Preparation method of clean and environment-friendly high-purity proline
By using a membrane separation and resin combined process, the pollution and energy consumption problems in the separation and purification of proline were solved, and high-purity, high-yield proline preparation was achieved, thus realizing the goal of clean and environmentally friendly production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- WUXI JINGHAI AMINO ACID
- Filing Date
- 2023-11-29
- Publication Date
- 2026-07-24
AI Technical Summary
Existing proline separation and purification processes suffer from severe pollution, high energy consumption, numerous impurities, and low yield. In particular, it is difficult to achieve high purity and high yield in the fermentation broth treatment and purification process.
Impurities are removed by flocculation technology, combined with ceramic membrane separation, cation exchange resin purification, ultrafiltration/nanofiltration membrane decolorization, reverse osmosis membrane concentration, and ultrasonic-assisted macroporous resin decolorization, forming a combined process of membrane separation and resin decolorization, which replaces the traditional activated carbon decolorization step and achieves continuous production and high-efficiency purification.
This method enables the preparation of high-purity proline with low pollution, low energy consumption, and low impurities. The product purity is higher than 99.0%, and the yield exceeds 89.0%, which significantly improves product quality and reduces solid waste emissions.
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Figure CN117624008B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a clean, environmentally friendly method for preparing high-purity proline, belonging to the field of amino acid technology. Background Technology
[0002] L-Proline (L-Proline for short) is a neutral amino acid containing an imine group. It is one of the 20 essential amino acids that make up proteins and is a non-essential amino acid for the human body. Proline has excellent biological activity and is widely used in medicine, chemical industry, agriculture, and food. Especially in medicine, it is an intermediate in the synthesis of many new drugs and is receiving increasing attention. The production of proline using microbial fermentation involves a reaction system containing pigments, sugars, other amino acids, organic acids, and inorganic salts. To obtain high-purity proline, extraction, separation, and purification are necessary.
[0003] In existing processes for separating, purifying, and refining proline, the fermentation broth is generally not pretreated with flocculants, resulting in high levels of impurities in the extracted amino acids. Plate and frame filtration for cell separation requires a large area and the filter inlet is prone to clogging. Ion exchange purification uses ammonia as the eluent, which can cause environmental pollution. Activated carbon decolorization involves repeated steps and requires heating, increasing energy consumption and solid waste. Evaporation and concentration methods are time-consuming and energy-intensive. Therefore, there is an urgent need to develop a new, clean, environmentally friendly method for preparing high-purity proline to overcome these existing technological shortcomings. Summary of the Invention
[0004] The technical problem this invention aims to solve is to overcome the shortcomings of existing methods and provide a low-pollution, low-ammonia-emission, low-energy-consumption, low-impurity, high-yield, and high-purity proline preparation method. This method employs flocculation technology to remove impurities, increase proline adsorption, and improve yield; ceramic membrane separation technology pretreatment of the fermentation broth; purification with high-capacity cation exchange resin, compared to commonly used resins, can improve the yield of amino acids in the eluent; ultrafiltration membrane decolorization and reverse osmosis membrane concentration enable continuous production, shorten the evaporation and crystallization cycle, and allow for the recycling of reverse osmosis water, saving nearly half the water consumption of the fermentation broth volume; the use of low-power pulsed ultrasonic-assisted weakly acidic macroporous resin decolorization technology, compared to macroporous resin decolorization without ultrasonic assistance, enhances pigment adsorption and improves decolorization efficiency; compared to activated carbon decolorization, it reduces the number of decolorization cycles, and the resin can be reused multiple times, reducing activated carbon solid waste emissions, thus achieving the goals of energy conservation, emission reduction, and improved product quality. The key point of this invention is that the combination of membrane separation and resin decolorization can achieve better results than activated carbon decolorization in separating proline from fermentation broth (higher purity, higher yield, and no solid-liquid waste discharge from activated carbon). To achieve the above objectives, this invention adopts the following technical solution:
[0005] (1) Adjust the pH of the proline fermentation broth, add flocculant chitosan and β-cyclodextrin, and treat with ultrasound to obtain the pretreated fermentation broth;
[0006] (2) The pretreated fermentation broth is filtered through a ceramic membrane to obtain the filtrate;
[0007] (3) The filtrate was purified by a high-adsorption-capacity cation exchange resin and eluted with low-concentration sodium hydroxide to obtain the eluent;
[0008] (4) The eluent is decolorized by ultrafiltration / nanofiltration membrane to obtain decolorized solution 1;
[0009] (5) Decolorizing solution 1 is concentrated through a reverse osmosis membrane to obtain a concentrated solution;
[0010] (6) Use ultrasound-assisted weak acid macroporous resin to decolorize, and obtain decolorized solution 2;
[0011] (7) After the decolorizing solution 2 is filtered through a fine filter membrane, it is poured into a reaction vessel for crystallization and concentrated under vacuum to obtain wet crystals;
[0012] (8) After the wet crystallizer is cooled to room temperature, it is filtered, washed, centrifuged and dried to obtain proline crystals.
