WO2020140388A1 - Glutamic acid green clean fermentation process - Google Patents

Abstract

A glutamic acid fermentation process, comprising the following steps: introducing Corynebacterium glutamicum into a fermentation tank filled with a clean fermentation medium for fermentation culture, performing ultrasonic treatment, and adjusting the pH value of the fermentation broth.

Description

Green clean fermentation process of glutamic acid Technical field

The invention belongs to the technical field of amino acid production, and specifically relates to a green clean fermentation process of glutamic acid.

Background technique

Glutamate is an acidic amino acid. The molecule contains two carboxyl groups and the chemical name is α-aminoglutaric acid. Glutamate was discovered by Risoxon in 1856. It is a colorless crystal with umami taste, slightly soluble in water, and soluble in hydrochloric acid solution. The isoelectric point is 3.22. It exists in a large amount in cereal proteins and is also abundant in animal brains. Glutamate occupies an important position in the process of protein metabolism in organisms and participates in many important chemical reactions in animals, plants and microorganisms. Sodium glutamate, commonly known as monosodium glutamate, is an important umami agent that enhances flavor. Sodium glutamate is widely used as a food flavoring agent. It can be used alone or in combination with other amino acids. Used in foods, it has a flavor enhancing effect. The concentration in food is 0.2%-0.5%, and the per capita daily intake per person is 0-120 μg/kg (as glutamic acid). The general dosage in food processing is 0.2-1.5 g/kg.

As the amino acid with the largest yield in the production of amino acids, glutamic acid is currently the most commonly used method for preparing glutamic acid by microbial fermentation. Corynebacterium glutamicum is a conventional strain of glutamic acid fermentation. There are many factors that affect the acid production efficiency of Corynebacterium glutamicum, and its improvement in this field mainly includes the following aspects: 1. Microorganisms use different substrates in different environmental conditions, different metabolic pathways, and purposeful Modification and transformation of the cell's metabolic pathways to change the original metabolic characteristics of the cell can increase the yield and yield of the target product; 2. By increasing the cell membrane permeability of the bacterial cell, increase glutamate secretion, and then eliminate the high bacterial cell The feedback regulation function of concentration glutamic acid improves the production of glutamic acid; 3. Optimize the fermentation medium and fermentation parameters, so that the proliferation rate of the bacteria is increased, thereby increasing the production of amino acids. The applicant has made considerable research on the fermentation of glutamic acid. For example, the Chinese invention patent "CN106148445A" discloses a new glutamic acid extraction process, in which the fermentation culture and fermentation parameters are improved to reduce the fermentation cost and solve The existing technology has high fermentation medium cost, low conversion rate, high consumption of sulfuric acid and liquid ammonia, etc.; Chinese invention patent "CN107227324A" discloses a sub-appropriate fermentation process of glutamic acid biotin, using fermentation tank and membrane coupling technology Filtration and dialysis in the fermentation process to separate the glutamic acid in the fermentation broth in a timely manner, eliminating the feedback regulation of the high concentration of glutamic acid in the fermentation broth; through the use of specific dialysis fermentation medium formula for re-fermentation, to improve the utilization efficiency of bacteria and Conversion rate of sugar and acid; in addition, filtration and dialysis after a certain stage of fermentation can timely separate toxic by-products in the fermentation broth, reducing the inhibition of acid production by the bacteria; the fermentation process of the present invention achieves the bacteria by dialysis fermentation The re-fermentation technology prolongs the acid production cycle of glutamic acid fermentation, improves the utilization rate of the bacteria, and improves the conversion rate of sugar and acid; the Chinese invention patent "CN104099382A" discloses a method for fermenting L-glutamine using cotton seed cake powder hydrolysate Acid method, which is: adding cottonseed cake powder hydrolysate to L-glutamic acid fermentation medium, the amino nitrogen concentration of the cottonseed cake powder hydrolysate is 0.5-3.0%, and the L-glutamic acid fermentation culture The base is a temperature-sensitive L-glutamic acid fermentation medium. The added amount of the cottonseed cake powder hydrolysate is 15-25 ml/l. It can effectively ensure the fermentation of L-glutamic acid by controlling the added amount of the cottonseed cake powder hydrolysate. With smooth progress, the utilization of cottonseed cake powder hydrolysate not only broadens organic nitrogen source resources but also reduces fermentation costs, and has broad application prospects in the fermentation industry.

