Method for producing polyglutamic acid by fermenting glutamic acid extract

Abstract

A method for producing polyglutamic acid by fermenting a glutamic acid extract, which method comprises: S1: inoculating a salt-tolerant bacillus seed solution into a fermentation tank containing a fermentation culture medium at an inoculation amount of 20%-25% to perform fermentation, with the liquid loading volume of the fermentation culture medium being 5 L, the culture temperature being 36-38°C, the stirring speed being 350-380 rpm / min and the culture time being 36-42 h, and collecting a polyglutamic acid fermentation liquid; and S2: extracting polyglutamic acid from the polyglutamic acid fermentation liquid by means of using a salt extraction method and a pH adjustment method. The fermentation culture medium has the following formula per 1 L: 15%-20% of concentrated molasses, 2%-5% of corn steep liquor, 55%-60% of a glutamic acid extract and 0.3%-0.5% of K2HPO4, which are dissolved in distilled water such that the system has a total volume of 1 L and a pH of 6.0-6.5. In the method for producing polyglutamic acid by fermenting a glutamic acid extract provided by the present application, the glutamic acid extract is used as the main raw material for the fermentation and production of polyglutamic acid for the first time in the industry, such that nutrient components in the glutamic acid extract are fully utilized, and high-quality utilization of the glutamic acid extract is realized.

Description

A method for producing polyglutamic acid by fermenting glutamic acid extract Technical Field

[0001] The present application belongs to the field of biological fermentation technology, and specifically relates to a method for producing polyglutamic acid by fermenting glutamic acid extract. Background Art

[0002] Polyglutamic acid (PGA) is a biopolymer material that is highly water-soluble, biodegradable, and non-toxic, offering promising applications in the environmental, pharmaceutical, food, and agricultural sectors. Currently, the main methods for synthesizing PGA include chemical synthesis, extraction, and microbial synthesis. Microbial synthesis, however, has become the primary method for producing PGA due to its stable yield and easily controllable production process.

[0003] Monosodium glutamate is produced through a biological fermentation process. Glutamic acid is extracted from the fermentation broth and then subjected to isoelectric treatment to obtain an isoelectric mother liquor, which is the glutamate extract. The glutamate content in the glutamate extract is relatively low, typically 0.5% to 1%. Re-extraction of the glutamate from this extract is costly, and direct disposal of the extract causes environmental pollution and waste of resources. Currently, most glutamate extracts are treated to meet standards before being discharged or manufactured into fertilizer. Therefore, a new method for processing glutamate extracts is urgently needed in the art.

[0004] Summary of the Invention

[0005] The present application provides a method for producing polyglutamic acid by fermenting glutamic acid extract. The method uses glutamic acid extract as the main raw material to ferment and produce polyglutamic acid, fully utilizing the nutrients in the fermentation liquid, thereby achieving high-quality utilization of the glutamic acid extract and producing agricultural polyglutamic acid at the same time.

[0006] The present application provides a method for producing polyglutamic acid by fermentation using a glutamic acid extract, the method comprising the following steps:

[0007] S1: Inoculate a 20-25% inoculum amount of a halodurable Bacillus seed solution into a fermentation tank containing a fermentation medium, wherein the fermentation medium is filled with 5 L, the culture temperature is 36-38°C, the stirring speed is 350-380 rpm / min, and the culture time is 36-42 hours. After the culture is completed, the polyglutamic acid fermentation liquid is collected;

[0008] S2: extracting polyglutamic acid from the polyglutamic acid fermentation broth by adding sulfate and organic solvent to obtain solid polyglutamic acid;

[0009] The fermentation medium has the following formula per 1 L: 15-20% concentrated molasses, 2-5% corn steep liquor, 55-60% glutamic acid extract and 0.3-0.5% K2HPO4, which are dissolved in distilled water to make the total volume of the system 1 L and the pH value 6.0-6.5.