[0013] In one embodiment, the pH of the fermentation broth in step (1) is adjusted by hydrochloric acid to a range of 3.00 to 3.50, the mass ratio of flocculant to fermentation broth is (2 to 4): 1000, and the mass ratio of chitosan to β-cyclodextrin is (5 to 10): 1.
[0014] In one embodiment, the ultrasonic temperature in step (1) is 25-40°C, the frequency is 40kHz, and the processing time is 10-20min.
[0015] In one embodiment, the ceramic membrane described in step (2) has a pore size of 50 nm, an operating pressure of 0.1 to 0.2 MPa, a flow rate of 5 to 10 L / h, and a temperature control of below 50 °C.
[0016] In one embodiment, the high-adsorption-capacity cation exchange resin mentioned in step (3) is HZ016 cation exchange resin, and the sodium hydroxide elution concentration is 1-2%.
[0017] In one embodiment, the ultrafiltration / nanofiltration membrane described in step (4) has a molecular weight cutoff of 500 to 1000D, an operating pressure of 0.6 to 3.0 MPa, a flow rate of 3 to 5 L / h, and a temperature below 45°C.
[0018] In one embodiment, the reverse osmosis membrane described in step (5) has a molecular weight cutoff of 50 to 100D, an operating pressure of 2 to 3.5 MPa, a flow rate of 3 to 5 L / h, and a temperature below 45°C.
[0019] In one embodiment, the macroporous decolorizing resin described in step (6) is a D155 weakly acidic resin.
[0020] In one embodiment, the ultrasonic power of the ultrasonic generator in step (6) is 100-200W, the ultrasonic time is 0.5-1h, and the ultrasonic temperature is below 40℃.
[0021] In one embodiment, the fine filtration membrane described in step (7) is a polyethersulfone spiral wound membrane with a pore size of 0.22 μm.
[0022] In one embodiment, the vacuum concentration in the reactor in step (7) is performed with a vacuum degree of 0.09 to 0.098 MPa, a concentration temperature of 55 to 65°C, and a stirring speed of 100 to 150 r / min.
[0023] In one embodiment, the wet crystallization in step (8) is cooled to room temperature, which is 20 to 30°C.
[0024] In one embodiment, the rinsing solution in step (8) is anhydrous ethanol, and the mass ratio of anhydrous ethanol to wet crystals is (1-2):1.
[0025] In one embodiment, the centrifugation temperature in step (8) is 4-10°C, the rotation speed is 3000-4000 r / min, and the centrifugation time is 10-20 min.
[0026] In one embodiment, the drying method described in step (8) uses a vacuum dryer with a vacuum degree of 0.12±0.01 MPa, a drying temperature of 55 to 65°C, and a drying time of 3 to 6 hours.
[0027] Beneficial effects:
[0028] This invention involves adding a flocculant to the proline fermentation broth for flocculation, followed by sterilization via a ceramic membrane, decolorization via a high-capacity cation exchange resin, ultrafiltration / nanofiltration membrane, concentration via reverse osmosis membrane, and ultrasonic-assisted macroporous resin, followed by fine filtration, crystallization, rinsing, centrifugation, and drying. The resulting product is proline with a purity >99.0%, transmittance >99.0%, yield >89.0%, and specific rotation between -84.5° and -86.0°.