Extensive use of fermented nitrogen source materials such as corn syrup, soybean meal hydrolysate, and molasses in the prior art for deep fermentation, high viscosity, and many impurities makes the fermentation process difficult to control, easily causing fermentation instability and separation and extraction. difficult. Therefore, by adjusting the fermentation medium and using nutrients with less impurities, the stability of fermentation and the cost of separation and extraction can be achieved. Clean fermentation technology mainly refers to the relatively clean fermentation medium. By adjusting and replacing the composition of the fermentation medium, nutrients with simple ingredients and few impurities are used to replace nutrients with complex ingredients and relatively many impurities, so that the fermentation broth The impurity content is reduced, the mass transfer and dissolved oxygen efficiency are improved, and the fermentation process is more stable. Corn syrup, soybean meal hydrolysate, etc. are often added to the fermentation medium of L-glutamic acid as a nitrogen source of fermentation and provide a small amount of vitamins, which can accelerate the growth rate of the bacteria. However, this type of nitrogen source contains a large amount of proteins, pigments, and insoluble impurities, which makes it easy to foam during fermentation, low dissolved oxygen efficiency, and large mass transfer resistance. In addition, due to the unstable biotin content in corn steep liquor, it is easy to Causes fluctuations in fermentation acid production. At the same time, the fermentation broth with more impurities adds difficulty to the subsequent separation and extraction. Therefore, based on the control fermentation medium, through the substitution and optimization of the fermentation nitrogen source, a clean fermentation medium with less impurity content was finally determined, which made the fermentation process easier to control and the fermentation acid production was more stable.

In the amino acid anabolic pathway, when the four carbon dicarboxylic acids are all supplied by the CO ₂ fixation reaction, the highest theoretical sugar-acid conversion rate is 81%; while when the CO ₂ fixation reaction does not work at all, the four carbon dicarboxylic acid can only pass through Glyoxylic acid supply, the highest theoretical conversion rate is 54%. The production process of glutamic acid has been relatively mature, the main technical indicators are glutamic acid concentration of 10%-12%, sugar conversion rate of 55%-60%. However, compared with foreign advanced fermentation technology, there is still much room for improvement. Effectively increasing the conversion rate will save raw material costs and increase the economic benefits of glutamic acid fermentation. There are not many metabolic by-products of glutamic acid fermentation using C. glutamicum, and the main by-product is CO ₂ . Therefore, strengthening the CO ₂ fixation reaction in the metabolic pathway of Corynebacterium glutamicum, and for this purpose, allowing key enzyme systems for glutamate synthesis to interact effectively and mutually will hopefully increase the CO ₂ reuse rate and sugar acid conversion rate, Save raw material costs and increase corporate profits.

An important point of glutamic acid fermentation is the specific changes in the structure and function of the cell membrane of the glutamic acid producing bacteria during the fermentation culture, so that the cell membrane is transformed into a membrane that facilitates the infiltration of glutamic acid out of the membrane, that is, the completion of the non-accumulative cells Transition to glutamate accumulation cells. In this way, because the final product of glutamic acid is continuously discharged from the cell, so that the glutamic acid in the cell cannot accumulate to the concentration that causes feedback regulation, glutamic acid will continue to be preferentially synthesized in the cell, and will continue to penetrate The cell membrane is secreted into the fermentation medium, thereby accumulating in large quantities. There are many substances to adjust the permeability of the cell membrane, and the structure of the cell membrane varies greatly between different strains, so there is no rule to follow. Choosing the appropriate method to adjust the permeability of the cell membrane is also a technology that needs to be solved in the glutamic acid fermentation process. problem.

Summary of the invention

The technical problem to be solved by the present invention is to provide a green clean fermentation process of glutamic acid, which effectively solves a variety of problems in the glutamic acid fermentation process, including the efficiency of glutamic acid synthesis, cell permeability, and fermentation broth culture medium Optimization improves the efficiency of fermentation and reduces the difficulty of separation and purification of glutamic acid.

The present invention is achieved by the following technical solutions.

The green clean fermentation process of glutamic acid includes the following steps: Corynebacterium glutamicum is inserted into a fermentation tank equipped with a clean fermentation medium for fermentation culture, the total fermentation time is 30-40h, wherein, the fermentation culture is 0- At 8h, ultrasonic treatment was performed, and the pH value of the fermentation broth was adjusted to 7.0-7.2 by adding ammonia water; after 8h, the fermentation regulator was added to the fermentor, and at the same time, the pH value of the fermentation broth was adjusted to 7.0-7.2 by adding liquid ammonia.

Further, the conditions of the ultrasonic treatment are: ultrasonic power 500W, frequency 20kHZ, ultrasonic time 50s, amplitude 65%, interval 5min.

Further, the clean fermentation medium is: glucose 80g/L, MnSO ₄ ·H ₂ O 3mg/L, FeSO ₄ ·7H ₂ O 3mg/L, MgSO ₄ ·7H ₂ O 2g/L, Na ₂ HPO ₄ 12H ₂ O 4g/L, KCl 2g/L, V _B1 10mg/L, Fulvic acid 1mg/L, Biotin 7μg/L, bacterial cell digest 80ml/L.

