A method for decolorizing γ-polyglutamic acid fermentation broth

Abstract

The present invention provides a method for decolorizing γ-polyglutamic acid fermentation broth, comprising: four steps of resin decolorization, activated carbon decolorization, glutamic acid sedimentation decolorization, and membrane filtration treatment decolorization. By means of the combined decolorization process of the present invention, the decolorization rate of the γ-polyglutamic acid fermentation broth can reach over 95%, the recovery rate of γ-polyglutamic acid can reach over 92%, the protein removal rate reaches over 67%, and the recovery rate of glutamic acid can reach over 90%. The decolorization method provided by the present invention has a novel route, uses less decolorizing agent, does not use organic solvents, has no high-temperature heating, has a low degree of damage to high-molecular-weight γ-PGA, and can realize the recycling of the fermentation raw material glutamic acid. The present invention is suitable for large-scale industrial production.

Description

Technical Field

[0001] The present invention belongs to the technical field of separation and extraction of γ-polyglutamic acid, and more specifically, relates to a method for decolorizing γ-polyglutamic acid fermentation broth. Background Art

[0002] γ-Polyglutamic acid is a biological macromolecular amino acid anionic polymer, and its relative molecular weight is generally 100 kDa - 2000 kDa. Due to its good water absorption and retention, ion adsorption, non-toxic and harmless properties, etc., it is widely used in the fields of food, medicine, agriculture, etc.

[0003] It is reported that there are two types of γ-polyglutamic acid-producing strains: strains that do not require the provision of glutamic acid and rely only on the self-metabolism of the strains to synthesize γ-polyglutamic acid are called glutamic acid-independent strains; strains that require the artificial provision of glutamic acid to synthesize γ-polyglutamic acid are called glutamic acid-dependent strains. Yang Qi et al. (CN118086418A) used glutamic acid-independent strains to produce γ-polyglutamic acid, and the final yield was 23.8 g / L, which was lower than the generally reported glutamic acid-dependent strains. For example, the glutamic acid-dependent strain CGMCC NO.23967 reported in CN114456980A had a yield of 86.3 g / L. Due to the high yield of glutamic acid-dependent strains, most factories currently use glutamic acid-dependent strains to produce γ-polyglutamic acid.

[0004] Due to the extremely large molecular weight of γ-polyglutamic acid, the viscosity of the fermentation broth is increased, which increases the difficulty in the separation and extraction process. Glutamic acid-dependent strains need to add a large amount of glutamic acid or sodium glutamate to the culture medium during the fermentation process, and a large amount of glutamic acid will remain after the fermentation, resulting in waste of raw materials and environmental pollution if not treated and discharged.

[0005] Zhang Linjun et al. (CN104804183A) used activated carbon for decolorization under acidic conditions at 80°C, and then adjusted the pH of the fermentation broth to 2.5 to collect the precipitate. Wang Qingbo et al. (CN114774488A) first heated the γ-polyglutamic acid fermentation broth to 121°C, cooled it, added 1% (w / v) activated carbon, and the decolorization time was 1 hour, and then used plate and frame filtration to obtain the feed liquid.

[0006] The decolorization methods reported above mainly have problems such as a large amount of decolorizing agent used, high decolorization temperature, and serious hydrolysis of high-molecular-weight γ-polyglutamic acid. None of the above methods involve the step of recycling glutamic acid.

[0007] At present, the research on γ-polyglutamic acid focuses on the fields of production and synthesis of γ-polyglutamic acid, optimization of fermentation conditions, and product technology applications, etc. There is relatively little research on the separation and extraction of γ-polyglutamic acid. Therefore, for the production of γ-polyglutamic acid by glutamate-dependent strains, a separation and extraction method with less decolorizing agent consumption, recyclable raw materials, and no damage to the original molecular weight is required. Summary of the Invention

[0008] The purpose of the present invention is to overcome the problems in the prior art and provide a method for decolorizing γ-polyglutamic acid fermentation broth.

[0009] Specifically, the method of the present invention can achieve extremely high decolorization rate, recovery rate, and protein removal rate under the conditions of not using organic solvents, without high-temperature heating, and low consumption of decolorizing agent, with low damage to high-molecular-weight γ-polyglutamic acid, and the recycling of glutamic acid can be realized.

[0010] In order to achieve the above purpose, a method for decolorizing γ-polyglutamic acid fermentation broth of the present invention includes the following steps:

[0011] Step 1: Resin decolorization: Adjust the pH value of the sterilized γ-polyglutamic acid fermentation broth, add resin, and separate the resin from the fermentation broth after decolorization.

[0012] Step 2: Activated carbon decolorization: Adjust the pH value of the fermentation broth decolorized in Step 1, add activated carbon, and separate the activated carbon from the fermentation broth after decolorization.

[0013] Preferably, any of the above-mentioned methods for decolorizing γ-polyglutamic acid fermentation broth further includes: Step 3: Glutamic acid sedimentation decolorization: Adjust the pH value of the fermentation broth decolorized in Step 2, cool down, stir, crystallize, and sediment to obtain wet glutamic acid, and redissolve the sedimented wet glutamic acid.