[0029] Compared with the traditional activated carbon decolorization process, this membrane separation and resin decolorization combined process greatly reduces the emission of solid pollutants (such as activated carbon), shortens the separation and purification cycle, and improves product yield (>89.0% vs 41.09%–82.61%) and product purity (>99.0% vs 85.39%–98.15%). Attached Figure Description
[0030] Figure 1 This is a process flow diagram of the present invention. Detailed Implementation
[0031] The present invention will be further described below with reference to specific embodiments and comparative examples.
[0032] Methods for detecting proline:
[0033] 1. Rapid acid spotting method
[0034] Dip a glass rod into the test solution and place it on filter paper. Dry the paper at 105°C. Add one drop of ninhydrin indicator solution and dry the paper at 105°C. Observe whether there is a color development. If there is a color development, it means that there are amino acids in the test solution. If there is no color development, it means that there are no amino acids in the test solution.
[0035] 2. Concentration Detection Method
[0036] According to GB 5009.124-2016 National Food Safety Standard "Determination of Amino Acids in Food", the amino acid content was determined using an amino acid analyzer in accordance with the law.
[0037] 3. Purity testing methods
[0038] According to GB 5009.124-2016 National Food Safety Standard "Determination of Amino Acids in Food", the amino acid content was determined using an amino acid analyzer in accordance with the law.
[0039] 4. Transmittance testing method
[0040] According to the 2020 edition of the Pharmacopoeia of the People's Republic of China, the transmittance was determined at a wavelength of 430 nm using ultraviolet-visible spectrophotometry (General Rule 0401).
[0041] 5. Specific curl testing method
[0042] According to the 2020 edition of the Pharmacopoeia of the People's Republic of China, accurately weigh this product and determine its composition according to the method described in General Rule 0621.
[0043] The HZ016 cation exchange resin used in the following embodiments was purchased from Shanghai Huazhen Technology Co., Ltd.; the D155 weakly acidic resin was purchased from Shanghai Huazhen Technology Co., Ltd.
[0044] The proline fermentation broth in the following examples contains: 43.78–48.15 g / L proline, 4.9–6.15 g / L glutamic acid, 3.5–4.0 g / L alanine, 1.2–1.6 g / L valine, and 4.0–5.0 g / L inorganic salts.
[0045] Example 1
[0046] The pH of a 45.65 g / L, 9.85 L proline fermentation broth was adjusted to 3.05 with hydrochloric acid. 16.40 g of chitosan flocculant and 3.26 g of β-cyclodextrin were added. The fermentation broth was then treated with ultrasound at a temperature of 28–30 °C, a frequency of 40 kHz, and a treatment time of 10 min.
[0047] The pretreated fermentation broth was filtered through a ceramic membrane with a pore size of 50 nm, with the pressure controlled at 0.1–0.15 MPa, the flow rate at 5–8 L / h, and the temperature at 25–40 °C. The filtrate was collected, and the remaining concentrate was diluted with water by half and filtered through a ceramic membrane again. The filtrate was then collected.
[0048] The filtrate was purified using 5 L of HZ016 cation exchange resin at a flow rate of 1.8 L / h, and the adsorption endpoint was determined by a rapid acid spot test. Unadsorbed amino acids and impurities were washed away with 10 L of purified water at a flow rate of 3.6 L / h. After washing, elution was performed with 2.0% NaOH eluent at a flow rate of 1.8 L / h. Eluent collection began after one column volume of eluent had been removed. The eluent with a pH ≤ 10 was used as the collected eluent.
[0049] The eluent is decolorized by an ultrafiltration membrane with a molecular weight cutoff of 1000D, with the pressure controlled at 0.6-1.0MPa, the flow rate controlled at 3-4L / h, and the temperature controlled at 25-38℃. Decolorized solution 1 is collected, and the remaining concentrate is diluted with water by one-fold and decolorized again by an ultrafiltration membrane.