Further, the fermentation regulator is an aqueous solution containing inositol and glycerin.

Further, the fermentation regulator is: inositol 1-2g/L, glycerol 10-20g/L.

Further, the method for preparing the enzymatic hydrolysis solution of the bacterial cell is to take the Corynebacterium glutamicum cell in the glutamic acid fermentation broth, dry it to a dry cell with a moisture content of less than 5 wt%, and dilute it to the concentration of the dry cell with water 50g/L, placed in a high-speed shear at a speed of 10,000rpm for 120s, and stirred to obtain a bacterial suspension. Add the same volume of 1mol/L hydrochloric acid solution to the bacterial suspension, mix well, at 95 After treatment at ℃ for 1h, trypsin is added for hydrolysis, and then the ceramic membrane is filtered, and the filtrate is collected, which is the bacterial enzymolysis solution.

Further, the hydrolysis conditions of the trypsin are: pH 8, temperature 37°C, hydrolysis time 6 h; the enzyme activity of the trypsin is 4000 U/g, and the addition amount is: dry mass ratio of enzyme to substrate 4%.

Further, the cut-off molecular weight of the ceramic membrane is 5000-10000Da.

Further, the shear speed of the high-speed shear is 10000 rpm, and the shear time is 120 s.

Further, the process includes the following steps:

Corynebacterium glutamicum is inoculated with 10-15% of the seed liquid into a fermentation tank equipped with a clean fermentation medium for fermentation culture, fermentation temperature 35-38 ℃, ventilation ratio 1:0.5-2, stirring speed 300 -700r/min, maintain dissolved oxygen at 10%-30%, add 80% glucose by mass concentration to maintain residual sugar at 1%-2%, add defoamer to defoam, total fermentation time is 33-34h; Among them, the fermentation culture is 0-8h. After the fermentation is started, the ultrasonic controller is turned on for ultrasonic treatment. The conditions of ultrasonic treatment are: ultrasonic power 500W, frequency 20kHZ, ultrasonic time 50s, amplitude 65%, interval time 5min, and flow through Add 25% ammonia water to adjust the pH of the fermentation broth to 7.0-7.2; after 8h, add a fermentation regulator to the fermentor at a time, the addition amount accounts for 1-5% of the volume of the fermentation broth, while adding liquid ammonia to adjust the fermentation broth pH value to 7.0-7.2.

Compared with the prior art, the beneficial effects achieved by the present invention mainly include but are not limited to the following aspects:

By adding bacterial proteolysis solution to replace corn steep liquor, research data found that with the addition of bacterial proteolysis solution, the bacterial concentration and glutamate content have increased, but adding too much bacterial proteolysis solution not only It causes waste, and makes it easy to foam during the fermentation process, low dissolved oxygen efficiency and large mass transfer resistance and other problems, resulting in a decline in fermentation efficiency. For the fermentation supernatant of clean fermentation, the substances that affect the light transmittance mainly come from the metabolism of the bacteria, which continuously declines as the impurities generated by the bacteria increase, but the overall light transmittance is much higher than that of corn syrup fermentation. For the subsequent separation and extraction of glutamic acid, the production cost can be greatly reduced. Fulvic acid contains a large number of phenolic hydroxyl groups, carbonyl groups and other groups, with a high degree of electrolysis, which promotes the use of O ₂ as a hydrogen acceptor in the process of glutamic acid synthesis, thereby reducing pyruvate as a hydrogen acceptor, so by-products lactic acid and alanine The amount of acid produced is reduced, thereby increasing the production of glutamic acid. The optimization of the medium of the present invention not only makes the glutamic acid fermentation process more stable and easy to control, but also improves the glutamic acid yield and sugar-acid conversion rate, improves the quality of the fermentation broth, reduces the cost of glutamic acid extraction, and comprehensive benefits Has been improved.

Realize a reasonable ratio of nutrients, maximize the acid production capacity of the bacteria to improve the fermentation conversion rate and acid production; when the glutamic acid-producing bacteria multiply to a larger value, when the glutamic acid-producing enzyme system is completely formed, add an appropriate amount The inositol can both strengthen the CO ₂ fixation reaction, weaken the glyoxylic acid cycle, ensure that the tricarboxylic acid cycle is not interrupted and continuously supply α-ketoglutaric acid. Through the reductive amination reaction, a large amount of glutamic acid accumulates and improves Fermentation conversion rate; glycerol provides a carbon skeleton, promotes the synthesis of glutamic acid, and can improve the permeability of the cell membrane, and promote the secretion of glutamic acid into the fermentation broth.

