Method for decoloring gamma-polyglutamic acid fermentation liquor

Abstract

The invention provides a method for decoloring gamma-polyglutamic acid fermentation liquor. The method comprises four steps of resin decoloring, activated carbon decoloring, glutamic acid sedimentation decoloring and membrane filtration treatment decoloring. Through the combined decolorization process, the decolorization rate of the gamma-polyglutamic acid fermentation liquor can reach 95% or above, the recovery rate of gamma-polyglutamic acid can reach 92% or above, the protein removal rate can reach 67% or above, and the recovery rate of glutamic acid can reach 90% or above. The decolorizing method provided by the invention has the advantages of novel route, less decolorizing agent dosage, no use of organic solvent, no high-temperature heating, low damage degree of high-molecular-weight gamma-PGA, realization of cyclic utilization of the fermentation raw material glutamic acid, and suitableness for large-scale industrial production.

Description

Technical Field

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

[0002] γ-Polyglutamic acid is a biological macromolecular amino acid anionic polymer with a relative molecular weight of generally 100kDa-2000kDa. It is widely used in food, medicine, agriculture and other fields due to its good water absorption and retention, ion adsorption and non-toxicity.

[0003] It is reported that there are two types of synthetic γ-polyglutamic acid strains: strains that do not need to provide glutamate and only rely on the strain's own metabolism to synthesize γ-polyglutamic acid are called glutamate-independent strains; strains that need to be artificially provided with glutamate to synthesize γ-polyglutamic acid are called glutamate-dependent strains. Yang Qi et al. (CN118086418A) used glutamate-independent strains to produce γ-polyglutamic acid, and the final yield was 23.8g / L, which was lower than the commonly reported glutamate-dependent strains, such as the glutamate-dependent strain CGMCC NO.23967 reported in CN114456980A, which had a yield of 86.3g / L. Because glutamate-dependent strains have a higher yield, most factories currently use glutamate-dependent strains to produce γ-polyglutamic acid.

[0004] Due to the extremely large molecular weight of γ-polyglutamic acid, the viscosity of the fermentation liquid is relatively high, which increases the difficulty of separation and extraction. During the fermentation process, glutamate-dependent strains need to add a large amount of glutamate or sodium glutamate to the culture medium. After the fermentation is completed, a large amount of glutamate will remain, causing a waste of raw materials. If it is discharged without treatment, it will pollute the environment.

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

[0006] The decolorization methods used in the above reports mainly have the problems of large amount of decolorization agent, high decolorization temperature and serious hydrolysis of high molecular weight γ-polyglutamic acid. The above methods and technologies do not involve the recycling step of glutamic acid.

[0007] At present, the research on γ-polyglutamic acid is concentrated in the fields of production and synthesis of γ-polyglutamic acid, optimization of fermentation conditions, and product technology application. 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 is needed that uses a small amount of decolorizing agent, can realize raw material recycling, and does not destroy the original molecular weight. 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 gamma-polyglutamic acid fermentation broth.

[0009] Specifically, the method of the present invention can achieve extremely high decolorization rate, recovery rate and protein removal rate without using organic solvents, high temperature heating and low decolorization agent dosage, with low degree of damage to high molecular weight γ-polyglutamic acid, and can realize the recycling of glutamic acid.

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

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

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

[0013] Any of the above is preferably that the method for decolorizing γ-polyglutamic acid fermentation broth further includes: Step 3: glutamic acid sedimentation decolorization: adjusting the pH value of the fermentation broth after decolorization in Step 2, cooling, stirring, crystallizing, and sedimenting to obtain wet glutamic acid, and re-dissolving the precipitated wet glutamic acid.

[0014] Any one of the above is preferably that the method for decolorizing γ-polyglutamic acid fermentation broth further comprises:

[0015] Step 4: Decolorization by membrane filtration: γ-polyglutamic acid is separated by membrane filtration equipment and further decolorized.