[0014] Preferably, any of the above-mentioned methods for decolorizing γ-polyglutamic acid fermentation broth further includes:

[0015] Step 4: Membrane filtration treatment for decolorization: Use membrane filtration equipment to separate γ-polyglutamic acid and achieve further decolorization.

[0016] Preferably, any of the above-mentioned methods for decolorizing γ-polyglutamic acid fermentation broth further includes Step 3 and Step 4. After glutamic acid sedimentation decolorization in Step 3, wet glutamic acid and decolorized liquid (supernatant) are obtained, and the sedimented wet glutamic acid is redissolved to obtain a wet glutamic acid redissolution solution. The membrane filtration treatment step in Step 4 is preferably:

[0017] Step 4: Separate glutamic acid from γ-polyglutamic acid using the wet glutamic acid redissolution solution in Step 3 with membrane filtration equipment; Separate the supernatant in Step 3 with membrane filtration equipment and achieve further decolorization.

[0018] In the present invention, the decolorization sequence of Step 1 and Step 2 can be swapped, and the present invention is equally applicable to the fermentation broth produced by glutamate-dependent strains and the fermentation broth produced by non-glutamate-dependent strains.

[0019] In a preferred embodiment of the present invention, for the decolorization and purification of the fermentation broth produced by glutamate-dependent strains, preferably the glutamate residue is greater than 20 g / L. The method for decolorizing a γ-polyglutamic acid fermentation broth includes the following steps:

[0020] Step 1: Resin decolorization: Adjust the pH value of the sterilized γ-polyglutamic acid fermentation broth, add resin, and separate the resin from the fermentation broth after decolorization;

[0021] Step 2: Activated carbon decolorization: Adjust the pH value of the fermentation broth decolorized in Step 1, add activated carbon, and separate the activated carbon from the fermentation broth after decolorization;

[0022] Step 3: Glutamate sedimentation decolorization: Adjust the pH value of the fermentation broth decolorized in Step 2, cool down, stir, crystalize, sediment to obtain wet glutamate, and redissolve the sedimented wet glutamate.

[0023] In Step 3, the glutamate sedimentation not only removes glutamate from the fermentation broth but also takes away pigments, achieving the technical effect of decolorization.

[0024] After Step 3, the decolorized fermentation broth (supernatant) is the purified γ-polyglutamic acid of the present invention.

[0025] Using this preferred embodiment, the decolorization rate of the fermentation broth is not less than 83.57%, the recovery rate of polyglutamic acid is not less than 89.78%, the protein removal rate is not less than 60.67%, and the glutamate recovery rate is not less than 52.94%.

[0026] In a preferred embodiment of the present invention, for the decolorization and purification of the fermentation broth produced by glutamate-dependent strains, preferably the glutamate residue is greater than 20 g / L. The method for decolorizing a γ-polyglutamic acid fermentation broth includes the following steps:

[0027] Step 1: Resin decolorization: Adjust the pH value of the sterilized γ-polyglutamic acid fermentation broth, add resin, and separate the resin from the fermentation broth after decolorization;

[0028] Step 2: Activated carbon decolorization: Adjust the pH value of the fermentation broth decolorized in Step 1, add activated carbon, and separate the activated carbon from the fermentation broth after decolorization;

[0029] Step 3: Glutamate sedimentation decolorization: Adjust the pH value of the fermentation broth decolorized in Step 2, cool down, stir, crystalize, sediment to obtain wet glutamate and decolorized liquid (supernatant), and redissolve the sedimented wet glutamate to obtain a wet glutamate redissolved solution;

[0030] Step 4: Decolorization by membrane filtration treatment: Use a membrane filtration device to separate γ-polyglutamic acid from the wet glutamic acid complex solution in Step 3; use a membrane filtration device to separate γ-polyglutamic acid from the supernatant of Step 3 and achieve further decolorization. Among them:

[0031] In order to recover a small amount of γ-polyglutamic acid that may remain in the wet glutamic acid obtained in Step 3.

[0032] In Step 4, use a membrane filtration device to separate γ-polyglutamic acid from the wet glutamic acid complex solution in Step 3; use a membrane filtration device to separate γ-polyglutamic acid from the supernatant of Step 3 and achieve further decolorization; after separation by the membrane filtration device, the retentate is γ-polyglutamic acid with a large molecular weight, and the permeate is glutamic acid. Add the permeate, that is, glutamic acid, to the γ-polyglutamic acid fermentation system for subsequent fermentation culture. The obtained retentate is the purified γ-polyglutamic acid product.

[0033] Using this preferred embodiment, the decolorization rate of the fermentation broth is not less than 95.23%, the recovery rate of polyglutamic acid is not less than 92.01%, the protein removal rate is not less than 67.86%, and the recovery rate of glutamic acid is not less than 90%.