Through single factor and orthogonal test, the invention determines the best ultrasonic parameters, including intensity, time, amplitude, etc., can improve the permeability of the cell membrane, and promote the improvement of bacterial vitality, glutamic acid production and dissolved oxygen efficiency. Pyruvate produced by glycolysis will not accumulate excessively, and will enter the tricarboxylic acid cycle more. Accordingly, the metabolic byproducts produced by pyruvate, lactic acid and alanine, will also be reduced. According to this study, ultrasound-assisted glutamic acid fermentation can effectively improve the permeability of bacterial cell membranes, increase glutamate secretion, increase glutamate production, and reduce fermentation vice in an appropriate ultrasound environment. The product realized the improvement of glutamic acid fermentation efficiency.

BRIEF DESCRIPTION

Figure 1: The effect of ultrasonic time on glutamic acid fermentation;

Figure 2: The effect of ultrasonic amplitude on glutamic acid fermentation;

Figure 3: The effect of interval time on glutamic acid fermentation;

Figure 4: Comparison of the optimal ultrasonic fermentation and control fermentation;

Figure 5: The effect of optimal ultrasound conditions on bacterial transformation time and by-products.

detailed description

In order to enable those skilled in the art to better understand the technical solutions in the present application, the technical solutions of the present application will be described clearly and completely in conjunction with specific embodiments of the present application. Obviously, the described embodiments are only the present application Some embodiments, not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.

Example 1

The strain selected in this experiment is Corynebacterium glutamicum GDK-9 (also known as Brevibacterium flavum GDK-9, from Tianjin University of Science and Technology, and the source of the strain can also be found in "L-Glutamate Fermentation Temperature Control Technology Research, Tianjin Chemical Industry" year 2010").

The green clean fermentation process of glutamic acid includes the following steps: the seed liquid (OD _600nm is 10) of Corynebacterium glutamicum GDK-9 at 15% inoculation volume is connected to a 50L fully automatic with 30L clean fermentation medium Fermentation culture is carried out in the fermenter, fermentation temperature is 38℃, ventilation ratio is 1:0.7, stirring speed is 500r/min, dissolved oxygen is maintained at 20%, glucose is added at a mass concentration of 80%, and residual sugar is maintained at 1.5%. Antifoaming agent defoaming, total fermentation time 33h; Among them, fermentation culture 0-8h (first stage), insert high temperature resistant ultrasonic probe in the fermentation tank, after fermentation starts, turn on the ultrasonic controller, under certain conditions Ultrasonic treatment, the conditions of ultrasonic treatment are: ultrasonic power 500W, frequency 20kHZ, ultrasonic time 50s, amplitude 65%, interval time 5min, and adjust pH of fermentation broth to 7.0-7.2 by adding 25% ammonia water; after 8h (the first In the second stage, choose to add the fermentation regulator at the beginning of the second stage), add the fermentation regulator to the fermentor at a time, the amount of the addition is 2% of the volume of the fermentation broth, and at the same time add liquid ammonia to adjust the pH of the fermentation broth to 7.0 -7.2.

The clean fermentation medium is: glucose 80g/L, MnSO ₄ · H ₂ O 3mg/L, FeSO ₄ · 7H ₂ O 3mg/L, MgSO ₄ · 7H ₂ O 2g/L, Na ₂ HPO ₄ · 12H ₂ O 4g/L, KCl 2g/L, V _B1 10mg/L, fulvic acid 1mg/L, biotin 7μg/L, bacterial enzymolysis solution 80ml/L; sterilized at 115℃ for 15min.

The clean fermentation medium improves the conventional medium, in which:

Use bacterial enzymolysis solution to replace corn syrup as a nitrogen source for fermentation;

Quantitative addition of biotin to replace biotin in corn steep liquor;

Add fulvic acid to fermentation medium;

The preparation method of the bacterial cell enzymolysis liquid is: taking the Corynebacterium glutamicum cell in the glutamic acid fermentation broth, drying it to a dry cell with a moisture content of less than 5 wt%, and diluting it with water to a concentration of 50 g per dry cell L, placed in a high-speed shear at a speed of 10,000 rpm for 120 s to obtain a bacterial suspension, add the same volume of 1 mol/L hydrochloric acid solution to the bacterial suspension, mix well, and process at 95°C for 1 h. After adding trypsin for hydrolysis, the ceramic membrane is filtered and the filtrate is collected; the molecular weight cut-off of the ceramic membrane is 10,000 Da; the macromolecular substances that are difficult to be used by the strain are removed by filtration, including cell wall components and macromolecular proteins.

That is, a bacterial proteolysis solution having a protein content of 231 mg/g (dried cells) and a total free amino acid content of 367 mg/g (dried cells) is obtained.