[0050] The collected decolorized solution 1 was concentrated using a reverse osmosis membrane with a molecular weight cutoff of 100D. The pressure was controlled at 2–3.0 MPa, the flow rate at 3–4 L / h, and the temperature at 25–40℃. No significant amino acid permeation was detected during the concentration process using a rapid acid spot test. The concentrated solution was collected; the permeate can be used as a cleaning solution for ceramic membranes (saving 20–50% of process water).
[0051] The collected concentrate was passed into an adsorption column packed with 2L of D155 weakly acidic macroporous decolorizing resin. An external ultrasonic generator was connected, and the ultrasonic power was adjusted to 100W, the ultrasonic time to 0.5h, and the ultrasonic temperature to 25-30℃. The resin was washed with 1L of purified water, and the decolorized solution 2 was collected.
[0052] After filtering the decolorizing solution 2 through a 0.22 μm fine filter membrane, it was added to a reaction vessel for concentration and crystallization. The vacuum degree was 0.09–0.098 MPa, the concentration temperature was 60–65 °C, and the stirring speed was 120 r / min. Concentration was stopped after crystal formation was observed. The wet crystals were poured out, cooled to room temperature, and filtered to obtain wet crystals. The filtered mother liquor was then used to repeat the ultrasonic-assisted resin decolorization step and concentrated for crystallization.
[0053] The wet crystals from the previous step were combined and cooled to 28°C, filtered through a 0.22 μm microporous membrane, rinsed with anhydrous ethanol, and centrifuged at 4°C, 3000 r / min for 10 min. The ethanol was recovered, and the wet crystals were dried in a vacuum dryer at 0.12 ± 0.01 MPa for 3 h at 65°C to obtain proline crystals.
[0054] The obtained proline crystals were tested for purity, transmittance, and specific rotation. The results were: purity 99.52%, transmittance 99.75%, and specific rotation -85.5°. Sampling and testing results for each step are shown in the table below.
[0055] Table 1
[0056]
[0057]
[0058] Example 2
[0059] The pH of the proline fermentation broth with a concentration of 43.78 g / L and a volume of 9.55 L was adjusted to 3.10 with hydrochloric acid. 25.55 g of chitosan flocculant and 2.85 g of β-cyclodextrin were added. The fermentation broth was then treated with ultrasound at a temperature of 33–40 °C and a frequency of 40 kHz for 15 min.
[0060] The pretreated fermentation broth was filtered through a ceramic membrane with a pore size of 50 nm, with the pressure controlled at 0.1–0.2 MPa, the flow rate at 8–10 L / h, and the temperature at 25–40 °C. The filtrate was collected, and the remaining concentrate was diluted with water by half and filtered through the ceramic membrane again. The filtrate was then collected.
[0061] The filtrate was purified using 5 L of HZ016 cation exchange resin at a flow rate of 1.8 L / h, and the adsorption endpoint was determined by a rapid acid spot method. Unadsorbed amino acids and impurities were washed away with 10 L of purified water at a flow rate of 3.6 L / h. After washing, elution was performed with 1.5% NaOH eluent at a flow rate of 1.8 L / h. Eluent was collected after one column volume of eluent had been removed. The eluent with a pH ≤ 10 was used as the collected eluent.
[0062] The eluent is decolorized by a nanofiltration membrane with a molecular weight cutoff of 800D, with the pressure controlled at 1.5-2.5MPa, the flow rate controlled at 3-4L / h, and the temperature controlled at 25-38℃. Decolorized solution 1 is collected, and the remaining concentrate is diluted with water by one-fold and decolorized again by an ultrafiltration membrane.
[0063] The collected decolorized solution 1 was concentrated using a reverse osmosis membrane with a molecular weight cutoff of 100D. The pressure was controlled at 2–3.0 MPa, the flow rate at 3–4 L / h, and the temperature at 25–40℃. No significant amino acid permeation was detected during the concentration process using a rapid acid spot test. The concentrated solution was collected; the permeate can be used as a cleaning solution for ceramic membranes.