[0034] In a preferred embodiment of the present invention, the method for decolorizing a γ-polyglutamic acid fermentation broth includes the following steps. Preferably, the following method is applied to the fermentation broth of non-dependent fermentation production; preferably, the following method is applied to the fermentation broth of glutamate-dependent fermentation production and the residual amount of glutamate is less than 20 g / L:

[0035] Step 1: Decolorization with resin: Adjust the pH value of the sterilized γ-polyglutamic acid fermentation broth, add resin, and separate the resin from the fermentation broth after decolorization;

[0036] Step 2: Decolorization with activated carbon: Adjust the pH value of the fermentation broth decolorized in Step 1, add activated carbon, and separate the activated carbon from the fermentation broth after decolorization;

[0037] Step 4: Decolorization by membrane filtration treatment: Use a membrane filtration device to separate γ-polyglutamic acid from the fermentation broth decolorized in Step 2 and achieve further decolorization.

[0038] After separation by the membrane filtration device, the retentate is γ-polyglutamic acid with a large molecular weight.

[0039] The decolorization mechanism of the present invention is as follows: there is an electrostatic attraction between acidic pigment molecules and weakly basic resins. The resin is a macroporous resin, and there is also van der Waals force to adsorb the pigments in the fermentation broth. The adsorption process fits better with the Freundlich model than the Langmuir model, so the pigments are adsorbed on the resin in multiple layers; the activated carbon surface has a microporous structure and a large adsorption surface area, which can quickly adsorb pigments. The adsorption process fits better with the Langmuir model than the Freundlich model, so the pigments are adsorbed on the surface of the activated carbon in a single layer; the decolorization of glutamic acid sedimentation relies on electrostatic force to carry some pigments to sediment together during the sedimentation of glutamic acid.

[0040] In a preferred embodiment of the present invention, the fermentation broth (supernatant) and the wet glutamic acid complex solution after decolorization and sedimentation in step 3 are preferably filtered through a membrane with a cut-off molecular weight of 1000D. The retentate is a polyglutamic acid solution, and the permeate is a glutamic acid solution.

[0041] Preferably, the resin described in step 1 is a macroporous adsorption resin or an anion exchange resin.

[0042] Preferably, any of the above, the resin model is at least one of LX-300C, HPD600, LX-68M, D-301, and more preferably LX-300C.

[0043] Preferably, any of the above, the resin addition amount in step 1 is 0.10 - 10.00% w / v. Preferably, the resin addition amounts are 0.10, 0.50, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0% w / v and the ranges therebetween; preferably, the resin addition amount is 0.33 - 7.05% w / v; more preferably, the resin addition amount is 0.50 - 2.00% w / v.

[0044] Preferably, any of the above, the decolorization time in step 1 is 4.0h - 24.0h. Further preferably, it is 4, 5, 6, 7, 8, 9, 10, 12, 18, 24h and the ranges therebetween; further preferably, it is 5.0h - 12.0h, 9.0h - 12.0h.

[0045] Preferably, any of the above, the pH value of the sterilized γ-polyglutamic acid fermentation broth in step 1 is adjusted to 3.0 - 10.0, and the pH values are further 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and the ranges therebetween; preferably, it is 4.0 - 7.0, preferably, it is 6.0 - 7.0.

[0046] Preferably, in any of the above, the decolorization temperature in Step 1 is 20°C - 50°C, more preferably 20, 25, 30, 35, 40, 45, 50°C and the ranges therebetween, and even more preferably 25°C.

[0047] Preferably, in any of the above, the activated carbon in Step 2 has a particle size of 100 - 325 mesh, preferably 100, 150, 200, 250, 300, 325 mesh and the ranges therebetween.

[0048] Preferably, in any of the above, the iodine value in Step 2 is 500 - 1200, and more preferably, the iodine value is 500, 800, 900, 1000, 1050, 1100, 1200 and the ranges therebetween.

[0049] Preferably, in any of the above, the material in Step 2 is any one of coal-based, wood-based, and fruit shell activated carbon.

[0050] Preferably, in any of the above, the activated carbon in Step 2 is coal-based activated carbon with a particle size of 200 mesh and an iodine value of 1000.

[0051] Preferably, in any of the above, the addition amount of the activated carbon in Step 2 is 0.10 - 5.00% w / v. More preferably, the addition amount of the activated carbon is 0.10, 0.50, 1.0, 2.0, 3.0, 4.0, 5.0% w / v and the ranges therebetween; preferably, the addition amount of the activated carbon is 0.13 - 0.25% w / v; more preferably, the addition amount of the activated carbon is 0.15 - 0.2% w / v.

[0052] Preferably, in any of the above, the decolorization time in Step 2 is 0.5 h - 8.0 h; preferably 0.5, 1, 2, 3, 4, 5, 6, 7, 8 h and the ranges therebetween; more preferably 1.0 - 3.0 h.

[0053] Preferably, in any of the above, the pH value of the fermentation broth after decolorization in Step 1 is adjusted to 3.0 - 10.0 in Step 2; the preferred pH values are 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and the ranges therebetween; more preferably the pH value is 5.0 - 7.0.

[0054] Preferably, in any of the above, the decolorization temperature in Step 2 is 20°C - 60°C; preferably 20, 30, 40, 50, 60°C and the ranges therebetween; more preferably 35°C - 45°C.

