A method for extracting and purifying γ-polyglutamic acid using γ-polyglutamic acid fermentation broth

Abstract

This invention relates to a method for extracting and purifying γ-polyglutamic acid (PGA) from fermentation broth. The process includes fermentation broth pretreatment, metal mesh filtration, adsorption, ceramic membrane filtration, concentration, crystallization, recrystallization, and drying. This invention uses a novel adsorbent instead of conventional adsorbent materials, eliminating the necessary alcohol precipitation extraction method in traditional processes. Compared with current domestic conventional processes, this method significantly improves impurity removal and effectively purifies γ-PGA, achieving a purity of over 97%. It features high extraction yield, stable product quality, and low production costs, thus enhancing the product's competitiveness in international markets.

Description

Technical Field

[0001] This invention belongs to the field of γ-polyglutamic acid production technology, and in particular relates to a method for extracting and purifying γ-polyglutamic acid using γ-polyglutamic acid fermentation broth. Background Technology

[0002] γ-polyglutamic acid (also known as polyglutamic acid) is a water-soluble extracellular glutamic acid polymer (γ-PGA) produced by microbial fermentation. Its molecular weight ranges from 50,000 to 2 million Daltons, so it can be widely used in food, industry, cosmetics, health care, water treatment, wastewater treatment, hygiene products, medical and hydrogel fields.

[0003] In China, γ-PGA is produced using a fermentation method. However, the high viscosity of the fermentation broth makes separation and purification difficult. Domestic literature discloses methods for extracting and purifying γ-PGA from fermentation broth, including organic solvent precipitation, chemical precipitation, and membrane separation.

[0004] The main problems are:

[0005] 1. In the extraction and purification process of γ-polyglutamic acid, lower alcohols (methanol, ethanol or isopropanol) or organic solvents such as acetone are used to precipitate and remove impurities. However, this method results in a large amount of organic solvent used, often 3-4 times the volume of the fermentation broth. At the same time, some reducing sugars will also be precipitated, which affects the quality of the finished product.

[0006] 2. Using chemical precipitation to extract and purify polyglutamic acid will introduce new impurities, which is not conducive to subsequent purification. In addition, the amount of salt used is too large and causes serious environmental pollution.

[0007] 3. In China, diatomaceous earth and activated carbon are used to adsorb proteins and pigments, and γ-polyglutamic acid is also adsorbed. The adsorption amount is 1-2%, which reduces the extraction yield.

[0008] 4. Domestic literature discloses the yield and purity of γ-polyglutamic acid extracted and purified using different methods. The yield ranges from 70-90%, and the purity ranges from 90-97%. The range varies greatly, which is not conducive to large-scale industrial production.

[0009] A domestic patent, "A Method for Separating and Purifying Polyglutamic Acid Without Added Organic Solvents" (Patent No.: 201310576404.0), discloses a method that uses a mixture of diatomaceous earth and red clay for plate and frame filtration for sterilization, and combines heating with hollow fiber membrane ultrafiltration to remove impurities such as proteins and large molecular active organic matter. After activated carbon decolorization, the solution is repeatedly filtered through a nanofiltration membrane to remove most of the amino acids and salt ions. The clarified solution after the above treatment is then subjected to pressurized circulating ultrafiltration-dilution-ultrafiltration-concentration steps using 10kDa and 100kDa ultrafiltration membranes, ultimately obtaining a concentrated γ-PGA solution with very few impurities. This solution is then freeze-dried under vacuum to obtain purified white γ-PGA. Problems with this process:

[0010] 1. This patent uses organic membrane filtration technology to separate and purify γ-polyglutamic acid, which is overly dependent on membrane filtration equipment. The cost of membrane modules and corresponding auxiliary equipment is high. The membrane is prone to clogging and fouling, which affects the separation efficiency. Moreover, the membrane equipment needs to be replaced regularly, resulting in high overall production costs.

[0011] 2. During the process of removing impurities and decolorizing using diatomaceous earth, red clay, and activated carbon, γ-PGA is easily adsorbed, which reduces the extraction yield.

[0012] 3. Verification based on the process described in the patent showed that the total yield of γ-PGA was <90%, which did not reach the technical level described in the patent. Summary of the Invention

[0013] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method with simple extraction and purification steps, no use of organic solvents (lower environmental impact), and high yield and purity.

[0014] The technical solution adopted to achieve the above objectives is as follows:

[0015] A method for extracting and purifying γ-polyglutamic acid using γ-polyglutamic acid fermentation broth, comprising the following steps:

[0016] (1) Fermentation broth pretreatment

[0017] Add water to the fermentation broth once, heat the fermentation broth to 70-80℃, adjust the pH to 2.8-3.2 with 30% phosphoric acid, stir for 20-40 minutes, and let stand for 60-100 minutes; add aluminum hydroxide, adjust the pH to 7-8, stir for 20-40 minutes, and let stand for 60-100 minutes.

[0018] (2) Metal mesh filter

[0019] After the fermentation broth pretreatment is completed, it is filtered through a metal mesh, and the filtrate is collected.

[0020] (3) Adsorption

[0021] Add the filtrate from step (2) to the adsorbent, stir for 20-40 minutes, let stand for 80-100 minutes, separate the solid and liquid and collect the filtrate;

[0022] (4) Ceramic membrane filtration

[0023] The filtrate from step (3) is filtered through a ceramic membrane and the filtrate is collected.

