CN110643547A - Brevibacterium flavum for producing L-valine and method for producing L-valine by using same - Google Patents
Brevibacterium flavum for producing L-valine and method for producing L-valine by using same Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention belongs to the technical field of bioengineering, and particularly relates to a Brevibacterium flavum for producing L-valine, which is preserved in China center for type culture collection (CCTCC M2019496) in 2019 at 07.01.M.with the preservation number of CCTCC M2019496 and the preservation address of eight Lopa of Wuchang district in Wuhan city, Hubei province. The invention also relates to a method for producing L-valine by using the brevibacterium flavum, which comprises the steps of carrying out activated culture and seed liquid culture on the brevibacterium flavum strain, inoculating the obtained seed liquid into a fermentation culture medium for fermentation culture, wherein the fermentation culture process comprises aerobic fermentation and then anaerobic fermentation. The strain preserved by the invention and the production method are adopted to produce the L-valine, the yield is high, the sugar-acid conversion rate is high, the time consumption in the fermentation process is short, and the production efficiency is greatly improved.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to Brevibacterium flavum for producing L-valine and a method for producing L-valine by using the Brevibacterium flavum.
Background
L-valine, also known as 2-amino-3-methylbutyric acid, belongs to branched chain amino acid, has various physiological functions, has obvious functions in the metabolic process of human bodies and other animals, and can be applied to the pharmaceutical industry, the food industry, the feed industry and the like.
In the field of medicine, L-valine is a raw material of compound amino acid infusion and amino acid injection, and the compound amino acid infusion prepared from the L-valine is widely applied to the treatment of blood brain barrier, hepatic coma, chronic cirrhosis and renal failure, the dietary treatment of congenital metabolic defect diseases, the treatment of septicemia and postoperative diabetes, the treatment of surgical wound healing and the nutrition support treatment of tumor patients.
With the rapid development of L-valine from traditional feed additives to high value-added industries such as food, medicine, cosmetics and the like, the market demand of L-valine is increasing year by year. In 2005-2010, the domestic market sales of the valine industry increased from 0.78 to 1.78 billion, and the annual increase rate reached 18%. After 2010, the valine market size still keeps about 20% of growth rate. In 2017, the market scale of the Chinese valine is 3.80 million yuan, and the output and sale amount of the valine should be more than 1 million tons. On the export side, the annual export quantity is increased from 3765 tons to 2 ten thousand tons from 2015 to 2018, and the increase is rapid.
In the prior art, methods for producing L-valine include extraction methods, synthesis methods, fermentation methods and the like. The content of L-valine in animal blood powder, silkworm pupa and hair hydrolysate is high, the valine is separated from the mixed amino acid by applying an ion exchange technology, the separation efficiency is high, the extraction operation is simple, the production period is short, but the cost is high, and the method is not suitable for modern industrial production; the chemical synthesis method is characterized in that isobutyraldehyde is used as a raw material, reacts with ammonia and hydrocyanic acid to generate cyanamide, is hydrolyzed to obtain DL-valine, and is separated to obtain the L-valine. The chemical synthesis method has high production cost, complex reaction, more steps and a plurality of byproducts; the fermentation method is a method for producing L-valine by utilizing a microbial fermentation method, has the advantages of low raw material cost, mild reaction conditions, large-scale production and the like, and is a very economic production method. Therefore, the microbial fermentation method is mostly adopted to produce L-valine at home and abroad.
The invention discloses a Chinese invention patent named ' a Brevibacterium flavum for producing L-valine and application thereof ' (application number is 201910349690.4) ', and discloses a Brevibacterium flavum and L-valine produced by culturing and fermenting the strain, wherein the L-valine yield can reach high yield of 80-90 g/L. However, the method needs to maintain a specific oxygen pressure in the fermentation culture process, the culture process takes longer time, about 90 hours, and the glucose yield (namely the saccharic acid conversion rate) is lower, and is only 25-30%. Therefore, the method for producing L-valine has low efficiency. Wherein the glucose yield (saccharic acid conversion rate) refers to: the ratio of the total mass of L-valine formed at the end of the fermentation to the total mass of glucose (without water) added at the beginning of the fermentation.