The hydrolysis conditions of the trypsin are: pH 8, temperature 37°C, hydrolysis time 6 h; the enzyme activity of the trypsin is 4000 U/g, and the added amount is: the dry mass ratio of enzyme to substrate is 4 %.

Example 2

The green clean fermentation process of glutamic acid includes the following steps: the seed liquid (OD _600nm is 14) of Corynebacterium glutamicum GDK-9 at a 10% inoculation volume is connected to a 50L fully automatic with 30L of clean fermentation medium Fermentation culture is carried out in the fermenter, the fermentation temperature is 37℃, the ventilation ratio is 1:0.8, the stirring speed is 300-700r/min, the dissolved oxygen is maintained at 25%, and the addition of glucose with a mass percentage concentration of 80% maintains the residual sugar at 1%. Adding defoaming agent to defoam, the total fermentation time is 34h; among them, fermentation culture is 0-8h, insert the high temperature resistant ultrasonic probe in the fermentation tank, after the fermentation starts, turn on the ultrasonic controller and perform ultrasonic treatment under certain conditions, The conditions of ultrasonic treatment are: ultrasonic power 500W, frequency 20kHZ, ultrasonic time 50s, amplitude 65%, interval time 5min, and adjust the pH of the fermentation broth to 7.0-7.2 by adding 25% ammonia water; after 8h, go to the fermentation tank The fermentation regulator is added at a time, the amount of which accounts for 3% of the volume of the fermentation broth, and liquid ammonia is added at the same time to adjust the pH of the fermentation broth to 7.0-7.2.

The clean fermentation medium is: glucose 80g/L, MnSO ₄ · H ₂ O 3mg/L, FeSO ₄ · 7H ₂ O 3mg/L, MgSO ₄ · 7H ₂ O 2g/L, Na ₂ HPO ₄ · 12H ₂ O 4g/L, KCl 2g/L, V _B1 10mg/L, fulvic acid 1mg/L, biotin 7μg/L, bacterial enzymolysis solution 80ml/L.

The clean fermentation medium improves the conventional medium, in which:

Use bacterial enzymolysis solution to replace corn syrup as a nitrogen source for fermentation;

Quantitative addition of biotin to replace biotin in corn steep liquor;

Add fulvic acid to fermentation medium;

The fermentation regulator is: inositol 1.5g/L, glycerol 15g/L.

Example 3

Optimization of glutamic acid fermentation medium.

1. The effect of the amount of bacterial proteolysis solution on the concentration of bacterial cells in the fermentation broth and the production of glutamic acid. Five concentration gradients of 20, 40, 60, 80, 100, 120 (ml/L) were selected. Increase the amount of bacteria in the fermentation broth

Table 1

Add amount ml/L	OD 600nm of bacteria in fermentation broth	Glutamate production g/L
20	60.7	143.6
40	68.9	152.1
60	75.3	159.8
80	81.8	167.0
100	80.1	163.5

120

76.9

156.9

By adding bacterial proteolysis solution to replace corn syrup (10g/L, OD _{600nm in} fermentation broth is 65.8, glutamic acid yield is 151g/L, light transmittance (T ₄₃₀ ) is 0.98), the data found that with the bacteria With the addition of proteolysis solution, the bacterial cell concentration and glutamic acid content are both improved. When added to 10mg/L, the bacterial cell concentration and glutamic acid content reach the peak. Adding too much bacterial cell proteolysis solution will not only cause waste And, it makes problems such as easy foaming, low dissolved oxygen efficiency and large mass transfer resistance during the fermentation process, resulting in a decrease in fermentation efficiency. As for the fermentation supernatant of clean fermentation, the substances that affect the light transmittance mainly come from the metabolism of the bacteria, and they continue to decline as the impurities generated by the bacteria increase. The light transmittance at the end of the fermentation is 6.1, but the overall light transmittance The rate is much higher than that of corn syrup fermentation. For the subsequent separation and extraction of glutamic acid, the production cost can be greatly reduced.

2. The effect of the addition amount of fulvic acid on the production of glutamic acid and alanine in the fermentation broth, choose the addition concentration as five concentration gradients of 0,0.5,1,2,4 (mg/L), as shown in Table 2:

Table 2

Addition mg/L	Alanine production g/L	Glutamate production g/L
0	3.3	160.5
0.5	2.4	163.8
1	1.8	167.0
1.5	1.7	166.8
2	1.7	167.1

Fulvic acid contains a large number of phenolic hydroxyl groups, carbonyl groups and other groups, with a high degree of electrolysis, which promotes the use of O ₂ as a hydrogen acceptor in the process of glutamic acid synthesis, thereby reducing pyruvate as a hydrogen acceptor, so by-products lactic acid and alanine The amount of acid produced is reduced, thereby increasing the production of glutamic acid. With the increase of the amount of fulvic acid, the content of alanine gradually decreased, accompanied by the increase in the production of glutamic acid. When it increased to 1 mg/L, the amount of alanine and glutamic acid did not change much, considering For cost factors, it is most appropriate to choose the addition amount of 1 mg/L.