[0024] (5) Concentration

[0025] Pour the filtrate from step (4) into a four-effect falling film evaporator for concentration and collect the concentrate;

[0026] (6) Crystallization

[0027] Start the stirring system and control the speed at 20-40 r / min. Reduce the temperature of the concentrate to 0-3℃ and control the cooling rate at 5-9℃ / h. When the solution becomes turbid, add pure γ-polyglutamic acid and continue stirring for 80-100 min. Then let it stand for 240-300 min. After solid-liquid separation, solid γ-polyglutamic acid is obtained.

[0028] (7) recrystallization

[0029] Solid γ-polyglutamic acid was added to purified water, and the concentration was controlled at 60-70 g / L. The process of step (6) was repeated to obtain solid γ-polyglutamic acid.

[0030] (8) Drying

[0031] The solid γ-polyglutamic acid in step (7) is dried at a temperature of 70-80°C until the moisture content is less than 0.5%, and then the drying is stopped to obtain pure γ-polyglutamic acid.

[0032] In step (1), the amount of water used at one time is 30-40% of the volume of the fermentation liquid.

[0033] In step (2), the metal mesh filter uses a sintered stainless steel filter element with a filter element precision of 40 μm and a filtration rate controlled at 1-3 m³ / h. During filtration, the temperature of the γ-polyglutamic acid fermentation broth is controlled at 30-35℃.

[0034] The adsorbent preparation method in step (3) includes the following steps:

[0035] 1) Weigh the clay and pass it through a 200-mesh sieve. Add purified water, stir for 20-30 minutes, and filter to obtain wet solid clay.

[0036] W clay : L purified water = 1 kg : 2-4 L.

[0037] 2) Add purified water and 80% concentrated sulfuric acid to the wet solid clay, heat to 80-85℃, stir for 300-350 min, and filter to obtain wet solid A.

[0038] W clay : L purified water = 1 kg : 1-2 L

[0039] The ratio of clay (W) to concentrated sulfuric acid (L) is 1 kg to 0.6-0.8 L.

[0040] 3) Wet solid A is washed once with purified water and filtered to obtain wet solid A.

[0041] W clay A ∶ L purified water = 1 kg ∶ 6-10 L.

[0042] 4) Add purified water, solid alcohol and acetic anhydride to wet solid A, adjust the pH to 3-3.5 with 80% concentrated sulfuric acid, stir at 40-45℃ for 450-500 min, and filter to obtain wet solid B.

[0043] W clay : L purified water = 1 kg : 1-2 L

[0044] W_clay : W_solid alcohol = 1kg : 0.4-0.6kg,

[0045] W clay : L acetic anhydride = 1 kg : 0.4-6 L.

[0046] 5) The wet solid B was washed twice with purified water, filtered, and dried to obtain solid B with a moisture content of less than 2%.

[0047] W clay : L purified water = 1 kg : 3-5 L.

[0048] 6) Chitosan is passed through a 200-mesh sieve.

[0049] 7) Add chitosan to solid B, stir until homogeneous, then add 40% glass aqueous solution and stir for 80-100 minutes. During stirring, add purified water every 20 minutes. After stirring, pulverize and sieve through a 200-mesh sieve, then dry at 70-80℃ until the moisture content is below 2%. Finally, subject the dried mixture to high-temperature heat treatment. Calcine at 250-300℃ for 6-10 minutes. After calcination, pulverize and sieve through a 200-mesh sieve again to obtain the adsorbent.

[0050] The adsorbent prepared by the above method has the following composition: W solid B : W chitosan = 1 kg : 0.3-0.5 kg, W solid B : L 40% glass water = 1 kg : 0.2-0.4 L, W solid B : L purified water = 1 kg : 60-100 ml.

[0051] Wherein, the amount of adsorbent used is: W_adsorbent (kg) = 0.29 × fermentation unit (g / L) × fermentation liquid volume (m³). 3 ).

[0052] Among them, the ceramic membrane filtration mentioned in step (4) refers to:

[0053] 1) Acidification: Heat the filtrate to 65-70℃, add 30% phosphoric acid, adjust the pH to 2-3, stir for 30-50 minutes, and keep the temperature constant.

[0054] 2) Ceramic membrane filtration: The ceramic membrane is made of inorganic materials such as lithium bromide, zirconium dioxide, and titanium dioxide, sintered at high temperature. It has a pore size of 5 nm and an operating pressure of 0.2-0.5 MPa. Add the filtrate to the feed hopper of the ceramic membrane filtration device, circulate for 2-6 minutes, begin filtration, and collect the filtrate.

[0055] In step (5), the concentration refers to the filtrate filtered through the ceramic membrane being kept at a temperature of 50-60°C, entering a four-effect falling film evaporator, with a flow rate of 6000-8000 L / h, and the concentration of the filtrate at the outlet being 2-3 times that at the inlet.

[0056] The amount of pure γ-polyglutamic acid used in step (6) is as follows:

[0057] W (pure γ-polyglutamic acid) (kg) = 0.01 × fermentation units (g / L) × fermentation broth volume (m³) 3 )

[0058] The beneficial effects of this invention patent are reflected in the following aspects:

[0059] 1. The process described in this invention does not use organic solvents, thus reducing the impact on the environment.

[0060] 2. Compared with organic membrane filtration, using composite inorganic ceramic membranes instead of organic membranes results in lower costs for membrane modules and related auxiliary equipment, while extending the maintenance and upkeep cycle of the equipment. During use, it was found that inorganic membrane filtration reduces membrane clogging and fouling, thus improving membrane efficiency.

[0061] 3. This patent uses a novel adsorbent with an adsorption rate of over 85% for impurities and around 80% for pigments, which is higher than the adsorption effect of conventional adsorbents, and the adsorption amount of γ-polyglutamic acid is less than 0.7%.