Therefore, through years of research, the inventor develops Brevibacterium flavum with high L-valine yield, high production efficiency and high sugar acid conversion rate.
Disclosure of Invention
The invention aims to provide Brevibacterium flavum (Brevibacterium flavum) HHVLA-002 for producing L-valine.
In order to achieve the purpose, the invention adopts the technical scheme that: brevibacterium flavum (Brevibacterium flavum) for producing L-valine, which is preserved in China center for type culture collection in 2019, 07 and 01 days, with the preservation number of CCTCC M2019496 and the preservation address of eight-way Lopa in Wuhan city, Wuchang city, Hubei province.
The Brevibacterium flavum HHVLA-002 is obtained by long-term anaerobic acclimation of commercially available Brevibacterium flavum as an original strain. The starting strain is purchased from Beijing Beinana Chuanglian Union of biology and technology research institute, and is numbered BNCC 184294. The starting strain metabolizes to produce 1-10 g/L of L-valine.
The anaerobic domestication process comprises the following steps: (1) inoculating and culturing a starting strain brevibacterium flavum to obtain a seed solution, carrying out anaerobic fermentation on the seed solution in a fermentation culture medium with the glucose concentration of A, stopping fermentation to obtain a fermentation broth after the glucose concentration in the fermentation culture medium is reduced to the concentration of B, carrying out second anaerobic fermentation on the fermentation broth serving as the seed solution, wherein the culture medium and the fermentation conditions are the same as those of the previous time, stopping fermentation to obtain the fermentation broth after the glucose concentration in the culture medium is reduced to the concentration of B during the second fermentation, repeating the fermentation culture process by using the fermentation broth obtained by the second fermentation as the seed solution, and recording the acid production rate of each fermentation culture process, wherein the concentration of B is 40-60% of the concentration of A;
(2) when the acid production rate and the sugar consumption rate of the Nth fermentation are obviously higher than those of the previous fermentation, the glucose concentration of the culture medium is increased during the (N + 1) th fermentation, and other fermentation conditions are the same as those of the previous fermentation; continuously monitoring the acid production rate in the fermentation process, and repeating the process of increasing the glucose concentration of the culture medium, wherein the glucose concentration increase value is 8-10% of the concentration A each time;
(3) when the glucose concentration of the fermentation medium is increased to 30-50 times of the concentration A, the anaerobic acclimation is completed, and the anaerobically acclimated brevibacterium flavum is obtained.
Specifically, the glucose concentration A is 2-10 g/L.
The culture medium further comprises: 1-20 g/L diammonium hydrogen phosphate, 1-20 g/L potassium dihydrogen phosphate, 1-5 g/L ammonium sulfate, 0.2-2 g/L magnesium sulfate heptahydrate, 0.01-0.05 g/L ferrous sulfate heptahydrate, 1-500 mu g/L biotin and vitamin B11-500 mu g/L, and continuously adjusting the pH value to 6.5-7.8 by using ammonia water;
fermentation conditions are as follows: the temperature is 25-35 ℃, and the rotating speed is 50-200 r/min.
In the anaerobic acclimation process, the brevibacterium flavum can not adapt to an anaerobic environment at the beginning, so the glucose consumption rate is very low, when the acid production rate begins to increase through the circulating fermentation culture process, strains which adapt to the anaerobic environment appear, the glucose concentration of a culture medium begins to be gradually increased at the moment, the bacteria which adapt to the anaerobic fermentation environment are gradually increased, the production rate is increased, the glucose consumption amount is increased, and the brevibacterium flavum can be used as a strain to be cultured and produced in batch when the acid production rate is increased to a proper range.
Another object of the present invention is to provide a method for producing L-valine by using the Brevibacterium flavum, which has high yield, high conversion rate of sugar and acid, high conversion rate and short fermentation time.