In short, the optimization of the medium of the present invention not only makes the glutamic acid fermentation process more stable and easy to control, but also improves the glutamic acid yield and sugar-acid conversion rate, improves the quality of the fermentation broth, and reduces the cost of glutamic acid extraction. Comprehensive benefits have been improved.

Example 4

Effects of fermentation regulators on conversion rate and glutamate production.

Set up a control group, where:

Control group 1: No fermentation regulator is added, the rest is the same as Example 1;

Control group 2: The fermentation regulator contains inositol only, the rest is the same as in Example 1;

Control group 3: The fermentation regulator contains only glycerin, and the rest is the same as in Example 1;

The experimental group is Example 1.

The conversion rate and glutamic acid concentration of each group are shown in Table 3.

table 3

Group	Sugar acid conversion rate%	Glutamate production g/L
Control group 1	60.4	151.7
Control 2	64.7	159.6
Control group 3	62.8	162.9
test group	68.5	169.2

Conclusion: Set up a control group to study the effects of inositol and glycerol in the fermentation regulator on the conversion rate and glutamate production. As can be seen from Table 3, the two have a certain synergistic effect and can significantly improve the conversion rate and glutamine Acid yield.

Example 5

The effect of ultrasound on glutamic acid fermentation.

1. The effect of ultrasonic time on glutamic acid fermentation

Under the condition that the ultrasonic amplitude is 70% and the interval time is 10 minutes, the effect of different ultrasonic time on glutamic acid fermentation is shown in Figure 1. It can be seen from Figure 1 that with the increase of ultrasound time, the OD _600nm value of bacteria, glutamate production and sugar acid conversion rate also continue to rise, and the maximum value is reached when the continuous ultrasound time is 50s, and then with the ultrasound time The extension of the three, the three began to decline, especially in the OD _600nm value of the bacteria and glutamate production fell faster. According to the analysis, the ultrasonic treatment with appropriate duration can improve the permeability of the cell membrane, increase the enzyme activity of the bacteria, accelerate the growth rate of the bacteria, and then can increase the secretion of glutamic acid and the conversion rate of sugar acid. However, if the continuous ultrasonic time is too long, it will inevitably cause a certain degree of damage to the cell membrane of the bacterial cell, and it will also disrupt the normal metabolic activity of the bacterial cell and reduce the growth capacity and acid production of the bacterial cell. Choose the best ultrasound time to achieve better results, so choose 50s as the continuous ultrasound time.

2. The effect of ultrasonic amplitude on glutamic acid fermentation

Under the condition that the optimal ultrasonic time is 50s and the interval time is 10min, the effect of different ultrasonic amplitudes on glutamic acid fermentation is shown in Figure 2. It can be clearly seen that when the ultrasonic amplitude is in the range of 55-65%, the OD _600nm value, glutamic acid production and sugar acid conversion rate continue to increase, especially the increase in bacterial volume and glutamic acid production is very obvious; when ultrasound After the amplitude is higher than 65%, there is a significant decrease in the three, especially the decrease in the amount of bacteria. The main reason is that the appropriate amplitude of ultrasonic amplitude can increase the growth rate and acid production rate of the bacteria, and excessively high ultrasonic amplitude will cause greater damage to the normal growth of the bacteria, reduce the vitality of the bacteria, and is not conducive to the growth of the bacteria And glutamate synthesis. Therefore, 65% is selected as the optimal ultrasonic amplitude.

3. The effect of interval time on glutamic acid fermentation

Under the condition that the optimal ultrasonic time is 50s and the optimal ultrasonic amplitude is 65%, the effect of different ultrasonic interval time on glutamic acid fermentation is shown in Figure 3. It can be seen from the figure that the effect of ultrasound time interval on the OD _600nm value of the bacterial cells, the production of glutamic acid and the conversion rate of sugar acid is very obvious. The interval time (4min) is too short, that is, the ultrasound frequency is high, the ultrasound intensity is large, and the damage to the bacteria is more serious. Not only can it not promote the growth of the bacteria and the secretion of glutamic acid, but it can also inhibit the bacteria to a certain extent. With the increase of the interval time (6min), the ultrasonic intensity is just right, and the OD _600nm value of the cells, the yield of glutamic acid and the conversion rate of sugar acid have been improved. When the interval time is too long, although the increase in OD _600nm value is not obvious, but the damage effect on the cell membrane is too low, and due to the cell membrane repair function of the cell itself, the permeability of the cell membrane has not been improved, not It has a substantial effect on the bacteria, so glutamate production and sugar-acid conversion rate begin to approach the normal fermentation level. Therefore, 6min was chosen as the best ultrasound interval.