[0062] 4. Using the process described in this patent, the yield of γ-polyglutamic acid is around 90%; the purity is 97-99%. Detailed Implementation

[0063] The invention is illustrated below with examples. It should be understood that these examples are for illustrative purposes only and not for limiting the invention. The scope and core content of the invention are defined by the claims.

[0064] Currently, polyglutamic acid is produced in China using a fermentation model, which utilizes Bacillus subtilis and employs a three-stage fermentation process. The nitrogen sources in the culture medium are mainly yeast powder, peptone, and corn steep liquor, while the carbon sources are mainly glucose, monosodium glutamate, and sodium citrate.

[0065] The ceramic membrane and membrane filtration equipment were provided by Shanghai Zhizheng Membrane Filtration Equipment Co., Ltd.

[0066] Example 1

[0067] 10ml of polyglutamic acid fermentation broth 3 Fermentation unit 45.8 g / L.

[0068] (1) Fermentation broth pretreatment

[0069] Add 3ml of water to the fermentation broth once. 3 Fermentation broth volume 13m³ 3 The fermentation broth was heated to 70°C; the pH was adjusted to 2.8 using 30% phosphoric acid, stirred for 20 minutes, and allowed to stand for 60 minutes; aluminum hydroxide was added, the pH was adjusted to 7, stirred for 20 minutes, and allowed to stand for 60 minutes.

[0070] (2) Metal mesh filter

[0071] After the fermentation broth pretreatment is completed, the temperature of the fermentation broth is controlled at 30℃, and it is filtered through a metal mesh at a filtration rate of 1m / s. 3 / h. Collect 11.2m of filtrate. 3 The content was 40.16 g / L, and the yield was 98.2%.

[0072] (3) Adsorption

[0073] 132.82 kg of adsorbent was added to the filtrate, stirred for 20 min, and allowed to stand for 80 min. Solid-liquid separation yielded 11.1 ml of filtrate. 3 The content was 40.28 g / L, and the yield was 99.4%.

[0074] (4) Ceramic membrane filtration

[0075] Heat the filtrate to 65°C, add 30% phosphoric acid, adjust the pH to 2, stir for 30 minutes, and maintain the temperature.

[0076] Ceramic membrane filtration: The ceramic membrane is made of inorganic materials such as lithium bromide, zirconium dioxide, and titanium dioxide, sintered at high temperature. It has a pore size of 5 nm and an operating pressure of 0.2-0.5 MPa. Add the filtrate to the feed hopper of the ceramic membrane filtration device, circulate for 2 minutes, begin filtration, and collect the filtrate. After ceramic membrane filtration is complete, collect 11.06 ml of filtrate. 3 The content was 39.78 g / L, and the yield was 98.4%.

[0077] (5) Concentration

[0078] The filtrate was concentrated twice in a quadruple-effect falling film evaporator, and 5.52 ml of the concentrate was collected. 3 .

[0079] (6) Crystallization

[0080] Start the stirring system and control the speed at 20 r / min. Reduce the temperature of the concentrate to 3℃ and control the cooling rate at 5℃ / h. When the solution becomes turbid, add 4.6 kg of pure γ-polyglutamic acid and continue stirring for 80 min. Then let it stand for 240 min. After solid-liquid separation, 476.5 kg of wet solid γ-polyglutamic acid is obtained.

[0081] (7) recrystallization

[0082] Solid γ-polyglutamic acid was added to purified water, and the concentration was controlled at 60 g / L.

[0083] Repeat step (6) to obtain 473.1 kg of wet solid γ-polyglutamic acid.

[0084] (8) Drying

[0085] The wet solid γ-polyglutamic acid was dried at a temperature controlled at 70-80℃ until the moisture content was less than 0.5%, yielding 413.6 kg of pure γ-polyglutamic acid (excluding the pure polyglutamic acid added during the crystallization process), with a yield of 90.3% and a purity of 97.4%.

[0086] Example 2

[0087] 10ml of polyglutamic acid fermentation broth 3 The fermentation unit is 39.6 g / L.

[0088] (1) Fermentation broth pretreatment: Add 3.3 ml of water to the fermentation broth once. 3 The fermentation broth volume was 13.3 m³. 3 The fermentation broth was heated to 72°C; the pH was adjusted to 2.9 using 30% phosphoric acid, stirred for 25 minutes, and allowed to stand for 70 minutes; aluminum hydroxide was added, the pH was adjusted to 7.2, stirred for 25 minutes, and allowed to stand for 70 minutes.

[0089] (2) Metal mesh filter

[0090] After the fermentation broth pretreatment was completed, the temperature of the fermentation broth was controlled at 31℃, and it was filtered through a metal mesh at a filtration rate of 1.5m / s. 3 / h. Collect 11.5m of filtrate. 3 The content was 33.85 g / L, and the yield was 98.3%.

[0091] (3) Adsorption

[0092] 114.84 kg of adsorbent was added to the filtrate, stirred for 25 min, and allowed to stand for 85 min. Solid-liquid separation yielded 11.4 ml of filtrate. 3 The content was 33.94 g / L, and the yield was 99.4%.

[0093] (4) Ceramic membrane filtration

[0094] Heat the filtrate to 66°C, add 30% phosphoric acid, adjust the pH to 2.2, stir for 35 minutes, and maintain the temperature.

[0095] Ceramic membrane filtration: The ceramic membrane is made of inorganic materials such as lithium bromide, zirconium dioxide, and titanium dioxide, sintered at high temperature. It has a pore size of 5 nm and an operating pressure of 0.2-0.5 MPa. Add the filtrate to the feed hopper of the ceramic membrane filtration device, circulate for 3 minutes, begin filtration, and collect the filtrate. After ceramic membrane filtration is complete, collect 11.31 ml of filtrate. 3 The content was 33.70 g / L, and the yield was 98.5%.

