 minutes.

[0019] Using the method of the present invention, pretreatment followed by centrifugation can significantly reduce the bacterial percentage in L-cystine precipitate and reduce subsequent separation steps. Preferably, in step (2), after treatment with a three-phase horizontal screw centrifuge, the density of the L-cystine precipitate is >1.36 g / mL, preferably >1.4 g / mL, and the bacterial percentage is <1.1%, preferably <1.0%. The density of the protein heavy liquid is 1.05-1.1 g / mL; The density of the saline solution is 1.00-1.02 g / mL, and the solid content of the saline solution is <0.5%.

[0020] Preferably, in step (2), the further purification includes: first adding acid to adjust the pH to <1 for dissolution, then adding alkali to adjust the pH to 3-5 for crystallization, and finally centrifuging and drying to obtain pure L-cysteine. The specific method used for centrifugation and drying is not particularly limited and can be a conventional choice in the art, as long as the water in the L-cysteine ​​crystals can be removed.

[0021] Compared with the prior art, the beneficial effects of the present invention are reflected in: This invention first breaks down the bacterial cells in a mixed solution of L-cysteine ​​precipitate, bacteria, and salt to disrupt the bacterial cell walls, then performs flocculation to coagulate the protein, and finally uses a three-phase horizontal screw centrifuge for separation. This process efficiently forms three phases: L-cysteine ​​precipitate, protein heavy liquid, and clear brine, achieving highly efficient separation of L-cysteine. The bacterial content in the resulting L-cysteine ​​precipitate is significantly reduced, eliminating the need for subsequent separation using ceramic membranes, shortening the separation steps, and making it easier to implement in industrial operations. Detailed Implementation

[0022] The method for preparing the L-cysteine ​​precipitate-bacteria-salt mixed solution obtained by fermentation catalytic oxidation in this invention is existing technology, and the specific process is as follows: Wet bacterial cells or crude enzyme solution with L-tryptophan synthase activity are mixed with a mixed amino acid solution containing L-serine. Hydrogen sulfide or sulfide is added, and the enzymatic reaction is carried out at 25-55°C and pH 6-11. The generated L-cysteine ​​is oxidized by aeration or by adding hydrogen peroxide to obtain an L-cysteine ​​precipitate-bacteria-salt mixed solution. For details, please refer to CN112813012 A, CN 102517352 A, etc., but the L-cysteine ​​precipitate-bacteria-salt mixed solution obtained by the above methods is not limited to any solution containing L-cysteine ​​product, post-fermentation bacterial cells, and various salts. Example 1

[0023] Three tons of L-cysteine ​​precipitate-bacteria-salt mixed solution (L-cysteine ​​content 7wt%) obtained from fermentation catalytic oxidation were added to a reactor. 5 kg of Triton X-100 was added, and the mixture was heated to 65℃ and stirred for 30 min. Then, 250 kg of ammonium sulfate solid was added, and the pH was adjusted to 5. Stirring continued for another 30 min, and flocculation and stratification occurred. The flocculation operation was terminated when the absorbance of the supernatant was <0.5 Abs. The above solution was centrifuged using a three-phase horizontal screw centrifuge with a separation factor of 1100 x g and a centrifugation residence time of 5 min. The resulting L-cysteine ​​precipitate had a density of approximately 1.46 g / mL, a bacterial percentage of 0.75%, and an L-cysteine ​​yield of 96.5%. The protein heavy liquid density was 1.07 g / mL, and the brine supernatant density was 1.01 g / mL. After the precipitate was dissolved by adjusting the pH to <1 with sulfuric acid, sodium hydroxide was added to adjust the pH to 4 for crystallization. After centrifugation and drying, 198.6 kg of pure L-cystine was obtained, with a yield of 94.6%, purity > 99%, and transmittance > 98%.

[0024] Examples 2-5 The preparation process is basically the same as in Example 1, except for the different centrifugation parameters. The specific conditions for the changes are listed in Table 1 below.

[0025] Table 1

[0026] a This yield is the yield obtained after centrifugation using a three-phase horizontal screw centrifuge.

[0027] Comparative Example 1 Three tons of L-cysteine ​​precipitate-bacteria-salt mixed solution (L-cysteine ​​content 7wt%) obtained from fermentation catalytic oxidation were centrifuged using a disc centrifuge with a separation factor of 3500xg and a slag discharge cycle of 3 min followed by 0.8 s s. The resulting L-cysteine ​​precipitate had a density of approximately 1.54 g / mL, a bacterial content of 9.65%, and an L-cysteine ​​yield of 96.75%. The density of the brine was 1.01 g / mL. After dissolving the precipitate by adjusting the pH to <1 with sulfuric acid, it was concentrated 15 times through a ceramic membrane at a flux of 7 L / (min*m²). An equal volume of sulfuric acid was added to wash the residue, resulting in a yield of 98.24%. Sodium hydroxide was added to the ceramic membrane filtrate to adjust the pH to 4 for crystallization. After centrifugation and drying, 195.6 kg of pure L-cysteine ​​was obtained, with a yield of 93.1%, a purity >99%, and a transmittance >98%.