4. Analysis of orthogonal test results

Based on the above single-factor test results, an orthogonal test design was conducted. The experimental results are shown in Table 4 and the analysis of variance is shown in Table 5.

Table 4 Ultrasonic-assisted orthogonal experiment design and results of glutamic acid fermentation

Table 5 ANOVA

On the one hand, from the orthogonal test results and range (R) analysis, it can be seen that the order of influence on the OD _600nm value of the bacteria is A>B>C, that is, ultrasound time>amplitude>interval time, and the optimal condition is A ₂ B ₂ C ₃ , that is, ultrasonic time 50s, amplitude 65%, interval time 7min; the order of influence on glutamate production is B>A>C, that is amplitude>ultrasound time>interval time, the optimal condition is A ₂ B ₂ C ₁ , That is, ultrasonic time 50s, amplitude 65%, interval time 5min; the order of influence on the conversion rate of sugar acid is B>A>C, that is amplitude>ultrasound time>interval time, the optimal condition is A ₂ B ₂ C ₁ , namely Ultrasound time 50s, amplitude 65%, interval time 5min. On the other hand, from the analysis of variance, it can be seen that the ultrasonic time and amplitude have a significant effect on the OD _600nm value and glutamate production, and the interval time has a significant effect on the OD _600nm value. Considering the above test results comprehensively, it can not only achieve a certain amount of OD _600nm value, but also increase glutamate production and sugar-acid conversion rate. Therefore, the optimal ultrasound condition is A ₂ B ₂ C ₁ , that is, ultrasound time 50s, amplitude 65%, interval 5min.

5. Analysis of verification experiment results

According to the best ultrasonic conditions obtained by the orthogonal test, three batches of parallel fermentation were verified, and the obtained test results are shown in Figure 4. From the analysis in Figure 4, it can be seen that, whether it is the OD _600nm value of the bacteria, glutamic acid production and sugar acid conversion rate, ultrasonic fermentation has a certain degree of improvement compared to the control fermentation. The acid production time of the control fermentation (that is, the glutamic acid cell begins to complete the conversion from the non-accumulating glutamic acid cell to the glutamic acid accumulating cell) is generally about 4h, while the ultrasonic fermentation can detect glutamic acid The fermentation time is 2h, and the rate of acid production rises rapidly. At the same time, combined with microscopic examination, it was found that part of the glutamic acid bacteria fermented by ultrasound had been elongated and expanded into acid-producing bacteria at 2h, and the transformation of the bacteria began. Therefore, compared with the control fermentation, the time of ultrasonic fermentation to start acid production was advanced 2h. The reason why the ultrasound end time is selected for fermentation is 8h. On the one hand, it is because the cell wall of Corynebacterium glutamicum is thick, which has a certain protective effect on the cell membrane. The ultrasound time is too short to damage the cell membrane to improve its permeability. Function; on the other hand, the bacteria themselves also have a certain ability to repair the cell membrane, so there must be enough ultrasound time to maintain the improvement of the permeability of the cell membrane. The microscopic examination of ultrasonic fermentation at 8h found that almost all the bacteria have completed the transformation of glutamic acid, and no more damage to the cell membrane is needed. At the same time, the OD _600nm value of the bacteria will soon reach the maximum value (about 14h) The vitality of the bacteria is about to reach the highest level. If you continue to perform ultrasonic treatment, it may cause serious damage to the bacteria and reduce the vitality of the bacteria. Therefore, you choose 8h to end the ultrasonic treatment. Overall, the OD _600nm value of ultrasonic fermentation is 82.5, which is an increase of 13.8% compared with the control fermentation of 72.5; the glutamic acid production is 168g/L, which is an increase of 11.3% compared with the control fermentation of 151g/L; sugar acid conversion The rate was 68.2%, which was an increase of 4.3% compared with 65.4% of the control fermentation. In Fig. 4, A, C and E are the OD600nm value of control fermentation, glutamic acid production and sugar acid conversion rate; B, D and F are the OD600nm value of optimal fermentation fermentation, glutamic acid production and sugar acid respectively Conversion rates.