[0010] Furthermore, the halotolerant Bacillus in step S1 is Bacillus halotolerans F29, with a preservation number of CGMCC NO.23662.

[0011] Furthermore, the halotolerant Bacillus seed solution in step S1 is obtained by the following method:

[0012] A1: Activation of Halotolerant Bacillus: Culture the Halotolerant Bacillus on a solid plate medium for 12-16 hours at a temperature of 35-39°C to obtain activated cells.

[0013] A2: Preparation of shake flask seed solution: Pick 1-2 loops of activated bacteria cultured on solid plates and inoculate them into shake flask culture medium. Incubate at 36-40°C on a shaker for 12-16 hours to obtain shake flask seed solution.

[0014] A3: Seed solution preparation: Inoculate the shake flask seed solution into the seed culture medium at an inoculum size of 3-5%, incubate at 32-40°C and 180-220 rpm / min for 6-10 hours to obtain the halotolerant Bacillus seed solution;

[0015] Furthermore, the solid plate culture medium is formulated as follows: 8-10 g / L tryptone, 3-4 g / L yeast powder, 7-8 g / L sodium chloride, 15-18 g / L glucose, 8-12 g / L sodium glutamate, and 15-18 g / L agar powder, dissolved in distilled water to a total volume of 1 L; and the pH is 6.8-7.2;

[0016] The shake flask culture medium formula is the same as the solid plate culture medium formula, except that agar powder is not added;

[0017] The seed culture medium comprises 6-8 g / L of tryptone, 4-6 g / L of yeast powder, 7-8 g / L of sodium chloride, 12-15 g / L of glucose, 8-12 g / L of sodium glutamate, and 15-18 g / L of agar powder, which are dissolved in distilled water to a total volume of 1 L; and the pH value is 6.8-7.2.

[0018] Furthermore, the specific steps of step S2 are: adding 300-400 g / L of sulfate to the polyglutamic acid fermentation broth, adjusting the pH of the polyglutamic acid fermentation broth to 6.5-7.5, letting it stand for 50-60 minutes, filtering with a metal mesh, and collecting the filtrate; adding 1-1.5 times the volume of an organic solvent to the filtrate, continuously stirring, collecting the polyglutamic acid precipitate by sedimentation filtration, adding water for redissolution, adding activated carbon for decolorization, and obtaining a polyglutamic acid clarified liquid; adding 1-1.5 times the volume of an organic solvent to the clarified liquid, stirring until the white precipitate no longer increases, and filtering to obtain solid polyglutamic acid.

[0019] Furthermore, the sulfate is anhydrous magnesium sulfate or ferrous sulfite; and the organic solvent is ethanol with a concentration of 60% (volume fraction).

[0020] Compared with the prior art, this application has the following beneficial effects:

[0021] (1) The present application provides a method for producing polyglutamic acid by fermentation using glutamic acid extract. The present application is the first in the industry to use glutamic acid extract as the main raw material to ferment and produce polyglutamic acid, making full use of the nutrients in the fermentation liquid, that is, achieving high-quality utilization of glutamic acid extract and producing agricultural polyglutamic acid at the same time.

[0022] (2) The components of the fermentation medium used in this application are significantly reduced compared to traditional strains, and there is no need to add fermentation medium during the process, thus achieving low-consumption production.

[0023] (3) The present application adds sulfate (anhydrous magnesium sulfate, ferrous sulfite) during the extraction of polyglutamic acid, which, on the one hand, reduces the production cost of agricultural polyglutamic acid, and on the other hand, sulfate can provide certain nutrients for plant growth. DETAILED DESCRIPTION

[0024] The present application is further described below with reference to the following embodiments. The present application includes but is not limited to the following embodiments.

[0025] Example 1

[0026] This embodiment provides a method for producing polyglutamic acid by fermentation using a glutamate extract. The glutamate extract used is obtained from the monosodium glutamate production process. Testing shows that the glutamate extract contains 0.5-0.6% residual sugar, 1-1.2% total sugar, 0.7-1.0% organic acids such as lactic acid and succinic acid, 0.05-0.07% keto acids, 0.6-0.8% ammonium ions, and some residual anions, cations, and other culture medium impurities.