[0064] The collected concentrate was passed into an adsorption column packed with 2L of D155 weakly acidic macroporous decolorizing resin. An external ultrasonic generator was connected, and the ultrasonic power was adjusted to 150W. The ultrasonic time was 0.5h, the ultrasonic temperature was 25-30℃, and the ultrasonic power was 0.5h. The resin was eluted with 1L of purified water, and the decolorized solution 2 was collected.
[0065] After filtering the decolorizing solution 2 through a 0.22μm fine filter membrane, it was added to a reaction vessel for concentration and crystallization under vacuum.
[0066] The concentration pressure was 0.09–0.098 MPa, the concentration temperature was 55–60 °C, and the stirring speed was 100 r / min. Concentration was stopped after crystallization was observed. The wet crystals were poured out, cooled to room temperature, and filtered to obtain wet crystals. The filtered mother liquor was then subjected to another ultrasonic-assisted resin decolorization step and concentrated for crystallization.
[0067] The wet crystals from the previous step were combined and cooled to 25°C, filtered through a 0.22 μm microporous membrane, rinsed with anhydrous ethanol, and centrifuged at 4°C, 4000 r / min for 10 min. The ethanol was recovered, and the wet crystals were dried in a vacuum dryer at 0.12 ± 0.01 MPa for 4 h at 60°C to obtain proline crystals.
[0068] The obtained proline crystals were tested for purity, transmittance, and specific rotation. The results were: purity 99.47%, transmittance 99.63%, and specific rotation -86.0°. The results of sampling and testing at each step are shown in the table below.
[0069] Table 2
[0070]
[0071] Example 3
[0072] The pH of a 10.2L proline fermentation broth with a concentration of 48.15 g / L was adjusted to 3.22 with hydrochloric acid. 36.21 g of chitosan flocculant and 3.68 g of β-cyclodextrin were added. The fermentation broth was then treated with ultrasound at a temperature of 35–40 °C and a frequency of 40 kHz for 20 min.
[0073] The pretreated fermentation broth was filtered through a 50 nm ceramic membrane at a pressure controlled at 0.1–0.2 MPa, a flow rate controlled at 8–10 L / h, and a temperature controlled at 25–40 °C. The filtrate was collected, and the remaining concentrate was diluted with water by half and filtered through the ceramic membrane again. The filtrate was then collected. This filtrate was purified using 5 L of HZ016 cation exchange resin at a flow rate of 1.8 L / h, and the adsorption endpoint was determined using a rapid acid spot method. Unadsorbed amino acids and impurities were washed away with 10 L of purified water at a flow rate of 3.6 L / h. After washing, elution was performed with 1.0% NaOH eluent at a flow rate of 1.8 L / h. Once one column volume of eluent had been eluted, the eluent was collected. The eluent with a pH ≤ 10 was used as the collected eluent.
[0074] The eluent was decolorized by a nanofiltration membrane with a molecular weight cutoff of 500D, with the pressure controlled at 2-3.0 MPa, the flow rate controlled at 3-4 L / h, and the temperature controlled at 25-38℃. Decolorized solution 1 was collected, and the remaining concentrate was diluted with water by one-fold and decolorized again by an ultrafiltration membrane.
[0075] The collected decolorized solution 1 was concentrated using a reverse osmosis membrane with a molecular weight cutoff of 50D. The pressure was controlled at 2–3.5 MPa, the flow rate at 3–4 L / h, and the temperature at 25–40℃. No significant amino acid permeation was detected during the concentration process using a rapid acid spot test. The concentrated solution was collected; the permeate can be used as a cleaning solution for ceramic membranes.
[0076] The collected concentrate was passed into an adsorption column packed with 2L of D155 weakly acidic macroporous decolorizing resin. An external ultrasonic generator was connected, and the ultrasonic power was adjusted to 1.0 h, the ultrasonic temperature to 25–40 °C, and the ultrasonic power to 200 W. The resin was eluted with 1L of purified water, and the decolorized solution 2 was collected.
