阅读说明：本技术 一种谷氨酸发酵提取工艺 (Glutamic acid fermentation extraction process ) 是由 杨雪 刘元涛 尤学波 罗玮 于 2019-12-13 设计创作，主要内容包括：本发明属于氨基酸生产技术领域,公开了一种谷氨酸发酵提取工艺,其包括如下步骤：步骤1)制备发酵培养基,步骤2)发酵工艺,步骤3)离心、过滤和浓缩,步骤4)等电、离心和干燥。本发明采用菌体蛋白酶解浸膏作为发酵氮源,发酵效率高,降低了成本；提取工艺中,采用超滤浓缩和浓缩等电技术,操作简单,产品纯度和收率高。(The invention belongs to the technical field of amino acid production, and discloses a glutamic acid fermentation extraction process, which comprises the following steps: step 1) preparing a fermentation medium, step 2) fermenting, step 3) centrifuging, filtering and concentrating, and step 4) isoelectric, centrifuging and drying. The invention adopts the mycoprotein enzymolysis extract as the fermentation nitrogen source, has high fermentation efficiency and reduces the cost; in the extraction process, the ultrafiltration concentration and concentration isoelectric technologies are adopted, the operation is simple, and the product purity and yield are high.)

1. A glutamic acid fermentation extraction process comprises the following steps: step 1) preparing a fermentation medium, step 2) fermenting, step 3) centrifuging, filtering and concentrating, and step 4) isoelectric, centrifuging and drying.

2. The process of claim 1, wherein step 1) of preparing a fermentation medium comprises the steps of: taking fermentation medium raw materials, preparing according to the following concentration of 80g/L glucose, 20g/L extract and K₂HPO₄2g/L，MgSO₄·7H₂O 50mg/L，MnSO₄·H₂O 3mg/L，FeSO₄·7H₂O 3 mg/L，VB₁10mg/L and 7 mu g/L biotin, uniformly stirring, adjusting the pH value to 6.5, sterilizing at 121 ℃ for 15min, and naturally cooling to prepare the fermentation medium.

3. The process of claim 2, wherein the step 2) fermentation process comprises the steps of: inoculating the seed solution into a fermentation culture medium according to the inoculation amount of 8-10% of brevibacterium flavum, performing fermentation culture for more than 36h, and collecting fermentation liquor; in the whole fermentation process, the fermentation temperature is controlled to be 35 ℃, the ventilation ratio is 1: 0.6, the stirring speed is 500r/min, the dissolved oxygen is maintained at 20 percent, glucose with the fed-batch mass percentage concentration of 50 percent is maintained at 1.0 percent of residual sugar, and the fed-batch defoaming agent is used for defoaming.

4. The process of claim 3, wherein the step 3) of centrifuging, filtering and concentrating comprises the steps of: centrifuging the fermentation liquor by a disc centrifuge at 4000rpm for 5min, collecting upper layer liquid, filtering by a ceramic membrane (molecular weight cut-off is 10000 Da), collecting filtrate, ultrafiltering by an ultrafiltration membrane (molecular weight cut-off is 300 Da), collecting filtrate, and concentrating by 3 times to obtain concentrated solution.

5. The process of claim 4, wherein the step 4) of isoelectric, centrifugation and drying comprises the steps of: feeding the concentrated solution obtained in the step 3) into a first-stage isoelectric tank, adding concentrated sulfuric acid to adjust the pH value to 3.5, controlling the temperature to be 22 ℃, sequentially passing the liquid passing through the first-stage isoelectric tank through a second-stage isoelectric tank, and simultaneously adding concentrated sulfuric acid to adjust the pH value, wherein the pH value of the second-stage isoelectric tank is controlled to be 3.3, and the temperature is 10 ℃; the liquid passing through the second-stage isoelectric point tank sequentially passes through the third-stage isoelectric point tank, and concentrated sulfuric acid is added to adjust the pH value, wherein the pH value of the third-stage isoelectric point tank is controlled to be 3.2, and the temperature is 5 ℃; centrifuging to obtain wet crystal, and drying at 80 deg.C to obtain glutamic acid product.