[0055] Preferably, in any of the above, the specific steps in step 3 include: adjusting the temperature of the γ-polyglutamic acid decolorized solution obtained in step 2 to 20-30°C, adding acid to a pH of 4.0-6.0, adding 0.2-0.4% w / v of α-glutamic acid crystal seeds and stirring for 1-3 hours; then continuously and slowly cooling and adding acid, the pH of the material liquid should be reduced to 3.2 within 3-5 hours and then stop adding acid, cool to 4°C within 8 hours and then stop cooling and continue stirring for more than 4 hours, and finally stop stirring and allow natural precipitation for more than 4 hours to obtain wet glutamic acid.

[0056] Preferably, in any of the above, the method for redissolving the wet glutamic acid in step 3 is: placing the obtained wet glutamic acid redissolution solution in deionized water, adding a NaOH solution to maintain the pH value of the system at 8.0-11.0, and continuously stirring.

[0057] Preferably, in any of the above, the membrane filtration equipment in step 4 is any one of a flat sheet membrane, a tubular membrane, a hollow fiber membrane or a spiral wound membrane.

[0058] Preferably, in any of the above, the cut-off molecular weight of the membrane filtration equipment in step 4 is 300-3000 D, the retentate is high molecular weight γ-polyglutamic acid, and the permeate is glutamic acid. The cut-off molecular weight is preferably 300, 500, 1000, 2000, 3000 D.

[0059] Preferably, in any of the above, the membrane filtration equipment in step 4 is a spiral wound membrane with a cut-off molecular weight of 1000 D.

[0060] In a preferred embodiment of the present invention, the resin is a macroporous adsorption resin or an anion exchange resin, the resin addition amount is 0.33%-7.05%, the decolorization time is 5 h-12 h, the pH value of the fermentation broth is 4.0-7.0, and the decolorization temperature is 20°C-30°C.

[0061] In a preferred embodiment of the present invention, the resin addition amount is 0.50%-2.00%, the decolorization time is 9 h-12 h, and the pH value of the fermentation broth is 6.0-8.0.

[0062] In a preferred embodiment of the present invention, the activated carbon addition amount in step 2 is 0.13%-0.25%, the decolorization time is 1.0 h-3.0 h, the pH value of the fermentation broth is 5.0-7.0, and the decolorization temperature is 35°C-45°C.

[0063] The present invention provides a method for decolorizing a glutamic acid-dependent γ-polyglutamic acid fermentation broth, having the following beneficial aspects:

[0064] (1) The present invention uses less decolorizing agent in the decolorization step, reduces the production cost, and meets the requirements of subsequent separation and extraction technologies.

[0065] (2) The present invention does not involve a high-temperature acidic environment, and to the greatest extent reduces the loss of γ-polyglutamic acid and the degree of damage to the molecular weight.

[0066] (3) The present invention can realize the recycling of glutamic acid that is not utilized during the γ-polyglutamic acid fermentation process, and does not involve the use of organic solvents. It can not only reduce production costs, but also reduce the generation and emission of waste, which is beneficial to environmental protection.

[0067] The resin and activated carbon decolorization step and the glutamic acid recycling step involved in the present invention can also remove impurities such as heteroproteins in the γ-polyglutamic acid fermentation broth, reducing the working difficulty of subsequent separation and extraction technologies. Brief Description of the Drawings

[0068] Figure 1 is a process flow chart of a method for decolorizing a γ-polyglutamic acid fermentation broth provided by the present invention.

[0069] Figure 2 is a linearized adsorption isotherm model for the resin to adsorb the pigments in the fermentation broth in Preferred Embodiment 6 of the present invention.

[0070] Figure 3 is a linearized adsorption isotherm model for the activated carbon to adsorb the pigments in the fermentation broth in Preferred Embodiment 6 of the present invention.

[0071] Figure 4 is a comparison diagram before and after decolorization in Preferred Embodiment 6 of the present invention. Detailed Description of the Invention

[0072] The present invention will be further described in detail below with reference to the drawings and specific embodiments. It should be understood that the following embodiments are only for explaining the present invention and do not limit the present invention.

[0073] Unless otherwise specified, the following embodiments are all conventional means well known to those skilled in the field of fermentation extraction technology. The γ-polyglutamic acid fermentation broth in the embodiments is sourced as follows. It should be noted that the following method for obtaining the γ-polyglutamic acid fermentation broth is only a preferred scheme, and the technical solution of the present invention is not limited thereto:

[0074] Fermentation conditions: Transfer the strain inoculated in LB medium to the seed medium and shake-culture it at 37°C for 12 hours as the primary seed solution. Transfer the primary seed solution to a secondary seed fermenter for culture. The volume of the secondary seed medium is 15 L, the inoculum size is 0.25%, the culture temperature is 37°C, the rotation speed is 300 rpm, the aeration rate is 2.2 vvm, the tank pressure is 0.03 MPa, and culture for 14 hours to obtain the secondary seed solution. Introduce the secondary seed solution into the fermenter at an inoculum size of 10%. The fermentation temperature is 37°C, the rotation speed is 300 rpm, the aeration rate is 0.6 vvm, the tank pressure is 0.03 MPa, and ferment for 72 hours to obtain the γ-PGA fermentation broth.