[0077] After filtering the decolorizing solution 2 through a 0.22 μm fine filter membrane, it was added to a reaction vessel for concentration and crystallization. The vacuum degree was 0.09–0.098 MPa, the concentration temperature was 55–65 °C, and the stirring speed was 150 r / min. Concentration was stopped after crystal formation was observed. The wet crystals were poured out, cooled to room temperature, and filtered to obtain wet crystals. The filtered mother liquor was then used to repeat the ultrasonic-assisted resin decolorization step and concentrated for crystallization.
[0078] The wet crystals from the previous step were combined and cooled to 25°C, filtered through a 0.22 μm microporous membrane, rinsed with anhydrous ethanol, and centrifuged at 4°C, 4000 r / min for 20 min. The ethanol was recovered, and the wet crystals were dried in a vacuum dryer at 55°C for 6 h under a vacuum of 0.12 ± 0.01 MPa to obtain proline crystals.
[0079] The obtained proline crystals were tested for purity, transmittance, and specific rotation. The results were: purity 99.33%, transmittance 99.49%, and specific rotation -86.0°. The results of sampling and testing at each step are shown in the table below.
[0080] Table 3
[0081]
[0082] Comparative Example 1: Flocculant addition, reverse osmosis concentration, and ultrasonic treatment omitted.
[0083] The specific implementation method is the same as in Example 1, except that no flocculant is added, there is no reverse osmosis concentration step, the resin decolorization is not connected to the ultrasonic generator, and there is no recycling process in the process route.
[0084] The pH of the proline fermentation broth with a concentration of 45.65 g / L and a volume of 9.85 L was adjusted to 3.05 using hydrochloric acid.
[0085] The pretreated fermentation broth was filtered through a ceramic membrane with a pore size of 50 nm. The pressure was controlled at 0.1–0.15 MPa, the flow rate at 5–8 L / h, and the temperature at 25–40 °C. The filtrate was collected, and the remaining concentrate was not recovered.
[0086] The filtrate was purified using 5 L of HZ016 cation exchange resin at a flow rate of 1.8 L / h, and the adsorption endpoint was determined by a rapid acid spot method. Unadsorbed amino acids and impurities were washed away with 10 L of purified water at a flow rate of 3.6 L / h. After washing, elution was performed with 2.0% NaOH eluent at a flow rate of 1.8 L / h. Once one column volume of eluent had been removed, the eluent was collected, and the eluent with a pH ≤ 10 was used as the collected eluent.
[0087] The eluent was decolorized using an ultrafiltration membrane with a molecular weight cutoff of 1000D. The pressure was controlled at 0.6–1.0 MPa, the flow rate at 3–4 L / h, and the temperature at 25–38℃. Decolorized solution 1 was collected, and the remaining concentrate was not recovered.
[0088] The collected decolorizing solution 1 was passed into an adsorption column filled with 2L of D155 weakly acidic macroporous decolorizing resin. The ultrasonic generator was not connected. The resin was washed with 1L of purified water, and the decolorizing solution 2 was collected.
[0089] After filtering the decolorizing solution 2 through a 0.22 μm fine filter membrane, it was added to a reaction vessel for concentration and crystallization. The vacuum degree was 0.09–0.098 MPa, the concentration temperature was 60–65 °C, and the stirring speed was 120 r / min. Concentration was stopped after crystal formation was observed. The wet crystals were poured out, cooled to room temperature, and filtered to obtain wet crystals. The mother liquor filtered out was not used for the resin decolorization step.
[0090] The wet crystals from the previous step were cooled to 28°C, filtered through a 0.22 μm microporous membrane, rinsed with anhydrous ethanol, and centrifuged at 4°C, 3000 r / min for 10 min. No ethanol was recovered. The wet crystals were then dried in a vacuum dryer at 0.12 ± 0.01 MPa for 3 h at 65°C to obtain proline crystals.
[0091] The obtained proline crystals were tested for purity and transmittance. The results were: purity 88.12% and transmittance 85.83%. The results of sampling and testing at each step are shown in the table below.