[0027] The method for producing polyglutamic acid by fermentation of glutamic acid extract comprises the following steps:

[0028] (1) Activating the salt-tolerant Bacillus: Cultivate the salt-tolerant Bacillus on a solid plate medium for 12 to 16 hours at an activation temperature of 35°C to obtain activated cells;

[0029] The solid plate culture medium is formulated as follows: 8 g / L tryptone, 3 g / L yeast extract, 7 g / L sodium chloride, 15 g / L glucose, 10 g / L sodium glutamate, and 16 g / L agar powder, dissolved in distilled water to a total volume of 1 L; and the pH is 6.8.

[0030] The halotolerant Bacillus selected in this example is Bacillus halotolerans F29, with a preservation number of CGMCC NO.23662.

[0031] (2) Preparation of shake flask seed solution: Pick one loop of activated bacteria cultured on a solid plate and inoculate it into a shake flask medium. Incubate at 37°C on a shaker for 14 h to obtain the shake flask seed solution.

[0032] The shake flask culture medium formula is consistent with the solid plate culture medium formula, except that agar powder is not added.

[0033] (3) Seed solution preparation: Inoculate the shake flask seed solution into the seed culture medium at an inoculum rate of 3% at a temperature of 32°C and a rotation speed of 200 rpm / min for 8 h to obtain the halotolerant Bacillus seed solution;

[0034] The seed culture medium comprises 6 g / L tryptone, 6 g / L yeast powder, 7 g / L sodium chloride, 12 g / L glucose, 8 g / L sodium glutamate, and 15 g / L agar powder, which are dissolved in distilled water to a total volume of 1 L; and the pH is 6.8.

[0035] (4) Fermentation tank culture: The seed liquid of the halodurable Bacillus was inoculated at an inoculum size of 20% into a fermentation tank containing a fermentation medium. The fermentation medium was loaded into a 5 L volume, cultured at a temperature of 36° C., stirred at a speed of 350 rpm / min, and cultured for 36 h. After the culture was completed, the fermentation liquid was collected to obtain a polyglutamic acid fermentation liquid;

[0036] The fermentation medium has the following formula per 1 L: 15% concentrated molasses, 3% corn steep liquor, 55% glutamic acid extract and 0.3% K2HPO4, which are dissolved in distilled water to make the total volume of the system 1 L and the pH value 6.0-6.5.

[0037] (5) Extraction of polyglutamic acid: 300 g / L of anhydrous magnesium sulfate was added to the polyglutamic acid fermentation broth, the pH of the polyglutamic acid fermentation broth was adjusted to 6.5, and the broth was allowed to stand for 50 min and filtered through a metal mesh to collect the filtrate; 1.2 times the volume of ethanol was added to the filtrate, and the mixture was stirred continuously. The polyglutamic acid precipitate was collected by sedimentation filtration, and redissolved in water. Activated carbon was added for decolorization to obtain a polyglutamic acid clarified liquid. 1.2 times the volume of ethanol was added to the clarified liquid, and the mixture was stirred until the white precipitate no longer increased. The solid polyglutamic acid was obtained by filtration.

[0038] In the fermentation process of polyglutamic acid, if the fermentation time is too long, the yield of polyglutamic acid will be low, because the fermentation time is too long and the fermentation bacteria will die; the rotation speed also has a greater impact on the production of polyglutamic acid. The higher the rotation speed, the faster the system transmission speed, and the cells can reach saturation in a shorter time, after which the bacteria die. At a lower rotation speed, the mass transfer of the system is slow and the overall fermentation rate is low. The present application has found through research that when a polyglutamic acid extract is used for fermentation production of polyglutamic acid, when the rotation speed is 350rpm / min and the fermentation time is controlled at 36h, the output value of polyglutamic acid is high, reaching 34.7g / L.