[0075] Among them:

[0076] Medium components:

[0077] LB medium: NaCl 10 g / L, tryptone 10 g / L, yeast extract powder 5 g / L, agar powder 20 g / L.

[0078] Seed medium: Glucose 30 g / L, yeast extract 7 g / L, trypsin 10 g / L, magnesium sulfate heptahydrate 0.5 g / L, dipotassium hydrogen phosphate 0.5 g / L.

[0079] Fermentation medium: Sodium glutamate 102.6 g / L, glucose 18.3 g / L, corn steep liquor 15.2 g / L, ferrous sulfate 0.7 g / L.

[0080] The initial pH values of the LB medium and the seed medium should be adjusted to 7.2 using NaOH solution, and the initial pH value of the fermentation medium should be adjusted to 6.8. The pH value is not adjusted during the seed culture and fermentation culture processes.

[0081] The source of the glutamate-independent fermentation broth refers to the scheme in the prior art, but the present invention is not limited thereto. For example, Effects of Fe 2+ addition to sugarcane molasses on poly-γ-glutamic acid production in Bacillus licheniformis CGMCC NO.23967, Lifei Guo, Liang Lu, Huichao Wang, Xiaoxing Zhang, Genan Wang, Tingbin Zhao, Guobao Zheng and Changsheng Qiao, Microbial Cell Factories, DOI: 10.1186 / s12934-023-02042-0.

[0082] Figure 1 Process flow chart of a method for decolorizing γ-polyglutamic acid fermentation broth provided by the present invention as shown

[0083] Example 1

[0084] Example 1 provides a method for decolorizing γ-polyglutamic acid fermentation broth, which specifically includes the following steps:

[0085] Step 1: Resin decolorization: Adjust the pH of the sterilized γ-polyglutamic acid fermentation broth to 7.0, add 10% (w / v) of LX-300C resin, separate the resin from the fermentation broth after 12.0 h, and the decolorization temperature is 25°C.

[0086] Step 3: Glutamic acid sedimentation decolorization: Adjust the pH value of the decolorized fermentation broth in step (1), cool down, stir, crystallize, and sediment to obtain wet glutamic acid, and redissolve the obtained wet glutamic acid;

[0087] The sedimentation step: Cool the γ-polyglutamic acid decolorized solution obtained in step (1) to 25°C, add acid to adjust the pH to 4.5, add 0.3% (w / v) of α-glutamic acid crystal seeds and stir for 2 hours; then continuously and slowly cool down and add acid, and the pH of the feed liquid should be reduced to 3.2 within 4 hours and then stop adding acid, cool down to 4°C within 8 hours and then stop cooling and continue stirring for 8 hours, and finally stop stirring, and wet glutamic acid can be obtained after natural precipitation for 6 hours;

[0088] The redissolution method is: Place the obtained wet glutamic acid in deionized water, add NaOH solution to maintain the pH of the system at 10.0, and continuously stir;

[0089] Step 4: Membrane filtration treatment for decolorization: Use a membrane filtration device to separate γ-polyglutamic acid in the supernatant of step (3) and achieve further decolorization, and use a membrane filtration device to separate the wet glutamic acid redissolution solution in step (3) to obtain γ-polyglutamic acid;

[0090] The membrane is a spiral wound membrane with a molecular weight cut-off of 1000 D. The retentate is macromolecular γ-polyglutamic acid, and the permeate is small molecular glutamic acid.

[0091] In this example, the decolorization rate of the fermentation broth is 86.03%, the recovery rate of γ-polyglutamic acid is 79.57%, and the protein removal rate is 62.43%.

[0092] In Example 1, there is no step of activated carbon decolorization, and the decolorization rate is still low even when using excessive resin. It shows that the combination of activated carbon decolorization and resin decolorization in the present invention is not a simple superposition of these two technologies, but has a synergistic effect.

[0093] Example 2

[0094] Example 2 provides a method for decolorizing γ-polyglutamic acid fermentation broth, which specifically includes the following steps:

[0095] Step 2: Decolorization with activated carbon: Adjust the pH of the sterilized γ-polyglutamic acid fermentation broth to 10.0, add 0.42% (w / v) of 200-mesh activated carbon with an iodine value of 1000 made from coal, separate the activated carbon from the fermentation broth after 0.5 h, and the decolorization temperature is 50 °C.

[0096] The steps of glutamic acid sedimentation decolorization in Step 3 and membrane filtration treatment decolorization in Step 4 are the same as those in Example 1.

[0097] In this example, the decolorization rate of the fermentation broth is 85.63%, the recovery rate of γ-polyglutamic acid is 94.83%, and the protein removal rate is 59.80%.

[0098] In Example 2, without the addition of resin decolorization and using a large amount of activated carbon, the decolorization rate is still low, indicating that the combination of activated carbon decolorization and resin decolorization in the present invention is not a simple superposition of these two technologies, but has a synergistic effect.