6. The process according to claim 2 or 4, wherein the extract is prepared according to the following process:

step (1) thallus separation: separating glutamic acid fermentation liquor by a disc separator, and collecting thallus precipitate;

step (2) high-pressure homogenization: adding a potassium dihydrogen phosphate aqueous solution into the thallus sediment to prepare a thallus cell suspension with the concentration of 100-;

centrifuging: stopping homogenizing, centrifuging at 1000rpm for 3-5min, and collecting upper layer liquid and precipitate;

and (4) ultrasonic treatment: adding 5-10 times of purified water into the precipitate, stirring, and treating with ultrasonic wave for 20-30 min;

step (5) enzymolysis: adjusting the temperature to 45 ℃, hydrolyzing by adopting neutral protease for 6-9 h;

and (6) evaporation and concentration: and (3) heating to 90-100 ℃, inactivating enzyme for 3-5min, mixing with the upper layer liquid obtained in the step (3), and finally evaporating and concentrating to obtain a mycoprotein extract with the dry weight part of 60-70%.

7. The process as claimed in claim 6, wherein the rotation speed of the disk centrifuge is 4000-5000rpm, and the centrifugation time is 3-5 min.

8. The process according to claim 6, characterized in that the homogenization parameters are: homogenizing at 25 deg.C under 90Mpa for 3 times.

9. The process according to claim 6, characterized in that the frequency of the ultrasound waves is 20 kHz.

10. The process according to claim 6, wherein the neutral protease is added in an amount of 500u enzyme activity/g dry matter.

Technical Field

The invention belongs to the technical field of amino acid production, and particularly relates to a glutamic acid fermentation extraction process.

Background

L-glutamic acid (L-glutamic acid) has a wide range of applications in food, medicine and industry, and is a precursor for producing monosodium glutamate, particularly in food. Since Kinoshita et al established microbial fermentation for glutamic acid production in 1957, there has been a history of over 50 years. The fermentation production of glutamic acid initiates a new era of the industrial production of amino acid by the fermentation method, so that the fermentation production of amino acid becomes one of the most important research fields of industrial microorganisms. In 1964, the glutamic acid is successfully produced by applying a fermentation method in China, a new fermentation process is firstly adopted in Shanghai, and then the method is gradually popularized in China, so that the rise of the monosodium glutamate industry in China is powerfully promoted. The yield of monosodium glutamate in China in 2003 is 118.9 ten thousand tons, which accounts for 53 percent of the world yield; yield in 2006 is 136 million tons, world first. In 2010, the yield of monosodium glutamate reaches 210 ten thousand tons, which is far ahead of other countries; the yield of monosodium glutamate in 2018 has broken through 300 ten thousand tons.

The optimization of a fermentation culture medium in a process for preparing glutamic acid by fermentation is an important factor for improving the fermentation efficiency, and the prior patent technology of the applicant 'a method for preparing the fermentation culture medium by fermenting waste thalli with glutamic acid' improves and optimizes the culture medium in the prior art, and comprises 15% of sorghum straw hydrolysate, 12% of thalli hydrolysate, 5% of glucose, 1.2% of rice bran extract, 1% of corn steep liquor, 0.02% of shell powder, 0.02% of ferrous sulfate heptahydrate, 0.02% of magnesium sulfate heptahydrate, 0.01% of potassium dihydrogen phosphate and the balance of water; the culture medium reduces the cost, but has fermentation nitrogen sources and carbon source substances with more pigments, high viscosity and more impurities, so that the fermentation process is difficult to control, and the instability of fermentation and the difficulty of separation and extraction are easily caused; in addition, hydrochloric acid is adopted in the method for treating mycoprotein in the patent document, which is easy to damage amino acid, and the nutritive value of hydrolysate is low. The present research continues to further discuss fermentation media, and aims to develop a fermentation medium with high fermentation efficiency and low cost.