[0039] Liquid polyglutamic acid has very stringent requirements on the storage environment, including storage temperature and time. There are also problems such as precipitation of polyglutamic acid during the storage process, and there are also many inconveniences in use and transportation. The present application makes it into a solid form, which can not only ensure the subsequent use of polyglutamic acid, but also avoid the impact of the storage period on its quality, thereby solving the drawbacks in the transportation of liquid products.

[0040] Example 2

[0041] This embodiment provides a method for producing polyglutamic acid by fermentation using a glutamic acid extract, which comprises the following steps:

[0042] (1) Activating the halogen-tolerant Bacillus: Cultivate the halogen-tolerant Bacillus on a solid plate medium for 15 h at an activation temperature of 37°C to obtain activated cells;

[0043] The solid plate culture medium is formulated as follows: 10 g / L tryptone, 4 g / L yeast extract, 8 g / L sodium chloride, 18 g / L glucose, 12 g / L sodium glutamate, and 17 g / L agar powder, dissolved in distilled water to a total volume of 1 L; and the pH is 7.2.

[0044] The halotolerant Bacillus selected in this example is Bacillus halotolerans F29, with a deposit number of CGMCC NO.23662.

[0045] (2) Preparation of shake flask seed solution: Pick 2 rings of activated bacteria cultured on solid plates and inoculate them into shake flask culture medium. Incubate at 40°C on a shaker for 16 h to obtain shake flask seed solution.

[0046] The shake flask culture medium formula is consistent with the solid plate culture medium formula, except that agar powder is not added.

[0047] (3) Seed solution preparation: Inoculate the shake flask seed solution into the seed culture medium at an inoculum rate of 5%, culture at 40°C and 180 rpm / min for 10 h to obtain the halotolerant Bacillus seed solution;

[0048] The seed culture medium comprises 8 g / L tryptone, 6 g / L yeast powder, 8 g / L sodium chloride, 15 g / L glucose, 10 g / L sodium glutamate, and 17 g / L agar powder, which are dissolved in distilled water to a total volume of 1 L; and the pH is 7.2.

[0049] (4) Fermentation tank culture: The seed liquid of the halodurable Bacillus was inoculated at an inoculum size of 25% into a fermentation tank containing a fermentation medium. The volume of the fermentation medium was 5 L, the culture temperature was 38° C., the stirring speed was 380 rpm / min, and the culture time was 40 h. After the culture was completed, the fermentation liquid was collected to obtain a polyglutamic acid fermentation liquid;

[0050] The fermentation medium has the following formula per 1 L: 20% concentrated molasses, 5% corn steep liquor, 60% glutamic acid extract and 0.5% K2HPO4, which are dissolved in distilled water to make the total volume of the system 1 L and the pH value 6.0-6.5.

[0051] (5) Extraction of polyglutamic acid: 350 g / L of anhydrous magnesium sulfate was added to the polyglutamic acid fermentation broth, and the pH of the polyglutamic acid fermentation broth was adjusted to 7.0. After standing for 55 minutes, the broth was filtered through a metal mesh and the filtrate was collected. 1.5 times the volume of ethanol was added to the filtrate, and the mixture was continuously stirred. The polyglutamic acid precipitate was collected by sedimentation filtration, and redissolved in water. Activated carbon was added for decolorization to obtain a polyglutamic acid clarified liquid. 1.5 times the volume of ethanol was added to the clarified liquid, and the mixture was stirred until the white precipitate no longer increased. The solid polyglutamic acid was obtained by filtration.

[0052] After testing, it was found that in the fermentation method provided in this embodiment, the glutamic acid content in the polyglutamic acid fermentation broth reached 33.5 g / L.