[0016] Any of the above is preferably that the method for decolorizing a γ-polyglutamic acid fermentation broth further comprises: step 3 and step 4, in step 3, wet glutamic acid and a decolorized solution (supernatant) are obtained after glutamic acid precipitation and decolorization, and the precipitated wet glutamic acid is redissolved to obtain a wet glutamic acid resolubilized solution; the membrane filtration treatment step of step 4 is preferably:

[0017] Step 4: Separate glutamic acid from γ-polyglutamic acid using a membrane filtration device in the wet glutamic acid reconstituted solution in step 3; separate the supernatant in step 3 using a membrane filtration device and further decolorize.

[0018] The decolorization order of step 1 and step 2 in the present invention can be exchanged, and the present invention is equally applicable to fermentation broth produced by glutamate-dependent strains and fermentation broth produced by non-glutamate-dependent strains.

[0019] In a preferred embodiment of the present invention, the fermentation broth produced by glutamate-dependent strains is decolorized and purified, and the preferred residual glutamate content is greater than 20 g / L. The method for decolorizing the γ-polyglutamic acid fermentation broth comprises the following steps:

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

[0021] Step 2: activated carbon decolorization: adjusting the pH value of the fermentation liquid after decolorization in step 1, adding activated carbon, and separating the activated carbon from the fermentation liquid after decolorization;

[0022] Step 3: Glutamic acid precipitation and decolorization: adjust the pH value of the fermentation liquid after decolorization in step 2, cool, stir, grow crystals, and precipitate to obtain wet glutamic acid, and redissolve the precipitated wet glutamic acid.

[0023] In step 3, glutamate is precipitated to remove the pigment from the fermentation liquid, thereby achieving the technical effect of decolorization.

[0024] In step 3, after the wet glutamic acid is precipitated, the decolorized fermentation liquid (supernatant) is the purified γ-polyglutamic acid of the present invention.

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

[0026] In a preferred embodiment of the present invention, the fermentation broth produced by glutamate-dependent strains is decolorized and purified, and the preferred residual glutamate content is greater than 20 g / L. The method for decolorizing the γ-polyglutamic acid fermentation broth comprises the following steps:

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

[0028] Step 2: activated carbon decolorization: adjusting the pH value of the fermentation liquid after decolorization in step 1, adding activated carbon, and separating the activated carbon from the fermentation liquid after decolorization;

[0029] Step 3: glutamic acid sedimentation decolorization: adjusting the pH value of the fermentation liquid after decolorization in step 2, cooling, stirring, growing crystals, and sedimentation to obtain wet glutamic acid and decolorized liquid (supernatant), and re-dissolving the precipitated wet glutamic acid to obtain a wet glutamic acid re-solution;

[0030] Step 4: Decolorization by membrane filtration: Separate the wet glutamic acid reconstituted solution in step 3 by membrane filtration equipment to obtain γ-polyglutamic acid; Separate the supernatant in step 3 by membrane filtration equipment to obtain γ-polyglutamic acid, and further decolorize. Wherein:

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

[0032] In step 4, the wet glutamic acid reconstituted solution in step 3 is separated by membrane filtration equipment to obtain γ-polyglutamic acid; the supernatant in step 3 is separated by membrane filtration equipment to obtain γ-polyglutamic acid, and further decolorization is achieved; after separation by the membrane filtration equipment, the retentate is high molecular weight γ-polyglutamic acid, and the permeate is glutamic acid. The permeate, that is, glutamic acid, is added to the γ-polyglutamic acid fermentation system and can be used for subsequent fermentation and culture. The obtained retentate is the purified γ-polyglutamic acid product.

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

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

[0035] Step 1: Resin decolorization: adjust the pH value of the sterilized γ-polyglutamic acid fermentation liquid, add the resin, and separate the resin from the fermentation liquid after decolorization;

[0036] Step 2: activated carbon decolorization: adjusting the pH value of the fermentation liquid after decolorization in step 1, adding activated carbon, and separating the activated carbon from the fermentation liquid after decolorization;

[0037] Step 4: Decolorization by membrane filtration: The fermentation liquid decolorized in step 2 is separated by membrane filtration equipment to obtain γ-polyglutamic acid, and further decolorized.

[0038] After separation by membrane filtration equipment, the retentate is high molecular weight γ-polyglutamic acid.