[0099] Example 3

[0100] Example 3 provides a method for decolorizing γ-polyglutamic acid fermentation broth, which specifically includes the following steps:

[0101] Step 1: Decolorization with resin: Adjust the pH of the sterilized γ-polyglutamic acid fermentation broth to 4.0, add 0.10% (w / v) of LX-300C resin, separate the resin from the fermentation broth after 24 h, and the decolorization temperature is 50 °C.

[0102] Step 2: Decolorization with activated carbon: Adjust the pH value of the fermentation broth decolorized in Step (1) to 7.0, add 0.10% (w / v) of 200-mesh activated carbon with an iodine value of 1000 made from coal, the decolorization temperature is 60 °C, and separate the activated carbon from the fermentation broth after 1.0 h.

[0103] The steps of glutamic acid sedimentation decolorization in Step 3 and membrane filtration treatment decolorization in Step 4 are the same as those in Example 1.

[0104] In this example, the decolorization rate of the fermentation broth is 48.37%, the recovery rate of γ-polyglutamic acid is 96.67%, and the protein removal rate is 32.71%.

[0105] The results of Example 3 show that when the addition amounts of resin and activated carbon are low, the decolorization effect cannot be achieved.

[0106] Example 4

[0107] Example 4 provides a method for decolorizing γ-polyglutamic acid fermentation broth, which specifically includes the following steps:

[0108] Step 1: Resin decolorization: Adjust the pH of the sterilized γ-polyglutamic acid fermentation broth to 4.0, add LX-300C resin at 1.48% (w / v), separate the resin from the fermentation broth after 12.0 h, and the decolorization temperature is 25°C.

[0109] Step 2: Activated carbon decolorization: Adjust the pH value of the fermentation broth decolorized in step (1) to 8.0, add coal-based activated carbon with 200 mesh and 1000 iodine value at 0.18% (w / v), the decolorization temperature is 25°C, and separate the activated carbon from the fermentation broth after 1.0 h.

[0110] Step 3: Glutamic acid sedimentation decolorization step and step 4: Membrane filtration treatment decolorization step are the same as those in Example 1.

[0111] In this example, the decolorization rate of the fermentation broth is 87.57%, the recovery rate of γ-polyglutamic acid is 96.33%, and the protein removal rate is 59.29%.

[0112] Example 4 shows that the adjustment of pH and temperature affects the decolorization effect of the present invention.

[0113] Example 5

[0114] Example 5 provides a method for decolorizing γ-polyglutamic acid fermentation broth, which specifically includes the following steps:

[0115] Step 1: Resin decolorization: Adjust the pH of the sterilized γ-polyglutamic acid fermentation broth to 7.0, add LX-300C resin at 1.48% (w / v), separate the resin from the fermentation broth after 4.0 h, and the decolorization temperature is 25°C.

[0116] Step 2: Activated carbon decolorization: Adjust the pH value of the fermentation broth decolorized in step (1) to 6.0, add coal-based activated carbon with 200 mesh and 1000 iodine value at 0.18% (w / v), the decolorization temperature is 40°C, and separate the activated carbon from the fermentation broth after 0.5 h.

[0117] Step 3: Glutamic acid sedimentation decolorization step and step 4: Membrane filtration treatment decolorization step are the same as those in Example 1.

[0118] In this example, the decolorization rate of the fermentation broth is 88.52%, the recovery rate of γ-polyglutamic acid is 93.54%, and the protein removal rate is 63.58%.

[0119] Example 5 shows that the decolorization time affects the decolorization effect of the present invention.

[0120] Example 6

[0121] Example 6 provides a method for decolorizing γ-polyglutamic acid fermentation broth, which specifically includes the following steps:

[0122] Step 1: Resin decolorization: Adjust the pH of the sterilized γ-polyglutamic acid fermentation broth to 7.0, add LX-300C resin at 1.48% (w / v), separate the resin from the fermentation broth after 12.0 h, and the decolorization temperature is 25°C.

[0123] Step 2: Activated carbon decolorization: Adjust the pH value of the fermentation broth decolorized in step (1) to 6.0, add 0.18% (w / v) of 200-mesh activated carbon with an iodine value of 1000, the decolorization temperature is 40°C, and separate the activated carbon from the fermentation broth after 2.5 h.

[0124] Step 3, the glutamic acid precipitation decolorization step and Step 4, the membrane filtration treatment decolorization step are the same as in Example 1.

[0125] In this example, the decolorization rate of the fermentation broth is 95.23%, the recovery rate of γ-polyglutamic acid is 92.01%, and the protein removal rate is 67.86%.

[0126] In Example 6, the contents of the resin and activated carbon, pH, reaction temperature, and decolorization time were optimized and adjusted. Through the synergistic effect of each parameter, the decolorization rate of the fermentation broth, the recovery rate of γ-polyglutamic acid, the protein removal rate, etc. were improved simultaneously. In Example 6, the obtained product was also detected as follows:

[0127] Figure 2 This is the kinetic model fitting curve for the resin to adsorb the pigment in the fermentation broth in the preferred Example 6 of the present invention. Among them, a is the linearized Langmuir isotherm of the pigment on the resin, and b is the linearized Freundlich isotherm.