In order to achieve the purpose, the invention adopts the technical scheme that: and (2) performing activation culture and seed liquid culture on the anaerobically domesticated brevibacterium flavum strain, and inoculating the obtained seed liquid into a fermentation culture medium for fermentation culture, wherein the fermentation culture process comprises aerobic fermentation and then anaerobic fermentation.
Specifically, the fermentation conditions for the aerobic fermentation are: the temperature is 25-35 ℃, the rotating speed is 50-200 r/min, the pressure is maintained by introducing sterile air into the tank to be 0.12-0.2 Mpa, the air volume is 0.1-1 v/vm, and the fermentation time is 8-12 h.
The fermentation conditions of the anaerobic fermentation are as follows: the temperature is 25-35 ℃, the rotating speed is 50-200 r/min, the fermentation tank is sealed to isolate air, and the fermentation time is 28-38 h.
Preferably, the fermentation medium is: the glucose concentration is 100-200 g/L.
The activation culture is cultured in an activation slant culture medium to a logarithmic phase; the activated slant culture medium comprises: 6.0-15.0 g/L of peptone, 3.0-7.0 g/L of sodium chloride, 3.0-10.0 g/L of yeast extract or yeast powder, 3.0-10.0 g/L of beef extract, 15.0-20.0 g/L of agar, and adjusting the pH value to 7.0-7.2.
The seed liquid culture is to culture 3-5 bacterial colonies in a seed culture medium after activating and culturing the bacterial strains to obtain a seed liquid; the seed culture medium is as follows: 10-30 g/L of glucose, 5-20 g/L of diammonium hydrogen phosphate, 5-20 g/L of potassium dihydrogen phosphate, 0.2-0.9 g/L of magnesium sulfate heptahydrate, 0.01-0.05 g/L of ferrous sulfate heptahydrate, 150-500 mu g/L of vitamin B, and continuously adjusting the pH value to 6.5-7.8 by using ammonia water.
The method has the advantages that the preserved yellow brevibacterium subjected to anaerobic domestication is adopted for L-valine production, the aerobic environment needs to be maintained at the early stage of the fermentation process, the propagation of the strain is accelerated, the anaerobic sealed environment is only needed at the middle and later stages, the glucose consumption in the strain propagation is reduced, the sugar-acid conversion rate is increased, the detection shows that the yield of the L-valine can reach 85-95 g/L, the sugar-acid conversion rate can reach more than 45%, the time for the fermentation process is only 36-46 hours, and the production efficiency is greatly improved.
Detailed Description
The technical scheme of the invention is further detailed by combining the embodiment.
An anaerobically acclimated Brevibacterium flavum, having microorganism preservation information:
microorganism classification nomenclature: brevibacterium flavum (Brevibacterium flavum)
The preservation number is: CCTCC M2019496;
the preservation date is as follows: 7 month 1 in 2019;
the preservation organization: china Center for Type Culture Collection (CCTCC);
address: eight-path Lojia mountain in Wuchang region of Wuhan city, Hubei province.
Example 1
The Brevibacterium flavum (Brevibacterium flavum) HHVLA-002 is inoculated into an activated slant culture medium and cultured to a logarithmic phase, and the activated slant culture medium: 6.0g/L of peptone, 3.0g/L of sodium chloride, 3.0g/L of yeast powder, 3.0g/L of beef extract and 15g/L of agar, and adjusting the pH value to 7.0-7.2; culturing at 30 + -1 deg.C for 20 h.
Inoculating the activated colony in a shake flask seed culture medium for seed culture, wherein the seed culture medium comprises the following components in percentage by weight: 10g/L of glucose, 5g/L of diammonium hydrogen phosphate, 5g/L of potassium dihydrogen phosphate, 0.2g/L of magnesium sulfate heptahydrate, 0.01g/L of ferrous sulfate heptahydrate and 150 mu g/L of vitamin B, and adjusting the pH value to 6.5-7.8 by using ammonia water; culturing at 28 + -1 deg.C and 180r/min for 24 hr to obtain seed solution.