[0053] Example 3

[0054] The preparation method comprises the following steps:

[0055] (1) Activating the halogen-tolerant Bacillus: Cultivate the halogen-tolerant Bacillus on a solid plate medium for 14 h at an activation temperature of 38°C to obtain activated cells;

[0056] The solid plate medium formula is: 7 g / L tryptone, 3 g / L yeast powder, 8 g / L sodium chloride, 15 g / L glucose, 10 g / L sodium glutamate, and 16 g / L agar powder, dissolved in distilled water to make the total volume of the system 1 L; and the pH is 7.0;

[0057] The halotolerant Bacillus selected in this example is Bacillus halotolerans F29, with a preservation number of CGMCC NO.23662.

[0058] (2) Preparation of shake flask seed solution: Pick 1 to 2 rings of activated bacteria cultured on solid plates and inoculate them into shake flask culture medium. Incubate at 38°C on a shaker for 14 h to obtain shake flask seed solution.

[0059] The shake flask culture medium formula is consistent with the solid plate culture medium formula, except that agar powder is not added.

[0060] (3) Seed solution preparation: Inoculate the shake flask seed solution into the seed culture medium at an inoculum rate of 4%, culture at 38°C and 220 rpm / min for 8 h to obtain the halotolerant Bacillus seed solution;

[0061] The seed culture medium comprises 7 g / L tryptone, 5 g / L yeast powder, 8 g / L sodium chloride, 13 g / L glucose, 10 g / L sodium glutamate, and 17 g / L agar powder, which are dissolved in distilled water to a total volume of 1 L; and the pH is 7.0.

[0062] (4) Fermentation tank culture: The seed liquid of the halodurable Bacillus was inoculated at an inoculum size of 22% into a fermentation tank containing a fermentation medium. The fermentation medium was loaded into a volume of 5 L, cultured at a temperature of 37° C., stirred at a speed of 370 rpm / min, and cultured for 40 h. After the culture was completed, the fermentation liquid was collected to obtain a polyglutamic acid fermentation liquid;

[0063] The fermentation medium has the following formula per 1 L: 18% concentrated molasses, 4% corn steep liquor, 60% glutamic acid extract and 0.4% K2HPO4, which are dissolved in distilled water to make the total volume of the system 1 L and the pH value 6.0-6.5.

[0064] (5) Extraction of polyglutamic acid: 400 g / L of ferrous sulfite was added to the polyglutamic acid fermentation broth, the pH of the polyglutamic acid fermentation broth was adjusted to 7.5, and the broth was allowed to stand for 60 min and filtered through a metal mesh to collect the filtrate; 1.2 times the volume of ethanol was added to the filtrate, and the mixture was stirred continuously. The polyglutamic acid precipitate was collected by sedimentation filtration, and redissolved in water. Activated carbon was added for decolorization to obtain a polyglutamic acid clarified liquid. 1.2 times the volume of ethanol was added to the clarified liquid, and the mixture was stirred until the white precipitate no longer increased. The solid polyglutamic acid was obtained by filtration.

[0065] After testing, it was found that in the fermentation method provided in this embodiment, the glutamic acid content in the polyglutamic acid fermentation broth reached 32.9 g / L.

[0066] This application provides a method for producing polyglutamic acid by fermentation using glutamic acid extract. This method is the first in the industry to use glutamic acid extract as the primary raw material for the fermentation production of polyglutamic acid, fully utilizing the nutrients in the fermentation broth. This method achieves high-quality utilization of the glutamic acid extract while simultaneously producing agricultural polyglutamic acid. The addition of sulfate (anhydrous magnesium sulfate, ferrous sulfite) during the polyglutamic acid extraction process reduces the production cost of agricultural polyglutamic acid while also providing nutrients for plant growth.

[0067] The above embodiment is only one of the preferred implementation methods of the present application and should not be used to limit the scope of protection of the present application. Any changes or modifications that are made to the main design concept and spirit of the present application and have no substantive significance, as long as the technical problems they solve are still consistent with those of the present application, should be included in the scope of protection of the present application.