Along with the development of the monosodium glutamate industry, the extraction process of glutamic acid is also continuously improved. In recent years, some monosodium glutamate production enterprises and research institutes in China carry out a great deal of research on the thallus-removing and glutamic acid-extracting of glutamic acid fermentation liquor, and the thallus-removing process comprises high-speed centrifugal separation, flocculant separation, membrane separation and the like, so that remarkable effect is achieved. According to different processes of the degerming body, the degerming rate reaches more than 70-90%, the membrane separation degerming rate can reach more than 95%, the obtained fermentation liquor is clear, the OD value is low, and the glutamic acid extraction operation is convenient. Because a large amount of impurities which affect the glutamic acid crystallization are removed, the glutamic acid crystallization has large particles, high purity, good quality, easy sedimentation separation and obviously improved extraction yield. The high-purity glutamic acid is beneficial to the refinement of monosodium glutamate, the consumption of activated carbon or resin can be reduced by neutralization, decoloration and filtration of monosodium glutamate, the crystallization quality of monosodium glutamate is improved, and the production cost of monosodium glutamate is greatly reduced. In addition, COD and BOD in the fermentation liquor after thallus removal and the waste liquor after isoelectric extraction are greatly reduced, the environmental pollution is reduced, the treatment load and difficulty of waste water are reduced, and the obtained thallus can be comprehensively utilized after being dried, such as being used as high-protein feed or being used as a production raw material of nucleotide.

Disclosure of Invention

The invention aims to solve the technical problem of providing a glutamic acid fermentation extraction process.

The invention is realized by the following technical scheme.

A glutamic acid fermentation extraction process comprises the following steps: step 1) preparing a fermentation medium, step 2) fermenting, step 3) centrifuging, filtering and concentrating, and step 4) isoelectric, centrifuging and drying.

The process of claim 1, wherein step 1) produces a fermentation cultureThe culture medium comprises the following steps: taking fermentation medium raw materials, preparing according to the following concentration of 80g/L glucose, 20g/L extract and K₂HPO₄2g/L，MgSO₄·7H₂O 50mg/L，MnSO₄·H₂O 3mg/L，FeSO₄·7H₂O 3 mg/L，VB₁10mg/L and 7 mu g/L biotin, uniformly stirring, adjusting the pH value to 6.5, sterilizing at 121 ℃ for 15min, and naturally cooling to prepare the fermentation medium.

The process of claim 2, wherein the step 2) fermentation process comprises the steps of: inoculating Brevibacterium flavum GDK-9 into fermentation culture medium according to inoculation amount of 8-10% to perform fermentation culture, wherein the inoculation concentration of thallus is OD_600nmFermenting for over 36h under 0.8, and collecting fermentation liquor; in the whole fermentation process, the fermentation temperature is controlled to be 35 ℃, the ventilation ratio is 1: 0.6, the stirring speed is 500r/min, the dissolved oxygen is maintained at 20 percent, glucose with the fed-batch mass percentage concentration of 50 percent is maintained at 1.0 percent of residual sugar, and the fed-batch defoaming agent is used for defoaming.

The process of claim 3, wherein the step 3) of centrifuging, filtering and concentrating comprises the steps of: centrifuging the fermentation liquor by a disc centrifuge at 4000rpm for 5min, collecting upper layer liquid, filtering by a ceramic membrane (molecular weight cut-off is 10000 Da), collecting filtrate, ultrafiltering by an ultrafiltration membrane (molecular weight cut-off is 300 Da), collecting filtrate, and concentrating by 3 times to obtain concentrated solution.

The process of claim 4, wherein the step 4) of isoelectric, centrifugation and drying comprises the steps of: feeding the concentrated solution into a first-stage isoelectric tank, adding concentrated sulfuric acid to adjust the pH value of the solution in the isoelectric tank to be 3.5, controlling the temperature to be 22 ℃, sequentially passing the liquid passing through the first-stage isoelectric tank through a second-stage isoelectric tank, and simultaneously adding concentrated sulfuric acid to adjust the pH value, wherein the pH value of the second-stage isoelectric tank is controlled to be 3.3, and the temperature is 10 ℃; the liquid passing through the second-stage isoelectric point tank sequentially passes through the third-stage isoelectric point tank, and concentrated sulfuric acid is added to adjust the pH value, wherein the pH value of the third-stage isoelectric point tank is controlled to be 3.2, and the temperature is 5 ℃; centrifuging to obtain wet crystal, and drying at 80 deg.C to obtain glutamic acid product.