[0039] The decolorization mechanism of the invention is as follows: electrostatic attraction exists between acidic pigment molecules and weakly alkaline resins; the resin is a macroporous resin and has van der Waals force to adsorb pigments in fermentation liquid; the Freundlich model fitting effect of the adsorption process is better 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, and can quickly adsorb pigments; the Langmuir model fitting effect of the adsorption process is better than the Freundlich model, so the pigments are adsorbed on the activated carbon surface in a single layer; and the glutamic acid precipitation decolorization relies on the electrostatic force to carry part of the pigments and precipitate together in the glutamic acid precipitation.

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

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

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

[0043] Any of the above is preferably that the amount of resin added in step 1 is 0.10-10.00% w / v. Preferably, the amount of resin added is 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 ranges therebetween; preferably, the amount of resin added is 0.33-7.05% w / v; more preferably, the amount of resin added is 0.50-2.00% w / v.

[0044] In any of the above, the decolorization time in step 1 is preferably 4.0 h to 24.0 h. More preferably, it is 4, 5, 6, 7, 8, 9, 10, 12, 18, 24 h and the range therebetween; and more preferably, it is 5.0 h to 12.0 h, 9.0 h to 12.0 h.

[0045] Any of the above items is preferably that in step 1, the pH value of the sterilized γ-polyglutamic acid fermentation broth is adjusted to 3.0-10.0, and the pH value is further 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and the range therebetween; preferably 4.0-7.0, preferably 6.0-7.0.

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

[0047] Any of the above is preferably that the activated carbon described in step 2 has a particle size of 100-325 mesh, preferably 100, 150, 200, 250, 300, 325 mesh and ranges therebetween.

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

[0049] Any of the above items is preferably that the material in step 2 is any one of coal, wood, and fruit shell activated carbon.

[0050] Preferably, in any of the above items, the activated carbon in step 2 is 200 mesh 1000 iodine value coal-made activated carbon.

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

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

[0053] Any of the above is preferably that in step 2, the pH value of the fermentation liquid after decolorization in step 1 is adjusted to 3.0-10.0; preferably, the pH value is 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and the range therebetween; further preferably, the pH value is 5.0-7.0.

[0054] Any of the above is preferably that the decolorization temperature in step 2 is 20°C-60°C; preferably 20, 30, 40, 50, 60°C and the range therebetween; further preferably 35°C-45°C.

[0055] Any of the above items is preferably that 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 α-glutamic acid seed crystals and stirring for 1-3 hours; then slowly cooling and adding acid, stopping the acid addition after the pH of the solution drops to 3.2 after 3-5 hours, stopping the cooling and continuing stirring for more than 4 hours after cooling to 4° C. within 8 hours, and finally stopping stirring and allowing natural precipitation for more than 4 hours to obtain wet glutamic acid.

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

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

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

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

[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 5h-12h, the pH value of the fermentation liquid is 4.0-7.0, and the decolorization temperature is 20℃-30℃.

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

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

[0063] The present invention provides a method for decolorizing glutamate-dependent γ-polyglutamic acid fermentation broth, which has the following benefits:

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

[0065] (2) The present invention does not involve a high temperature acidic environment, thereby minimizing the loss of γ-polyglutamic acid and the degree of destruction of the molecular weight.

[0066] (3) The present invention can realize the recycling of glutamic acid that is not utilized in the γ-polyglutamic acid fermentation process without involving the use of organic solvents, which can not only reduce production costs, but also reduce the generation and discharge 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 foreign proteins in the gamma-polyglutamic acid fermentation liquid, thereby reducing the difficulty of subsequent separation and extraction technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 The present invention provides a process flow chart of a method for decolorizing γ-polyglutamic acid fermentation broth.

[0069] Figure 2 This is the linearized adsorption isotherm model of the resin adsorbing the pigment in the fermentation broth in the preferred embodiment 6 of the present invention.

[0070] Figure 3 This is the linearized adsorption isotherm model of the activated carbon adsorbing the pigment in the fermentation broth in the preferred embodiment 6 of the present invention.