[0096] (5) Concentration

[0097] The filtrate was concentrated 2.2 times in a quadruple-effect falling film evaporator, and 5.14 ml of the concentrate was collected. 3 .

[0098] (6) Crystallization

[0099] Start the stirring system and control the speed at 25 r / min. Reduce the temperature of the concentrate to 2℃ and control the cooling rate at 6℃ / h. When the solution becomes turbid, add 3.96 kg of pure γ-polyglutamic acid and continue stirring for 85 min. Then let it stand for 250 min. After solid-liquid separation, 398.4 kg of wet solid γ-polyglutamic acid is obtained.

[0100] (7) recrystallization

[0101] Wet solid γ-polyglutamic acid was added to purified water, and the concentration was controlled at 62 g / L.

[0102] Repeat step (6) to obtain 392.2 kg of wet solid γ-polyglutamic acid.

[0103] (8) Drying

[0104] The wet solid γ-polyglutamic acid was dried at 72°C until the moisture content reached 0.4%, yielding 358 kg of pure γ-polyglutamic acid (excluding the pure polyglutamic acid added during the crystallization process), with a yield of 90.4% and a purity of 97.6%.

[0105] Example 3

[0106] 10ml of polyglutamic acid fermentation broth 3 Fermentation unit 41.4 g / L.

[0107] (1) Fermentation broth pretreatment

[0108] Add 3.5 ml of water to the fermentation broth once. 3 Fermentation broth volume: 13.5 m³ 3 The fermentation broth was heated to 75°C; the pH was adjusted to 3.0 with 30% phosphoric acid, stirred for 30 min, and allowed to stand for 80 min; aluminum hydroxide was added, the pH was adjusted to 7.5, stirred for 30 min, and allowed to stand for 80 min.

[0109] (2) Metal mesh filter

[0110] After the fermentation broth pretreatment is completed, the temperature of the fermentation broth is controlled at 32℃, and it is filtered through a metal mesh at a filtration rate of 2m / s. 3 / h. Collect 11.7m of filtrate. 3 The content was 34.89 g / L, and the yield was 98.6%.

[0111] (3) Adsorption

[0112] 120 kg of adsorbent was added to the filtrate, stirred for 30 min, and allowed to stand for 90 min. Solid-liquid separation yielded 11.6 ml of filtrate. 3 The content was 35.01 g / L, and the yield was 99.5%.

[0113] (4) Ceramic membrane filtration

[0114] Heat the filtrate to 67°C, add 30% phosphoric acid, adjust the pH to 2.5, stir for 40 minutes, and maintain the temperature.

[0115] Ceramic membrane filtration: The ceramic membrane is made of inorganic materials such as lithium bromide, zirconium dioxide, and titanium dioxide, sintered at high temperature. It has a pore size of 5 nm and an operating pressure of 0.2-0.5 MPa. Add the filtrate to the feed hopper of the ceramic membrane filtration device, circulate for 4 minutes, begin filtration, and collect the filtrate. After ceramic membrane filtration is complete, collect 11.51 ml of filtrate. 3 The content was 34.82 g / L, and the yield was 98.7%.

[0116] (5) Concentration

[0117] The filtrate was concentrated 2.5 times in a quadruple-effect falling film evaporator, and 4.6 ml of the concentrated liquid was collected. 3 .

[0118] (6) Crystallization

[0119] Start the stirring system and control the speed at 30 r / min. Reduce the temperature of the concentrate to 1℃ and control the cooling rate at 7℃ / h. When the solution becomes turbid, add 4.14 kg of pure γ-polyglutamic acid and continue stirring for 90 min. Then let it stand for 260 min. After solid-liquid separation, 410.34 kg of wet solid γ-polyglutamic acid is obtained.

[0120] (7) recrystallization

[0121] Wet solid γ-polyglutamic acid was added to purified water, and the concentration was controlled at 65 g / L.

[0122] Repeat step (6) to obtain 413.8 kg of wet solid γ-polyglutamic acid.

[0123] (8) Drying

[0124] The wet solid γ-polyglutamic acid was dried at 72°C until the moisture content reached 0.3%, yielding 378 kg of pure γ-polyglutamic acid (excluding the pure polyglutamic acid added during the crystallization process), with a yield of 91.3% and a purity of 98.8%.

[0125] Example 4

[0126] 10ml of polyglutamic acid fermentation broth 3 Fermentation unit 42.6 g / L.

[0127] (1) Fermentation broth pretreatment

[0128] Add 3.8 ml of water to the fermentation broth once. 3 Fermentation broth volume: 13.8 m³ 3 The fermentation broth was heated to 77°C; the pH was adjusted to 3.1 using 30% phosphoric acid, stirred for 35 min, and allowed to stand for 90 min; aluminum hydroxide was added, the pH was adjusted to 7.8, stirred for 35 min, and allowed to stand for 90 min.

[0129] (2) Metal mesh filter

[0130] After the fermentation broth pretreatment was completed, the temperature of the fermentation broth was controlled at 34℃, and it was filtered through a metal mesh at a filtration rate of 2.5m / s. 3 / h. Collect 12m of filtrate. 3 The content was 34.79 g / L, and the yield was 98%.

[0131] (3) Adsorption

[0132] 123 kg of adsorbent was added to the filtrate, stirred for 35 min, and allowed to stand for 95 min. Solid-liquid separation yielded 11.9 ml of filtrate. 3 The content was 34.87 g / L, and the yield was 99.4%.