The process according to claim 2 or 4, wherein the extract is prepared according to the following process:

step (1) thallus separation: separating glutamic acid fermentation liquor by a disc separator, and collecting thallus precipitate;

step (2) high-pressure homogenization: adding a potassium dihydrogen phosphate aqueous solution into the thallus sediment to prepare a thallus cell suspension with the concentration of 100-;

centrifuging: stopping homogenizing, centrifuging at 1000rpm for 3-5min, and collecting upper layer liquid and precipitate;

and (4) ultrasonic treatment: adding 5-10 times of purified water into the precipitate, stirring, and treating with ultrasonic wave for 20-30 min;

step (5) enzymolysis: adjusting the temperature to 45 ℃, hydrolyzing by adopting neutral protease for 6-9 h;

and (6) evaporation and concentration: heating to 90-100 ℃, inactivating enzyme for 3-5min, mixing with the upper layer liquid obtained in the step 3), and finally evaporating and concentrating to obtain a mycoprotein extract with the dry weight part of 60-70%.

The process as claimed in claim 6, wherein the rotation speed of the disk centrifuge is 4000-5000rpm, and the centrifugation time is 3-5 min.

The process according to claim 6, characterized in that the homogenization parameters are: homogenizing at 25 deg.C under 90Mpa for 3 times.

The process according to claim 6, characterized in that the frequency of the ultrasound waves is 20 kHz.

The process according to claim 6, wherein the neutral protease is added in an amount of 500u enzyme activity/g dry matter.

Compared with the prior art, the invention has the advantages that the following aspects are mainly included but not limited:

the invention does not adopt an acid or alkali hydrolysis mode for the mycoprotein, reduces pollution, avoids corrosion to equipment, only adopts an enzymolysis mode, has mild conditions, does not damage nutrient substances, has stable components of enzymolysis products and controllable quality, and is beneficial to the quality control of a large-scale fermentation system.

When the thalli is treated, firstly, high-pressure homogenization treatment is adopted, cell walls can be fully crushed, intracellular proteins are released, a monopotassium phosphate aqueous solution is adopted as a buffer solution, the crushing rate can be improved, and monopotassium phosphate can also be used as a component of a fermentation culture medium, so that two purposes are achieved; the operation pressure of the homogenization treatment is crucial to cell disruption and protein release, the disruption dynamics and the disruption process effect homogenization pressure are integrated, and multiple tests are carried out, preferably 90 MPa; the cell breakage rate is rapidly increased 2 times before homogenization, the cell breakage rate is not obviously improved after the homogenization again, but after the homogenization for the 3 rd time, macromolecular protein is also cracked into micromolecular short peptide or free amino acid under the action of the homogenization, the content of amino acid nitrogen is increased by a certain amount, the degree of proteolysis is increased, the number of times of homogenization is continuously increased, the degree of proteolysis is not obviously increased, and certain damage is caused to equipment; the supernatant liquid is centrifuged after homogenization, the level of amino acid nitrogen contained in the supernatant liquid is higher, and the partial product is not required to be subjected to enzymolysis treatment, so that the use amount of enzyme is reduced, and the cost is reduced; the precipitate is cell fragment and macromolecular protein, still contains a certain amount of protein in the cell wall fragment moreover, and partial protein can adhere on the cell wall fragment, through carrying out ultrasonic treatment to the precipitate for protein and polysaccharide on the cell fragment release, and the polysaccharide is utilized as the nutrient substance by the bacterial strain, and after ultrasonic treatment, and then adopt neutral protease to carry out enzymolysis, the use amount and the action time of enzyme reduce by a wide margin, have practiced thrift the cost.

Compared with the conventional yeast extract nitrogen source, the fermentation culture medium disclosed by the invention is slightly improved in terms of fermentation thallus concentration and glutamic acid yield, so that the thallus protein extract prepared by the invention is richer and more comprehensive in nutrient content and is beneficial to utilization of microorganisms and acid production compared with the conventionally used nitrogen source yeast extract (the dry matter content is 65-70%).

In the extraction process, the ultrafiltration concentration, the concentration and other electric technologies are adopted, the operation is simple, the purity of the glutamic acid is more than 90 percent through the separation and purification process, and the yield is more than 95 percent.

Drawings

FIG. 1: influence of high-pressure homogenization times on the cell breakage rate of thalli;

FIG. 2: the effect of the number of high pressure homogenizations on the degree of hydrolysis.

Detailed Description

Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the products and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate alterations and combinations, of the products and methods described herein may be made and utilized without departing from the spirit, scope, and spirit of the invention. For a further understanding of the present invention, reference will now be made in detail to the following examples.