[0028] This comparative example uses a conventional centrifugation method, which requires a larger separation factor to achieve separation. Furthermore, the L-cysteine ​​precipitate after centrifugation has a high bacterial content, requiring dissolution with sulfuric acid before separation with a ceramic membrane. This makes the separation process more complex and reduces the final yield.

[0029] Comparative Example 2 Three tons of a mixed solution of L-cysteine ​​precipitate, bacteria, and salt (7 wt% L-cysteine ​​content) obtained from fermentation and catalytic oxidation was added to a reactor. 5 kg of Triton X-100 was added, and the mixture was heated to 65°C and stirred for 30 min. The solution was then centrifuged using a three-phase horizontal screw centrifuge with a separation factor of 1100 x g and a centrifugation residence time of 5 min. The resulting L-cysteine ​​precipitate had a density of approximately 1.51 g / mL, a bacterial content of 1.98%, and an L-cysteine ​​yield of 94.66%. The protein heavy liquid density was 1.02 g / mL, and the brine supernatant density was also 1.02 g / mL. After dissolving the precipitate by adjusting the pH to <1 with sulfuric acid, sodium hydroxide was added to adjust the pH to = 4 for crystallization. The crystals were then centrifuged and dried, yielding 198.6 kg of pure L-cysteine, with a yield of 93.71%, a purity of 91%, and a transmittance of <70%.

[0030] This comparative example only involved cell wall disruption followed by separation using a three-phase horizontal screw centrifuge. The resulting L-cysteine ​​precipitate also had a high bacterial content, requiring prior dissolution with sulfuric acid followed by separation using a ceramic membrane. If the ceramic membrane is not used and the subsequent processes of the previous example are repeated, the resulting product will have lower purity, poor light transmittance, and a lower yield. The separated protein heavy liquid and the brine effluent do not separate into phases when mixed; they can be easily separated by sedimentation for more than 2 hours. However, the COD and ammonia nitrogen levels in the brine effluent are significantly higher than in the previous example.

Claims

1. A method for preparing L-cystine using a three-phase horizontal decanter centrifuge, characterized in that, Includes the following steps: (1) The L-cystine precipitate-bacteria-salt mixed solution was subjected to cell disruption and flocculation operations in sequence to obtain a multiphase mixed system; (2) The multiphase mixture obtained in step (1) is separated by a three-phase horizontal screw centrifuge to obtain L-cystine precipitate, protein heavy liquid and saline solution respectively. The L-cystine precipitate is further purified to obtain L-cystine.

2. The method for preparing L-cysteine ​​using a three-phase horizontal decanter centrifuge according to claim 1, characterized in that, In step (1), the cell wall disruption method is as follows: First, add the surfactant, then heat and stir.

3. The method for preparing L-cysteine ​​using a three-phase horizontal decanter centrifuge according to claim 2, characterized in that, The surfactants mentioned include one or more of the Triton series surfactants and the Tween series surfactants.

4. The method for preparing L-cysteine ​​using a three-phase horizontal decanter centrifuge according to claim 2 or 3, characterized in that, The amount of surfactant added is 0.1~0.5 wt% of the L-cystine precipitate-bacteria-salt mixed solution.

5. The method for preparing L-cysteine ​​using a three-phase horizontal decanter centrifuge according to claim 2, characterized in that, The heating and stirring temperature is 60~80℃.

6. The method for preparing L-cysteine ​​using a three-phase horizontal decanter centrifuge according to claim 1, characterized in that, In step (1), the flocculation operation includes one or more of the following: salting out, isoelectric point precipitation, heating, adding flocculant, and adding coagulant.

7. The method for preparing L-cysteine ​​using a three-phase horizontal decanter centrifuge according to claim 1, characterized in that, In step (1), the flocculation operation ends when the absorbance of the supernatant is <0.5 Abs.

8. The method for preparing L-cysteine ​​using a three-phase horizontal decanter centrifuge according to claim 1, characterized in that, In step (2), the separation factor of the three-phase horizontal screw centrifuge is 1000~2000 xg.

9. The method for preparing L-cysteine ​​using a three-phase horizontal decanter centrifuge according to claim 1, characterized in that, In step (2), after treatment with a three-phase horizontal decanter centrifuge, the L-cysteine ​​precipitate density is >1.36 g / mL, and the bacterial percentage is <1.1%; The density of the protein heavy liquid is 1.05~1.10 g / mL; The density of the saline solution is 1.00~1.02 g / mL, and the solid content of the saline solution is <0.5%.

10. The method for preparing L-cysteine ​​using a three-phase horizontal decanter centrifuge according to claim 1, characterized in that, In step (2), the further purification includes: first adding acid to adjust the pH to <1 for dissolution, then adding alkali to adjust the pH to 3-5 for crystallization, and then centrifuging and drying to obtain pure L-cystine.

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