[0092] Table 4
[0093]
[0094] Comparative Example 2: Flocculant addition, ultrafiltration membrane decolorization, reverse osmosis concentration, and ultrasonic treatment were omitted.
[0095] The specific implementation method is the same as in Example 1, except that no flocculant is added, there is no ultrafiltration decolorization step, no reverse osmosis concentration step, the resin decolorization is not connected to an ultrasonic generator, and there is no recycling process in the process route.
[0096] The pH of the proline fermentation broth with a concentration of 46.65 g / L and a volume of 9.85 L was adjusted to 3.05 using hydrochloric acid.
[0097] The pretreated fermentation broth was filtered through a ceramic membrane with a pore size of 50 nm. The pressure was controlled at 0.1–0.15 MPa, the flow rate at 5–8 L / h, and the temperature at 25–40 °C. The filtrate was collected, and the remaining concentrate was not recovered.
[0098] The filtrate was purified using 5 L of HZ016 cation exchange resin at a flow rate of 1.8 L / h, and the adsorption endpoint was determined by a rapid acid spot method. Unadsorbed amino acids and impurities were washed away with 10 L of purified water at a flow rate of 3.6 L / h. After washing, elution was performed with 2.0% NaOH eluent at a flow rate of 1.8 L / h. Once one column volume of eluent had been removed, the eluent was collected, and the eluent with a pH ≤ 10 was used as the collected eluent.
[0099] The collected liquid was passed into an adsorption column packed with 2L of D155 weakly acidic macroporous decolorizing resin. The ultrasonic generator was not connected. The resin was eluted with 1L of purified water, and the decolorized liquid was collected.
[0100] The decolorizing solution was filtered through a 0.22 μm fine filter membrane and then added to a reaction vessel for concentration and crystallization. The vacuum degree was 0.09–0.098 MPa, the concentration temperature was 60–65 °C, and the stirring speed was 120 r / min. Concentration was stopped after crystal formation was observed. The wet crystals were poured out, cooled to room temperature, and filtered to obtain wet crystals. The mother liquor was not used for the resin decolorization step.
[0101] The wet crystals from the previous step were cooled to 28°C, filtered through a 0.22 μm microporous membrane, rinsed with anhydrous ethanol, and centrifuged at 4°C, 3000 r / min for 10 min. No ethanol was recovered. The wet crystals were then dried in a vacuum dryer at 0.12 ± 0.01 MPa for 3 h at 65°C to obtain proline crystals.
[0102] The obtained proline crystals were tested for purity and transmittance. The results were: purity 85.39%, transmittance 78.24%. The results of sampling and testing at each step are shown in the table below.
[0103] Table 5
[0104]
[0105]
[0106] Comparative Example 3: Changing the dosage of flocculant
[0107] The specific implementation method is the same as in Example 1, except that the amount of flocculant was changed. 3.50g of chitosan and 1.50g of β-cyclodextrin were added to the fermentation broth. The purity and transmittance of the obtained proline crystals were tested. The results were: purity 91.20%, transmittance 88.15%. Sampling and testing were performed at each step, and the results are shown in the table below.
[0108] Table 6
[0109]
[0110] Comparative Example 4: Replacing the cation exchange resin
[0111] The specific implementation method is the same as in Example 1, except that 732 cation exchange resin is used instead of HZ016 cation exchange resin. The purity and transmittance of the obtained proline crystals were tested. The results were: purity 93.45% and transmittance 98.51%. Sampling and testing were performed at each step, and the results are shown in the table below.
[0112] Table 7
[0113]
[0114] Comparative Example 5: Decolorization using only activated carbon, without ultrafiltration / nanofiltration membranes or resins.
[0115] The specific implementation method is the same as in Example 1, except that ultrafiltration / nanofiltration membranes and resins are not used for decolorization. Only pharmaceutical activated carbon is added for decolorization. The amount of activated carbon added is 10% of the amino acid mass. The rotation speed is 200 r / min, and the decolorization is carried out at 55℃ for 1 hour. This decolorization step is repeated twice. The purity and transmittance of the obtained proline crystals are tested. The test results are: purity 90.47%, transmittance 91.73%. Samples were taken and tested at each step, and the results are shown in the table below:
[0116] Table 8
[0117]
[0118]
[0119] Comparative Example 6: Ultrafiltration membrane decolorization + activated carbon decolorization, without resin decolorization.