[0128] Figure 3 This is the fitting curve of the kinetic model for the activated carbon to adsorb the pigment in the fermentation broth in the preferred Example 6 of the present invention. Among them, a is the linearized Langmuir isotherm of the pigment on the activated carbon, and b is the linearized Freundlich isotherm.

[0129] Figure 4 This is the comparison diagram before and after decolorization in the preferred Example 6 of the present invention.

[0130] Among them, Figure a is the ultraviolet-visible spectrogram diluted 30 times. In Figure a, A is the fermentation broth without decolorization treatment, B is the fermentation broth treated only by glutamic acid precipitation, and C is the fermentation broth decolorized by the process provided by the present invention; Figure b is the visible spectrogram of the undiluted fermentation broth, and the meanings of the letters A, B, and C in Figure b are the same as those in Figure a; Figure c is the comparison photo of the fermentation broth, and the meanings of the letters A, B, and C in Figure c are the same as those in Figure a.

[0131] In Examples 1-6, the recovery rate of glutamic acid is not less than 90%.

[0132] Example 7

[0133] Example 7 provides a method for decolorizing γ-polyglutamic acid fermentation broth, which specifically includes the following steps:

[0134] Step 1: Resin decolorization: Adjust the pH of the sterilized γ-polyglutamic acid fermentation broth to 7.0, add 1.48% (w / v) of LX-300C resin, separate the resin from the fermentation broth after 12.0 h, and the decolorization temperature is 25°C.

[0135] Step 2: Activated carbon decolorization: Adjust the pH of the fermentation broth in step (1) to 6.0, add 0.18% (w / v) of 200-mesh activated carbon with an iodine value of 1000 made from coal, separate the activated carbon from the fermentation broth after 2.5 h, and the decolorization temperature is 40°C.

[0136] Step 3: Glutamic acid sedimentation decolorization is the same as in Example 1.

[0137] In this example, the decolorization rate of the fermentation broth is 83.57%, the recovery rate of γ-polyglutamic acid is 89.78%, the protein removal rate is 60.67%, and the recovery rate of glutamic acid is 52.94%.

[0138] In Example 7, after resin decolorization, activated carbon decolorization, and glutamic acid sedimentation decolorization with optimized conditions, the decolorization rate of the fermentation broth and the recovery rate of γ-polyglutamic acid have been significantly improved. At the same time, without membrane filtration operation, the recovery rate of glutamic acid is low and the decolorization rate is on the low side, indicating that the membrane filtration operation can further improve the technical effect of the present invention.

[0139] Example 8

[0140] Example 8 provides a method for decolorizing γ-polyglutamic acid fermentation broth, which specifically includes the following steps:

[0141] Step 1: Resin decolorization: Adjust the pH of the sterilized γ-polyglutamic acid fermentation broth to 7.0, add 1.48% (w / v) of LX-300C resin, separate the resin from the fermentation broth after 12.0 h, and the decolorization temperature is 25°C.

[0142] Step 2: Activated carbon decolorization: Adjust the pH in step (1) to 6.0, add 0.18% (w / v) of 200-mesh activated carbon with an iodine value of 1000 made from coal, separate the activated carbon from the fermentation broth after 2.5 h, and the decolorization temperature is 40°C.

[0143] Step 4: Membrane filtration treatment decolorization is the same as in Example 1.

[0144] In this example, the decolorization rate of the fermentation broth is 85.78%, the recovery rate of γ-polyglutamic acid is 92.68%, the protein removal rate is 63.09%, and the recovery rate of glutamic acid is 87.92%.

[0145] In Example 8, glutamic acid sedimentation decolorization was not carried out, and the decolorization rate was low. Examples 1 to 8 of the present invention are all fermentation broths produced by glutamic acid-dependent strains and fermentation broths with a glutamic acid residue content greater than 20 g / L produced by glutamic acid-dependent strains. Example 8 shows that for fermentation broths with a glutamic acid residue content greater than 20 g / L, glutamic acid sedimentation is beneficial to decolorization.

[0146] Example 9

[0147] Example 9 provides a method for decolorizing γ-polyglutamic acid fermentation broth, which specifically includes the following steps:

[0148] Step 1: Resin decolorization: Adjust the pH of the sterilized γ-polyglutamic acid fermentation broth to 7.0, add 1.48% (w / v) of LX-300C resin, separate the resin from the fermentation broth after 12.0 h, and the decolorization temperature is 25°C.

[0149] Step 2: Activated carbon decolorization: Adjust the pH value of the fermentation broth decolorized in step (1) to 6.0, add 0.18% (w / v) of 200-mesh activated carbon with an iodine value of 1000 made from coal, the decolorization temperature is 40°C, and separate the activated carbon from the fermentation broth after 2.5 h.