In addition, due to the increase in cell viability, glutamate production, and dissolved oxygen efficiency, it can be seen that the rate and flux of the TCA cycle have also been improved, and pyruvate produced by glycolysis does not accumulate excessively, but instead It will enter the tricarboxylic acid cycle more, so the metabolic byproducts produced by pyruvic acid, lactic acid and alanine, will also be reduced. As shown in Fig. 5, from the control fermentation (without ultrasound) of 3.6g/L and 2.54g/L to the ultrasound fermentation of 2.3g/L and 1.75g/L, respectively, the reduction was 36.11% and 31.10% respectively.

From the above research, it can be seen that ultrasound-assisted glutamic acid fermentation can effectively improve the permeability of bacterial cell membranes, increase glutamate secretion, increase the production of glutamic acid, and reduce the fermentation side in an appropriate ultrasound environment. The product realized the improvement of glutamic acid fermentation efficiency.

The above list is only the best embodiment of the present invention. Obviously, the present invention is not limited to the above embodiments, and there can be many variations. All variations that can be directly derived or associated with the disclosure of the present invention by those of ordinary skill in the art should be considered as the protection scope of the present invention.

Claims (10)

1. A green clean fermentation process for glutamic acid, characterized in that the process includes the following steps: Corynebacterium glutamicum is inserted into a fermentation tank equipped with a clean fermentation medium for fermentation culture, and the total fermentation time is 30-40h; Among them, fermentation culture 0-8h, ultrasonic treatment, and adjust the pH value of the fermentation broth to 7.0-7.2 by adding ammonia water; after 8h, add a fermentation regulator to the fermentation tank, while adding liquid ammonia to adjust the pH value of the fermentation broth To 7.0-7.2.
2. The process according to claim 1, characterized in that the conditions of the ultrasonic treatment are: ultrasonic power 500W, frequency 20kHZ, ultrasonic time 50s, amplitude 60-65%, interval time 5-6min, total ultrasonic time 8h; Preferably, the amplitude is 65% and the interval is 5 min.
3. The process according to any one of claims 1-2, wherein the clean fermentation medium is: glucose 80g/L, MnSO ₄ · H ₂ O 3mg/L, FeSO ₄ · 7H ₂ O 3mg/L , MgSO ₄ ·7H ₂ O 2g/L, Na ₂ HPO ₄ ·12H ₂ O 4g/L, KCl 2g/L, V _B1 10mg/L, fulvic acid 1mg/L, biotin 7μg/L, bacterial enzyme Solution 80ml/L.
4. The process according to any one of claims 1 to 3, wherein the fermentation regulator is an aqueous solution containing inositol and glycerin.
5. The process according to any one of claims 1 to 4, characterized in that the fermentation regulator is: inositol 1-2g/L, glycerol 10-20g/L.
6. The process according to claim 3, characterized in that the method for preparing the enzymatic hydrolysis solution of the bacterial cell is: taking the Corynebacterium glutamicum cell in the fermentation broth of glutamic acid and drying to a dry content of less than 5wt% Bacteria, diluted with water to a concentration of 50g/L of dried cells, placed in a high-speed shear to obtain a bacterial suspension, add the same volume of 1mol/L hydrochloric acid solution to the bacterial suspension, and mix well After treatment at 95℃ for 1h, trypsin was added for hydrolysis, and then the ceramic membrane was filtered, and the filtrate was collected, which was the bacterial enzymolysis solution.
7. The process according to claim 6, characterized in that the hydrolysis conditions of the trypsin are: a pH of 8, a temperature of 37°C, and a hydrolysis time of 6h.
8. The process according to claim 6, characterized in that the molecular weight cut-off of the ceramic membrane is 5000-10000Da.
9. The process according to claim 6, characterized in that the shear speed of the high-speed shear is 10000 rpm and the shear time is 120 s.
10. The process according to any one of claims 1-9, wherein the process includes the following steps:

    Corynebacterium glutamicum is inoculated with 10-15% of the seed liquid into a fermentation tank equipped with a clean fermentation medium for fermentation culture, fermentation temperature 35-38 ℃, ventilation ratio 1:0.5-2, stirring speed 300 -700r/min, maintain dissolved oxygen at 10%-30%, add 80% glucose by mass concentration to maintain residual sugar at 1%-2%, add defoamer to defoam, total fermentation time is 33-34h; Among them, the fermentation culture is 0-8h. After the fermentation is started, the ultrasonic controller is turned on for ultrasonic treatment. The conditions of ultrasonic treatment are: ultrasonic power 500W, frequency 20kHZ, ultrasonic time 50s, amplitude 65%, interval time 5min, and flow through Add 25% ammonia water to adjust the pH of the fermentation broth to 7.0-7.2; after 8h, add a fermentation regulator to the fermentor at a time, the addition amount accounts for 1-5% of the volume of the fermentation broth, while adding liquid ammonia to adjust the fermentation broth pH value to 7.0-7.2.

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