[0133] (4) Ceramic membrane filtration

[0134] Heat the filtrate to 68°C, add 30% phosphoric acid to adjust the pH to 2.8, stir for 45 minutes, and maintain the temperature.

[0135] Ceramic membrane filtration: The ceramic membrane is made of inorganic materials such as lithium bromide, zirconium dioxide, and titanium dioxide, sintered at high temperature. It has a pore size of 5 nm and an operating pressure of 0.2-0.5 MPa. Add the filtrate to the feed hopper of the ceramic membrane filtration device, circulate for 5 minutes, begin filtration, and collect the filtrate. After ceramic membrane filtration is complete, collect 11.82 ml of filtrate. 3 The content was 34.58 g / L, and the yield was 98.5%.

[0136] (5) Concentration

[0137] The filtrate was concentrated 2.8 times in a quadruple-effect falling film evaporator, and 4.2 ml of the concentrated liquid was collected. 3 .

[0138] (6) Crystallization

[0139] Start the stirring system and control the speed at 35 r / min. Reduce the temperature of the concentrate to 1℃ and control the cooling rate at 8℃ / h. When the solution becomes turbid, add 4.26 kg of pure γ-polyglutamic acid and continue stirring for 95 min. Then let it stand for 270 min. After solid-liquid separation, 415.2 kg of wet solid γ-polyglutamic acid is obtained.

[0140] (7) recrystallization

[0141] Wet solid γ-polyglutamic acid was added to purified water, and the concentration was controlled at 67 g / L.

[0142] Repeat step (6) to obtain 416.4 kg of wet solid γ-polyglutamic acid.

[0143] (8) Drying

[0144] The wet solid γ-polyglutamic acid was dried at 78°C until the moisture content reached 0.3%, yielding 384.2 kg of pure γ-polyglutamic acid (excluding the pure polyglutamic acid added during the crystallization process), with a yield of 90.2% and a purity of 98.4%.

[0145] Example 5

[0146] 10ml of polyglutamic acid fermentation broth 3 Fermentation unit 37.5 g / L.

[0147] (1) Fermentation broth pretreatment

[0148] Add 4ml of water to the fermentation broth once. 3 Fermentation broth volume 14m³ 3 The fermentation broth was heated to 80°C; the pH was adjusted to 3.2 using 30% phosphoric acid, stirred for 40 minutes, and allowed to stand for 100 minutes; aluminum hydroxide was added, the pH was adjusted to 8.0, stirred for 40 minutes, and allowed to stand for 100 minutes.

[0149] (2) Metal mesh filter

[0150] After the fermentation broth pretreatment is completed, the temperature of the fermentation broth is controlled at 35℃, and it is filtered through a metal mesh at a filtration rate of 3m / s. 3 / h. Collect 12.1m of filtrate. 3 The content was 30.43 g / L, and the yield was 98.2%.

[0151] (3) Adsorption

[0152] Add 108.8 kg of adsorbent to the filtrate, stir for 40 min, let stand for 100 min, and then separate the solid and liquid to obtain 12 ml of filtrate. 3 The content was 30.53 g / L, and the yield was 99.5%.

[0153] (4) Ceramic membrane filtration

[0154] Heat the filtrate to 70°C, add 30% phosphoric acid, adjust the pH to 3.0, stir for 50 minutes, and maintain the temperature.

[0155] Ceramic membrane filtration: The ceramic membrane is made of inorganic materials such as lithium bromide, zirconium dioxide, and titanium dioxide, sintered at high temperature. It has a pore size of 5 nm and an operating pressure of 0.2-0.5 MPa. Add the filtrate to the feed hopper of the ceramic membrane filtration device, circulate for 6 minutes, begin filtration, and collect the filtrate. After ceramic membrane filtration is complete, collect 11.93 ml of filtrate. 3 The content was 30.13 g / L, and the yield was 98.1%.

[0156] (5) Concentration

[0157] The filtrate was concentrated three times in a quadruple-effect falling film evaporator, and 4 ml of the concentrate was collected. 3 .

[0158] (6) Crystallization

[0159] Start the stirring system and control the speed at 40 r / min. The temperature of the concentrate drops to 0℃ and the cooling rate is controlled at 9℃ / h. When the solution becomes turbid, add 3.75 kg of pure γ-polyglutamic acid and continue stirring for 100 min. Then let it stand for 280 min. After solid-liquid separation, 367.3 kg of wet solid γ-polyglutamic acid is obtained.

[0160] (7) recrystallization

[0161] Wet solid γ-polyglutamic acid was added to purified water, and the concentration was controlled at 70 g / L.

[0162] Repeat step (6) to obtain 368.5 kg of wet solid γ-polyglutamic acid.

[0163] (8) Drying

[0164] The wet solid γ-polyglutamic acid was dried at 80℃ until the moisture content reached 0.2%, yielding 340.1 kg of pure γ-polyglutamic acid (excluding the pure polyglutamic acid added during the crystallization process), with a yield of 90.7% and a purity of 98.1%.

[0165] Example 6

[0166] 10ml of polyglutamic acid fermentation broth 3 The fermentation unit is 38.6 g / L.

[0167] (1) Fermentation broth pretreatment

[0168] Add 4ml of water to the fermentation broth once. 3 Fermentation broth volume 14m³ 3 The fermentation broth was heated to 70°C; the pH was adjusted to 3 using 30% phosphoric acid, stirred for 30 minutes, and allowed to stand for 100 minutes; aluminum hydroxide was added, the pH was adjusted to 7.6, stirred for 35 minutes, and allowed to stand for 100 minutes.