[0071] Figure 4 This is a comparison diagram before and after decolorization in preferred embodiment 6 of the present invention. DETAILED DESCRIPTION

[0072] The present invention is further described in detail below through the accompanying 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 examples are all conventional means known to those skilled in the art of fermentation and extraction. The γ-polyglutamic acid fermentation broth in the examples is obtained from the following sources. It should be noted that the following method for obtaining the γ-polyglutamic acid fermentation broth is only a preferred solution, and the technical solution of the present invention is not limited thereto:

[0074] Fermentation conditions: The strain inoculated in LB medium was transferred to the seed medium and cultured as the primary seed solution at 37°C with shaking for 12 hours. The primary seed solution was transferred to the secondary seed fermenter for culture. The volume of the secondary seed medium was 15L, the inoculation amount was 0.25%, the culture temperature was 37°C, the rotation speed was 300rpm, the ventilation volume was 2.2vvm, the tank pressure was 0.03MPa, and the culture was carried out for 14 hours to obtain the secondary seed solution. The secondary seed solution was introduced into the fermenter at an inoculation amount of 10%. The fermentation temperature was 37°C, the rotation speed was 300rpm, the ventilation volume was 0.6vvm, the tank pressure was 0.03MPa, and the fermentation was carried out for 72 hours to obtain the γ-PGA fermentation liquid.

[0075] in:

[0076] Medium ingredients:

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

[0078] Seed culture medium: glucose 30 g / L, yeast extract 7 g / L, trypsin 10 g / L, magnesium sulfate heptahydrate 0.5 g / L, potassium dihydrogen 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 of LB medium and seed medium should be adjusted to 7.2 using NaOH solution, and the initial pH of fermentation medium should be adjusted to 6.8. The pH value was not adjusted during seed culture and fermentation culture.

[0081] The source of glutamate-independent fermentation broth can be referred to the scheme in the prior art, but the present invention is not limited thereto. 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 The figure shows a process flow chart of a method for decolorizing γ-polyglutamic acid fermentation broth provided by the present invention.

[0083] Example 1

[0084] Example 1 provides a method for decolorizing γ-polyglutamic acid fermentation broth, which specifically comprises 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) LX-300C resin, separate the resin from the fermentation broth after 12.0 hours, and the decolorization temperature is 25°C.

[0086] Step 3: glutamic acid sedimentation decolorization: adjusting the pH value of the fermentation liquid decolorized in step (1), cooling, stirring, growing crystals, sedimentation to obtain wet glutamic acid, and re-dissolving the obtained wet glutamic acid;

[0087] The sedimentation step comprises cooling the γ-polyglutamic acid decolorized solution obtained in step (1) to 25° C., adding acid to a pH of 4.5, adding 0.3% (w / v) α-glutamic acid seed crystals and stirring for 2 hours; then slowly cooling and adding acid, stopping the acid addition after the pH of the solution drops to 3.2 within 4 hours, stopping the cooling and continuing stirring for 8 hours after cooling to 4° C. within 8 hours, and finally stopping stirring. After natural precipitation for 6 hours, wet glutamic acid can be obtained;

[0088] The re-dissolving method is as follows: placing the obtained wet glutamic acid in deionized water, adding NaOH solution thereto to maintain the pH of the system at 10.0, and continuously stirring;

[0089] Step 4: Decolorization by membrane filtration: using a membrane filtration device to separate the supernatant γ-polyglutamic acid in step (3) and further decolorize it, and using a membrane filtration device to separate the wet glutamic acid reconstituted solution in step (3) to obtain γ-polyglutamic acid;

[0090] The membrane is a roll membrane with a molecular weight cutoff of 1000D, a retentate liquid is macromolecular gamma-polyglutamic acid, and a permeate liquid is micromolecular glutamic acid.

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

[0092] In Example 1, there is no activated carbon decolorization step, and the decolorization rate is still low when using an excess of resin, which indicates that the combination of activated carbon decolorization and resin decolorization in the present invention is not a simple superposition of the 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 comprises the following steps:

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

[0096] Step 3, the glutamate precipitation decolorization step and step 4, the membrane filtration decolorization step are the same as those in Example 1.

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

[0098] In Example 2, no resin decolorization is added, and a large amount of activated carbon is used, but 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 the 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 comprises the following steps:

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

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

[0103] Step 3, the glutamate precipitation decolorization step and step 4, the membrane filtration decolorization step are the same as those in Example 1.

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

[0105] The results of Example 3 show that the decolorization effect cannot be achieved when the amount of resin and activated carbon added is too low.