[0120] The specific implementation method is the same as in Example 1, except that pharmaceutical activated carbon is used instead of D155 macroporous resin for decolorization. The activated carbon addition amount is 10% of the amino acid mass, the rotation speed is 200 r / min, and the decolorization is carried out at 55℃ for 1 hour. This decolorization step is repeated twice. The purity and transmittance of the obtained proline crystals are tested. The test results are: purity 98.15% and transmittance 99.17%. Samples were taken and tested at each step, and the results are shown in the table below:
[0121] Table 9
[0122]
[0123] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. A method for preparing clean, environmentally friendly, high-purity proline, characterized in that, The method includes the following steps: (1) Adjust the pH of the proline fermentation broth, add flocculant, and treat with ultrasound to obtain a pretreated fermentation broth; the flocculant includes chitosan and β-cyclodextrin, and the mass ratio of chitosan to β-cyclodextrin is (5~10):1; (2) The pretreated fermentation broth is filtered through a ceramic membrane to obtain filtrate; the ceramic membrane has a pore size of 50~100 nm, an operating pressure of 0.1~0.2 MPa, a flow rate of 5~10 L / h, and a temperature controlled below 50 ℃. (3) The filtrate is purified by cation exchange resin and eluted with sodium hydroxide solution to obtain eluent; the cation exchange resin is HZ016 cation exchange resin. (4) The eluent is decolorized by ultrafiltration / nanofiltration membrane to obtain decolorized solution 1; (5) Decolorizing solution 1 is concentrated through a reverse osmosis membrane to obtain a concentrated solution; (6) Use ultrasound-assisted macroporous resin to decolorize, and obtain decolorized solution 2; (7) After the decolorizing solution 2 is filtered through a fine filter membrane, it is poured into a reaction vessel for crystallization and concentrated under vacuum to obtain wet crystals; (8) After the wet crystals are cooled to room temperature, they are filtered, washed, centrifuged and dried to obtain proline crystals.
2. The preparation method according to claim 1, characterized in that, In step (1), the pH of the fermentation broth is adjusted to 3.00~3.50 with hydrochloric acid, and the mass ratio of flocculant to fermentation broth is (2~4):1000.
3. The preparation method according to claim 1, characterized in that, In step (1), the ultrasonic treatment temperature is 25~40℃, the frequency is 40 kHz, and the treatment time is 10~20 min.
4. The preparation method according to claim 1, characterized in that, The sodium hydroxide elution concentration is 1-2%.
5. The preparation method according to claim 1, characterized in that, The ultrafiltration / nanofiltration membrane described in step (4) has a molecular weight cutoff of 500~1000 D, an operating pressure of 0.6~3.0 MPa, a flow rate of 3~5 L / h, and a temperature below 45 ℃.
6. The preparation method according to claim 1, characterized in that, The reverse osmosis membrane described in step (5) has a molecular weight cutoff of 50~100 D, an operating pressure of 2~3.5 MPa, a flow rate of 3~5 L / h, and a temperature below 45℃.
7. The preparation method according to claim 1, characterized in that, The macroporous resin mentioned in step (6) is D155 weakly acidic resin.
8. The preparation method according to claim 1, characterized in that, The ultrasonic power in step (6) is 100~200 W, the ultrasonic time is 0.5~1 h, and the ultrasonic temperature is below 40 ℃.
9. The preparation method according to claim 1, characterized in that, The vacuum concentration in step (7) has a vacuum degree of 0.09~0.098 MPa, a concentration temperature of 55~65 ℃, and a stirring speed of 100~150 r / min.
10. The preparation method according to claim 1, characterized in that, The rinsing in step (8) uses anhydrous ethanol as the rinsing solution, and the mass ratio of anhydrous ethanol to wet crystals is (1~2):1.