[0169] (2) Metal mesh filter

[0170] After the fermentation broth pretreatment was completed, the temperature of the fermentation broth was controlled at 33℃, and it was filtered through a metal mesh at a filtration rate of 2.3 m / s². 3 / h. Collect 12m of filtrate. 3 The content was 31.62 g / L, and the yield was 98.3%.

[0171] (3) Adsorption

[0172] 112 kg of adsorbent was added to the filtrate, stirred for 35 min, and allowed to stand for 100 min. Solid-liquid separation yielded 11.92 ml of filtrate. 3 The content was 31.64 g / L, and the yield was 99.4%.

[0173] (4) Ceramic membrane filtration

[0174] Heat the filtrate to 66°C, add 30% phosphoric acid, adjust the pH to 2.9, stir for 45 minutes, and maintain the temperature.

[0175] Ceramic membrane filtration: The ceramic membrane is made of inorganic materials such as lithium bromide, zirconium dioxide, and titanium dioxide, sintered at high temperature. It has a pore size of 5 nm and an operating pressure of 0.2-0.5 MPa. Add the filtrate to the feed hopper of the ceramic membrane filtration device, circulate for 5 minutes, begin filtration, and collect the filtrate. After ceramic membrane filtration is complete, collect 11.89 ml of filtrate. 3 The content was 31.15 g / L, and the yield was 98.2%.

[0176] (5) Concentration

[0177] The filtrate was concentrated 2.7 times in a quadruple-effect falling film evaporator, and 4.4 ml of the concentrated liquid was collected. 3 .

[0178] (6) Crystallization

[0179] Start the stirring system and control the speed at 40 r / min. Reduce the temperature of the concentrate to 1℃ and control the cooling rate at 8℃ / h. When the solution becomes turbid, add 3.86 g of pure γ-polyglutamic acid and continue stirring for 80 min. Then let it stand for 250 min. After solid-liquid separation, 379.7 kg of wet solid γ-polyglutamic acid is obtained.

[0180] (7) recrystallization

[0181] Wet solid γ-polyglutamic acid was added to purified water, and the concentration was controlled at 70 g / L.

[0182] Repeat step (6) to obtain 380.2 kg of wet solid γ-polyglutamic acid.

[0183] (8) Drying

[0184] The wet solid γ-polyglutamic acid was dried at 80℃ until the moisture content reached 0.2%, yielding 348.9 kg of pure γ-polyglutamic acid (excluding the pure polyglutamic acid added during the crystallization process), with a yield of 90.4% and a purity of 98.3%.

[0185] Comparative Example 1

[0186] 1m of polyglutamic acid fermentation broth 3 Fermentation unit 40.6 g / L.

[0187] The fermentation broth was centrifuged at 5000 rpm for 20 min to remove bacterial cells, yielding 0.74 ml of supernatant. 3 The test results showed that the content of γ-polyglutamic acid in the supernatant was 51.41 g / L, with a yield of 93.7%.

[0188] Reverse micelle extraction:

[0189] (a) Preparation of reverse micelle solution: The pre-extraction solution was: 100 mmol / L hexadecyltrimethylammonium bromide (CTAB) / 80% (v / v) isooctane / 5% (v / v) n-hexanol / 15% (v / v) n-butanol; the reverse extraction solution was: acetate buffer solution with pH 4.2 containing 0.5 mol / L KBr;

[0190] (b) Supernatant pretreatment: Add 0.1 mol / L NaCl to the supernatant containing γ-polyglutamic acid obtained in step 2, and adjust the pH to 6 with 6 mol / L dilute hydrochloric acid;

[0191] (c) Reverse micelle extraction: Mix an equal volume of the pre-extraction solution with the pretreated supernatant for 5 min; then add the reverse extraction solution and mix for 10 min. After phase separation (25℃), an aqueous phase containing γ-polyglutamic acid is obtained.

[0192] The aqueous phase was placed in a 7000Dal dialysis bag and dialyzed in distilled water for 48 hours, with the distilled water being changed every 4 hours. The concentrated dialysis solution was then vacuum dried to obtain crude γ-polyglutamic acid.

[0193] Crude γ-polyglutamic acid was dissolved in distilled water, and the resulting solution was placed in a dialysis bag and dialyzed repeatedly with distilled water for 48 hours to remove small molecules and organic solvents. The concentrated solution was then freeze-dried under vacuum to obtain pure γ-polyglutamic acid with a yield of 307 kg, an extraction rate of 75.5%, and a purity of 96.1%.

[0194] Comparative Example 2

[0195] 500L of γ-polyglutamic acid fermentation broth (fermentation unit 29.4g / L) was transferred to a jacketed stirred tank. 1mol / L dilute sulfuric acid was added to adjust the pH to 5.0. After stirring evenly, the mixture was heated to 50℃. Then, 2 times the volume of food-grade ethanol was added, and the mixture was stirred at 1500rpm for 3 hours. After natural sedimentation, the supernatant was removed. The granular precipitate was washed twice with food-grade ethanol to remove floating color. At this point, a light yellow granular crude product was obtained.

[0196] The pale yellow granular crude product was dissolved in twice the volume of distilled water of the fermentation broth by rapid stirring at 60°C. Then, 25 kg of a composite diatomaceous earth (15 kg of 200-mesh diatomaceous earth and 10 kg of 100-mesh diatomaceous earth) and 200 g of 150-mesh activated carbon were added for adsorption. After thorough mixing, the mixture was filtered while hot. A filter cake of approximately 1 cm thickness was prepared using 200-mesh diatomaceous earth in a positive pressure filter. The mixture was then added to the filter for filtration at a pressure ≤0.5 MPa. The filtrate was collected at a filtration rate of 30 L / h. After filtration, 236 L of filtrate was obtained, containing 51.14 g / L of γ-polyglutamic acid, with a yield of 82.1%.