[0106] Example 4

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

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

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

[0110] Step 3, the glutamate precipitation decolorization step and step 4, the membrane filtration decolorization step are the same as those in Example 1.

[0111] In this example, the decolorization rate of the fermentation broth was 87.57%, the recovery rate of γ-polyglutamic acid was 96.33%, and the protein removal rate was 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 comprises the following steps:

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

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

[0117] Step 3, the glutamate precipitation decolorization step and step 4, the membrane filtration decolorization step are the same as those in Example 1.

[0118] In this example, the decolorization rate of the fermentation broth was 88.52%, the recovery rate of γ-polyglutamic acid was 93.54%, and the protein removal rate was 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 comprises the following steps:

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

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

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

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

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

[0127] Figure 2 This is the kinetic model fitting curve of the resin adsorption of the fermentation liquid pigment in the preferred embodiment 6 of the present invention. Wherein, a is the linearized Langmuir isotherm of the pigment on the resin, and b is the linearized Freundlich isotherm.

[0128] Figure 3 It is the fitting curve of the kinetic model of the adsorption of the pigment of the fermentation broth by activated carbon in the preferred embodiment 6 of the present invention. Wherein, 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 a comparison diagram before and after decolorization in preferred embodiment 6 of the present invention.

[0130] Among them, Figure a is a 30-fold diluted UV-visible spectrum, Figure a A is a fermentation broth that has not been decolorized, Figure a B is a fermentation broth that has only been treated with glutamate precipitation, and Figure a C is a fermentation broth decolorized using the process provided by the present invention; Figure b is a visible spectrum of the undiluted fermentation broth, and the letters A, B, and C in Figure b have the same meanings as Figure a; Figure c is a comparison photo of the fermentation broth, and the letters A, B, and C in Figure c have the same meanings as Figure a.

[0131] In Examples 1-6, the glutamic acid recovery rates were all not less than 90%.

[0132] Example 7

[0133] Example 7 provides a method for decolorizing γ-polyglutamic acid fermentation broth, which specifically comprises 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) LX-300C resin, separate the resin from the fermentation broth after 12.0 hours, and the decolorization temperature is 25°C.

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

[0136] Step 3: The glutamic acid precipitation and decolorization step is the same as in Example 1.

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

[0138] In Example 7, after resin decolorization and activated carbon decolorization under optimal conditions, as well as glutamic acid sedimentation decolorization, the fermentation broth decolorization rate and γ-polyglutamic acid recovery rate have been significantly improved. At the same time, without membrane filtration operation, the glutamic acid recovery rate is low and the decolorization rate is low, which shows that 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 comprises 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) LX-300C resin, separate the resin from the fermentation broth after 12.0 hours, 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) 200 mesh 1000 iodine value coal-made activated carbon, separate the activated carbon from the fermentation liquid after 2.5 hours, and the decolorization temperature is 40°C.

[0143] Step 4: The decolorization step is the same as in Example 1.

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

[0145] In Example 8, glutamate precipitation decolorization is not performed, and the decolorization rate is low. Examples 1 to 8 of the present invention are all fermentation broths produced by glutamate-dependent strains and fermentation broths with a glutamate residual content greater than 20 g / L produced by glutamate-dependent strains. Example 8 shows that for fermentation broths with a glutamate residual content greater than 20 g / L, glutamate precipitation is beneficial to decolorization.

[0146] Example 9

[0147] Example 9 provides a method for decolorizing γ-polyglutamic acid fermentation broth, which specifically comprises 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) LX-300C resin, separate the resin from the fermentation broth after 12.0 hours, and the decolorization temperature is 25°C.

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

[0150] Step 4: Decolorization by membrane filtration: using a membrane filtration device to separate the γ-polyglutamic acid in step (2) and achieve further decolorization;

[0151] The membrane is a roll membrane with a molecular weight cutoff of 1000D, a retentate liquid is macromolecular gamma-polyglutamic acid, and a permeate liquid is micromolecular glutamic acid.