Claims

1. A method for producing polyglutamic acid by fermentation using glutamic acid extract, comprising: S1: Inoculate the seed liquid of halotolerant Bacillus with an inoculum amount of 20-25% in a fermenter containing a fermentation medium for fermentation, wherein the volume of the fermentation medium is 5 L, the culture temperature is 36-38° C., the stirring speed is 350-380 rpm / min, the culture time is 36-42 h, and the polyglutamic acid fermentation liquid is collected after the culture is completed; S2: extracting polyglutamic acid from polyglutamic acid fermentation broth by adding sulfate and organic solvent to obtain solid polyglutamic acid; The fermentation medium has the following formula per 1L: 15-20% concentrated molasses, 2-5% corn steep liquor, 55-60% glutamic acid extract and 0.3-0.5% K2HPO4, which are dissolved in distilled water to make the total volume of the system 1L and the pH value 6.0-6.

5.

2. The method for producing polyglutamic acid by fermentation of glutamic acid extract according to claim 1, wherein: The halotolerans described in step S1 is halotolerans F29, with a preservation number of CGMCC NO.23662.

3. The method for producing polyglutamic acid by fermentation of glutamic acid extract according to claim 1 or 2, wherein: The halotolerant Bacillus seed solution in step S1 is obtained by the following method: A1: Activation of halotolerant Bacillus: Culture halotolerant Bacillus on a solid plate medium for 12 to 16 hours at an activation temperature of 35 to 39°C to obtain activated cells; A2: Preparation of shake flask seed solution: Pick 1-2 rings of activated bacteria cultured on solid plates and inoculate them into shake flask culture medium. Culture them on a shaker at 36-40°C for 12-16 hours to obtain shake flask seed solution. A3: Seed solution preparation: Inoculate the shake flask seed solution into the seed culture medium at an inoculum rate of 3-5%, culture at a temperature of 32-40°C and a rotation speed of 180-220 rpm / min for 6-10 hours to obtain the halotolerant Bacillus seed solution.

4. The method for producing polyglutamic acid by fermentation of glutamic acid extract according to claim 3, wherein: The solid plate culture medium is formulated as follows: 8-10 g / L tryptone, 3-4 g / L yeast powder, 7-8 g / L sodium chloride, 15-18 g / L glucose, 8-12 g / L sodium glutamate and 15-18 g / L agar powder, which are dissolved in distilled water and the total volume of the system is 1 L; and the pH is 6.8-7.2; The shake flask medium formula is consistent with the solid plate medium formula, except that agar powder is not added; The seed culture medium has a formula of 6-8 g / L of tryptone, 4-6 g / L of yeast powder, 7-8 g / L of sodium chloride, 12-15 g / L of glucose, 8-12 g / L of sodium glutamate and 15-18 g / L of agar powder, which are dissolved in distilled water and fixed to 1 L; and the pH is 6.8-7.

2.

5. The method for producing polyglutamic acid by fermentation of glutamic acid extract according to claim 1, wherein: The specific steps of step S2 are: adding 300-400 g / L of sulfate to the polyglutamic acid fermentation liquid, adjusting the pH of the polyglutamic acid fermentation liquid to 6.5-7.5, filtering with a metal mesh after standing for 50-60 minutes, and collecting the filtrate; adding 1-1.5 times the volume of organic solvent to the filtrate, continuously stirring, collecting the polyglutamic acid precipitate by sedimentation filtration, adding water for redissolution, adding activated carbon for decolorization to obtain a polyglutamic acid clarified liquid, adding 1-1.5 times the volume of organic solvent to the clarified liquid, stirring until the white precipitate no longer increases, and filtering to obtain solid polyglutamic acid.

6. The method for producing polyglutamic acid by fermentation of glutamic acid extract according to claim 5, wherein: The sulfate is anhydrous magnesium sulfate or ferrous sulfite; the organic solvent is ethanol with a concentration of 60% (volume fraction).