[0197] The filtered filtrate was directly subjected to vacuum distillation. In addition to concentration, this process also removed the trace amounts of ethanol contained in the filtrate. The liquid volume in the apparatus was 50%, and the temperature was controlled at 55°C until the polyglutamic acid content in the filtrate reached 8% (w / v). After removal, it was freeze-dried to obtain 11.4 kg of solid polyglutamic acid, with a total yield of 77.5% and a purity of 91.4%.

[0198] Comparative Example 3

[0199] 1m of polyglutamic acid fermentation broth 3 The fermentation unit is 37.2 g / L.

[0200] ① Acidification treatment of fermentation broth: Adjust the pH of fermentation broth to 3.0;

[0201] ② Sterilization of fermentation broth: Microfiltration membrane was used to remove bacterial cells; 0.76 ml of filtrate was obtained. 3 The content was 46.25 g / L.

[0202] ③ Ultrafiltration concentration: Add 3-10 times the volume of pure water to dilute, use an ultrafiltration membrane to concentrate and remove impurities from the diluted sterile fermentation broth, and repeat the dilution and concentration steps 3 times until the volume of the concentrated broth is 20% of the original fermentation broth.

[0203] ④ Separation and purification of γ-polyglutamic acid: After adjusting the pH of the ultrafiltration concentrated liquid to 3.0, precipitation was directly carried out using 95% ethanol at a volume of 2.1 mg / L. 3 The precipitate was collected, vacuum dried, and finally 34.12 kg of γ-polyglutamic acid product was obtained, with a yield of 88.4% and a purity of 96.2%.

[0204] Comparison Implementation 4

[0205] 1m of polyglutamic acid fermentation broth 3 Fermentation unit 35.5 g / L.

[0206] The pH of the fermentation broth was adjusted to 3.0, and 2% (20 kg) of diatomaceous earth (by volume percentage) of the fermentation broth was added. The mixture was then filtered through an 800-mesh filter cloth using a plate and frame filter to remove bacteria, yielding a filtrate of 0.72 ml.3 The content was 46.89 g / L, and the yield was 95.1%.

[0207] The decolorization method for polyglutamic acid fermentation broth is as follows:

[0208] Add 1% (10 kg) activated carbon, stir slowly at room temperature, decolorize for 90 min, filter under vacuum until the liquid is basically clear and colorless; then dilute the decolorized solution by 2 times and pass it through a nanofiltration membrane to obtain a 0.6 m solution of polyglutamic acid. 3 The content was 53.5 g / L, and the yield was 94.3%.

[0209] The concentration of the polyglutamic acid solution was adjusted to 10 g / L, and ultrafiltration was performed using a 10 kDa ultrafiltration membrane.

[0210] After ultrafiltration, freeze-drying was performed to obtain 30 kg of solid γ-polyglutamic acid product, with a yield of 84.5% and a purity of 95.7%.

[0211] Comparative Example 5

[0212] 1m of polyglutamic acid fermentation broth 3 Fermentation unit 42.7 g / L.

[0213] (1)Bacterial removal

[0214] The pH of the fermentation broth was adjusted to 4.5. Diatomaceous earth and red clay were mixed in a 2:1 ratio and added at a rate of 2% (20 kg). The mixture was filtered through an 800-mesh filter cloth at a pressure of 0.22 MPa using a plate and frame filter press to remove bacteria. The filtrate volume was 0.71 m³. 3 The content was 56.71 g / L, and the yield was 94.3%.

[0215] (2) Remove impurities such as proteins and macromolecular active organic matter.

[0216] The filtrate was adjusted to pH 6.5 with sodium hydroxide, heated to 90°C and held for 20 minutes, then cooled to room temperature and ultrafiltered through a 0.45µm hollow fiber membrane to remove more than 80% of impurities and proteins. The filtrate volume was 0.69 ml. 3 The content was 57.3 g / L, and the yield was 98.2%.

[0217] (3) Decolorization

[0218] The filtrate was treated with 1% activated carbon (6.9 kg) and slowly stirred at room temperature for 90 min to decolorize. The solution was then filtered to obtain 0.69 ml of filtrate. 3 The content was 56.5 g / L, and the yield was 98.6%.

[0219] (4) Remove small molecule amino acids and ionic impurities

[0220] The filtrate was diluted 2.5 times and passed through a 0.01 μm nanofiltration membrane. The permeate was then passed through a 0.01 μm nanofiltration membrane again, yielding 1.7 m of clear liquid. 3 The content was 22.34 g / L, and the yield was 97.4%.

[0221] (5) Ultrafiltration-concentration-ultrafiltration purification

[0222] The filtrate was subjected to 100 kDa ultrafiltration to obtain a filtrate volume of 1.67 m³. 3 Continue with 100 kDa ultrafiltration to obtain 1.65 ml of filtrate. 3 Continue ultrafiltration using a 100 kDa ultrafiltration membrane, ultimately obtaining a 1.62 m... 3 Dilute to 3m 3 Ultrafiltration was performed using a 10 kDa ultrafiltration membrane, yielding a filtrate of 2.5 ml. 3 And concentrated to 1m 3 After vacuum freeze-drying, 34.7 kg of solid γ-polyglutamic acid was obtained, with a yield of 81.3% and a purity of 94.6%.