[0152] Example 9 is applicable to fermentation broth with a glutamate residual content of less than 20 g / L produced by glutamate-dependent strains and fermentation broth produced by glutamate-independent strains. The fermentation broth decolorization rate is 88.85%, the γ-polyglutamic acid recovery rate 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 the glutamic acid recovery rate in the present invention is:

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

[0157] Glutamic acid recovery rate = recovered glutamate content / initial fermentation broth glutamate content = (step 3 decolorization permeate glutamate concentration × step 3 permeate volume + step 4 reconstituted permeate glutamate concentration × step 4 permeate volume) / (initial fermentation broth glutamate concentration × initial fermentation broth volume).

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

[0159] Table 1:

[0160]

[0161] The above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled 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 list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for decolorizing a γ-polyglutamic acid fermentation broth, comprising the following steps: Step 1: Resin decolorization: adjust the pH value of the sterilized γ-polyglutamic acid fermentation liquid, add the resin, and separate the resin from the fermentation liquid after decolorization; Step 2: Activated carbon decolorization: adjust the pH value of the fermentation liquid after decolorization in step 1, add activated carbon, and separate the activated carbon from the fermentation liquid after decolorization.

2. The method for decolorizing γ-polyglutamic acid fermentation broth according to claim 1, characterized in that: Also includes step 3 and / or step 4: Step 3: glutamic acid precipitation decolorization: adjusting the pH value of the fermentation liquid after decolorization in step 2, cooling, stirring, growing crystals, and precipitation to obtain wet glutamic acid, and re-dissolving the precipitated wet glutamic acid; Step 4: Decolorization by membrane filtration: γ-polyglutamic acid is separated by membrane filtration equipment and further decolorized.

3. The method for decolorizing γ-polyglutamic acid fermentation broth according to claim 1 or 2, characterized in that: The resin described in step 1 is a macroporous adsorption resin or an anion exchange resin.

4. The method for decolorizing glutamate-dependent γ-polyglutamic acid fermentation broth according to claim 3, characterized in that: The resin addition amount described in step 1 is 0.10-10.00% w / v.

5. The method for decolorizing γ-polyglutamic acid fermentation broth according to claim 4, characterized in that: In step 1, the decolorization time is 4.0h-24.0h; the pH value of the sterilized γ-polyglutamic acid fermentation liquid is adjusted to 3.0-10.0; and the decolorization temperature is 20°C-50°C.

6. The method for decolorizing γ-polyglutamic acid fermentation broth according to claim 1 or 2, characterized in that: The activated carbon described in step 2 has a particle size of 100-325 meshes, an iodine value of 500-1200, and is made of any one of coal, wood, and fruit shell activated carbon. The added amount of the activated carbon is 0.10-5.00% w / v.

7. The method for decolorizing γ-polyglutamic acid fermentation broth according to claim 6, characterized in that: The decolorization time in step 2 is 0.5h-8.0h, the pH value of the fermentation liquid after decolorization in step 1 is adjusted to 3.0-10.0, and the decolorization temperature is 20°C-60°C.

8. The method for decolorizing γ-polyglutamic acid fermentation broth according to claim 2, characterized in that: The specific steps in step 3 include: adjusting the temperature of the gamma-polyglutamic acid decolorized solution obtained in step 2 to 20-30° C., adding acid to a pH value of 4.0-6.0, adding 0.2-0.4% w / v of alpha-glutamic acid seed crystals and stirring for 1-3 hours; then slowly cooling and adding acid, stopping adding acid after the pH value of the solution is reduced to 3.2 after 3-5 hours, stopping cooling and continuing stirring for more than 4 hours after cooling to 4° C. within 8 hours, and finally stopping stirring and allowing natural precipitation for more than 4 hours to obtain wet glutamic acid.

9. The method for decolorizing γ-polyglutamic acid fermentation broth according to claim 8, characterized in that: The wet glutamic acid re-dissolving method described in step 3 is: placing the obtained wet glutamic acid re-dissolving solution in deionized water, adding NaOH solution thereto to maintain the pH value of the system at 8.0-11.0, and continuously stirring.

10. The method for decolorizing γ-polyglutamic acid fermentation broth according to claim 2, characterized in that: The membrane filtration device described in step 4 is any one of a flat membrane, a tubular membrane, a hollow fiber membrane or a spiral membrane; the molecular weight cutoff of the membrane filtration device is 300-3000D.

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