[0150] Step 4: Membrane filtration treatment for decolorization: Use a membrane filtration device to separate the γ-polyglutamic acid in step (2) and achieve further decolorization;

[0151] The membrane described is a spiral wound membrane with a molecular weight cut-off of 1000 D. The retentate is macromolecular γ-polyglutamic acid, and the permeate is small molecule glutamic acid.

[0152] Example 9 is applicable to fermentation broths with a glutamic acid residue content less than 20 g / L produced by glutamic acid-dependent strains and fermentation broths produced by glutamic acid-independent strains. The decolorization rate of the fermentation broth is 88.85%, the recovery rate of γ-polyglutamic acid is 90.58%, and the protein removal rate is 57.43%.

[0153] Example 10

[0154] Example 10 is a comparison of Examples 1 to 9.

[0155] The calculation method of glutamic acid recovery rate in the present invention is:

[0156] Recovered glutamic acid = glutamic acid contained in the permeate of the membrane filtration of the decolorized liquid in step 3 + glutamic acid contained in the permeate of the membrane filtration after glutamic acid re-dissolution in step 4;

[0157] Glutamic acid recovery rate = content of recovered glutamic acid / content of glutamic acid in the initial fermentation broth = (glutamic acid concentration in the permeate of the decolorized solution in step 3 × volume of the permeate in step 3 + glutamic acid concentration in the permeate after reconstitution in step 4 × volume of the permeate in step 4) / (glutamic acid concentration in the initial fermentation broth × volume of the initial fermentation broth).

[0158] Table 1 summarizes the decolorization rate of the fermentation broth, the recovery rate of polyglutamic acid, the protein removal rate, and the glutamic acid recovery rate in Examples 1-9.

[0159] Table 1:

[0160]

[0161] The above examples are merely illustrative for clear explanation and not limitations on the implementation modes. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation modes here. The obvious changes or modifications derived therefrom still fall within the protection scope of the present invention.

Claims

1. A method for decolorizing γ-polyglutamic acid fermentation broth, comprising the following steps: Step 1: Resin decolorization: Adjust the pH value of the sterilized γ-polyglutamic acid fermentation broth to 3.0 - 10.0, add resin, the decolorization temperature is 20°C - 50°C, the decolorization time is 4.0 h - 24.0 h, and after decolorization, separate the resin from the fermentation broth; Step 2: Activated carbon decolorization: Adjust the pH value of the fermentation broth after decolorization in Step 1 to 3.0 - 10.0, add activated carbon, the decolorization temperature is 20°C - 60°C, the decolorization time is 0.5 h - 8.0 h, and after decolorization, separate the activated carbon from the fermentation broth; Step 3: Glutamic acid sedimentation decolorization: Adjust the pH value of the fermentation broth after decolorization in Step 2 to 4.0 - 6.0, cool it to 20 - 30°C, add 0.2 - 0.4% w / v of α-glutamic acid crystal seeds and stir for 1 - 3 hours, then continuously and slowly cool and add acid, the pH of the feed liquid should be reduced to 3.2 after 3 - 5 hours and then stop adding acid, cool it to 4°C within 8 hours and then stop cooling and continue stirring for more than 4 hours, finally stop stirring, let it settle naturally for more than 4 hours to obtain wet glutamic acid and redissolve the settled wet glutamic acid; Step 4: Membrane filtration treatment for decolorization: Use a membrane filtration device to separate γ-polyglutamic acid and achieve further decolorization; For the fermentation broth with a glutamic acid residue content greater than 20 g / L produced by glutamate-dependent strains, the method includes Step 1, Step 2, Step 3, and Step 4; For the fermentation broth with a glutamic acid residue content less than 20 g / L produced by glutamate-dependent strains or the fermentation broth produced by glutamate-independent strains, the method includes Step 1, Step 2, and Step 4.

2. The method for decolorizing the γ-polyglutamic acid fermentation broth according to claim 1, wherein The resin described in Step 1 is macroporous adsorption resin or anion exchange resin.

3. The method for decolorizing the γ-polyglutamic acid fermentation broth according to claim 2, wherein The resin addition amount described in Step 1 is 0.10 - 10.00% w / v.

4. The method for decolorizing a γ-polyglutamic acid fermentation broth according to claim 1, characterized in that, The activated carbon described in Step 2 has a particle size of 100 - 325 mesh, an iodine value of 500 - 1200, and the material is any one of coal-based, wood-based, and fruit shell activated carbon, and the activated carbon addition amount is 0.10 - 5.00% w / v.

5. The method for decolorizing γ-polyglutamic acid fermentation broth according to claim 1, characterized in that, The method for redissolving the wet glutamic acid described in Step 3 is: Place the obtained wet glutamic acid redissolution solution in deionized water, add NaOH solution to maintain the system pH value at 8.0 - 11.0, and continuously stir.

6. The method for decolorizing γ-polyglutamic acid fermentation broth according to claim 1, characterized in that, The membrane filtration device described in Step 4 is any one of flat sheet membrane, tubular membrane, hollow fiber membrane, or spiral wound membrane; the cut-off molecular weight of the membrane filtration device is 300 - 3000 D.

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