Claims

1. A method for extracting and purifying γ-polyglutamic acid from a γ-polyglutamic acid fermentation broth, characterized by Process steps are: (1) fermentation broth pretreatment Add water to the fermentation broth, heat to 70-80℃, adjust pH to 2.8-3.2 with 30% phosphoric acid, stir for 20-40min, stand for 60-100min; add aluminum hydroxide, adjust pH to 7-8, stir for 20-40min, stand for 60-100min; (2) metal mesh filtration After the fermentation broth pretreatment is completed, filter through a metal mesh, and collect the filtrate; (3) adsorption Add the filtrate in step (2) to the adsorbent, stir for 20-40min, stand for 80-100min, separate the solid and liquid and collect the filtrate, wherein the preparation method of the adsorbent comprises the following steps: 1) weigh the clay and pass it through a 200 mesh sieve, add purified water, stir for 20-30min, filter to obtain wet solid clay; Wclay:Lpurified water=1kg:2-4L; 2) add purified water and 80% concentrated sulfuric acid to the wet solid clay, heat to 80-85℃, stir for 300-350min, filter to obtain wet solid A; Wclay:Lpurified water=1kg:1-2L, Wclay:Lconcentrated sulfuric acid=1kg:0.6-0.8L; 3) wash the wet solid A with purified water once and filter to obtain wet solid A; WclayA:Lpurified water=1kg:6-10L; 4) add purified water, solid alcohol and acetic anhydride to the wet solid A, add 80% concentrated sulfuric acid to adjust pH to 3-3.5, stir at 40-45℃ for 450-500min, filter to obtain wet solid B; Wclay:Lpurified water=1kg:1-2L, Wclay:Wsolid alcohol=1kg:0.4-0.6kg, Wclay:Lacetic anhydride=1kg:0.4-6L; 5) wash the wet solid B with purified water twice, filter and dry to obtain solid B, with moisture content less than 2%; Wclay:Lpurified water=1kg:3-5L; 6) pass the chitosan through a 200 mesh sieve; 7) add chitosan to the solid B, stir evenly, then add 40% glass water solution, stir for 80-100min; during the stirring process, add purified water every 20min, after the stirring is completed, crush and sieve through a 200 mesh sieve, dry at 70-80℃ until the moisture content is less than 2%; finally, heat treat the dried mixture at high temperature; calcine at 250-300℃ for 6-10min; after the calcination is completed, crush and sieve through a 200 mesh sieve to obtain the adsorbent; (4) ceramic membrane filtration Filter the filtrate in step (3) through a ceramic membrane, and collect the filtrate; (5) concentration Pour the filtrate in step (4) into a four-effect falling film evaporator to concentrate and collect the concentrated liquid; (6) crystallization Start the stirring system, control the rotation speed at 20-40r / min, reduce the temperature of the concentrated liquid to 0-3℃, control the cooling speed at 5-9℃ / h, when the solution appears turbid, add pure γ-polyglutamic acid, continue to stir for 80-100min, then stand for 240-300min, separate the solid and liquid to obtain solid γ-polyglutamic acid; (7) recrystallization Solid γ-polyglutamic acid is added to purified water, and the concentration is controlled at 60-70 g / L, and the process of step (6) is repeated to obtain solid γ-polyglutamic acid; (8) drying The solid γ-polyglutamic acid in step (7) is subjected to drying treatment, and the temperature is controlled at 70-80℃ until the moisture content is less than 0.5%, and the drying is stopped to obtain pure γ-polyglutamic acid.

2. The method for extracting and purifying γ-polyglutamic acid according to claim 1, characterized by The amount of water used in step (1) is 30-40% of the volume of the fermentation broth.

3. The method for extracting and purifying γ-polyglutamic acid according to claim 1, characterized by In step (2), the metal mesh filter uses a sintered mesh stainless steel filter element with a precision of 40um, and the filtration rate is controlled at 1-3 m3 / h; during filtration, the temperature of the γ-polyglutamic acid fermentation broth is controlled at 30-35℃.

4. The method for extracting and purifying γ-polyglutamic acid according to claim 1, characterized by In step (3), the adsorbent composition is: W solid B: W chitosan = 1 kg: 0.3-0.5 kg, W solid B: L 40% glass water = 1 kg: 0.2-0.4 L, W solid B: L purified water = 1 kg: 60-100 ml.

5. The method for extracting and purifying γ-polyglutamic acid according to claim 1, characterized by In step (3), the amount of adsorbent used is: W adsorbent (kg) = 0.29 x fermentation unit (g / L) x fermentation broth volume (m3).

6. The method for extracting and purifying γ-polyglutamic acid according to claim 1, characterized by In step (4), the ceramic membrane filtration refers to: 1) Acidification: heat the filtrate to 65-70℃, add 30% phosphoric acid, adjust the pH to 2-3, stir for 30-50 min, and maintain the temperature; 2) Ceramic membrane filtration: the ceramic membrane material is one or more inorganic materials such as lithium bromide, zirconium dioxide, and titanium dioxide sintered at high temperature, with a pore size of 5 nm and an operating pressure of 0.2-0.5 MPa; add the filtrate to the feed hopper of the ceramic membrane filtration device, and circulate for 2-6 min, start filtration and collect the filtrate.

7. The method for extracting and purifying γ-polyglutamic acid according to claim 1, characterized by In step (5), the concentration refers to the temperature of the filtrate after ceramic membrane filtration being controlled at 50-60℃, entering the four-effect falling film evaporator, and the flow rate being controlled at 6000-8000 L / h, and the concentration of the filtrate at the outlet being 2-3 times that of the inlet.

8. The method for extracting and purifying γ-polyglutamic acid according to claim 1, characterized by In step (6), the amount of pure γ-polyglutamic acid used is: W pure γ-polyglutamic acid (kg) = 0.01 x fermentation unit (g / L) x fermentation broth volume (m3).