Taking the obtained seed liquid, carrying out fermentation culture under the following fermentation culture medium and culture conditions to produce L-valine, wherein the fermentation culture medium comprises: 160g/L glucose, 20g/L diammonium hydrogen phosphate, 5g/L potassium dihydrogen phosphate, 1g/L ammonium sulfate, 0.9g/L magnesium sulfate heptahydrate, 0.05g/L ferrous sulfate heptahydrate, 1500 mu g/L vitamin B and continuously adjusting the pH to 6.5 by using ammonia water;
fermentation culture conditions: the culture conditions of 0 to 10 hours are: the temperature is 30 ℃, the tank pressure is 0.12-0.13 Mpa, the air volume is 0.1v/vm, and the rotating speed is 200 r/min; the culture conditions for 10 to 36 hours are: the temperature is 30 ℃, the anaerobic fermentation is carried out under the condition of air isolation, and the rotating speed is 200 r/min.
The yield of the obtained fermentation liquor is 87.36g/L through HPLC detection, and the saccharic acid conversion rate is 55.2%.
Example 2
The Brevibacterium flavum (Brevibacterium flavum) HHVLA-002 is inoculated into an activated slant culture medium and cultured to a logarithmic phase, and the activated slant culture medium: 7.0g/L of peptone, 4.0g/L of sodium chloride, 4.0g/L of yeast powder, 4.0g/L of beef extract and 15g/L of agar, and adjusting the pH value to 7.0-7.2. Culturing at 30 + -1 deg.C for 20 h.
Inoculating the activated colony in a shake flask seed culture medium for seed culture, wherein the seed culture medium comprises the following components in percentage by weight: 13g/L glucose, 6g/L diammonium hydrogen phosphate, 6g/L potassium dihydrogen phosphate, 0.3g/L magnesium sulfate heptahydrate, 0.01g/L ferrous sulfate heptahydrate and 180 mu g/L vitamin B, and adjusting the pH value to 6.5-7.2 by using ammonia water; culturing at 28 + -1 deg.C and 180r/min for 24 hr to obtain seed solution.
Taking the obtained seed liquid, and carrying out fermentation culture under the following fermentation culture medium and culture conditions to produce the L-valine:
fermentation medium: 180g/L glucose, 20g/L diammonium hydrogen phosphate, 15g/L potassium dihydrogen phosphate, 2g/L ammonium sulfate, 0.7g/L magnesium sulfate heptahydrate, 0.05g/L ferrous sulfate heptahydrate, 150 mu g/L vitamin B and continuously adjusting the pH to 6.9 by using ammonia water;
fermentation culture conditions: the culture conditions for 0 to 12 hours are: the temperature is 30 ℃, the tank pressure is 0.15-0.18 Mpa, the air volume is 0.2v/vm, and the rotating speed is 200 r/min; the culture conditions for 12 to 43 hours are: the temperature is 30 ℃, the anaerobic fermentation is carried out under the condition of air isolation, and the rotating speed is 100 r/min.
The yield of the obtained fermentation liquor is 90.01g/L through HPLC detection, and the gluconic acid conversion rate is 50.2%.
Example 3
The Brevibacterium flavum (Brevibacterium flavum) HHVLA-002 is inoculated into an activated slant culture medium and cultured to a logarithmic phase, and the activated slant culture medium: 6.0g/L of peptone, 7.0g/L of sodium chloride, 10.0g/L of yeast extract, 4.0g/L of beef extract and 15g/L of agar, and the pH value is adjusted to be 7.0-7.2. Culturing at 30 + -1 deg.C for 20 h.
Inoculating the activated colony in a shake flask seed culture medium for seed culture, wherein the seed culture medium comprises the following components in percentage by weight: 20g/L of glucose, 15g/L of diammonium hydrogen phosphate, 6g/L of potassium dihydrogen phosphate, 0.3g/L of magnesium sulfate heptahydrate, 0.01g/L of ferrous sulfate heptahydrate and 1120 mu g/L of vitamin B, and adjusting the pH value to 6.5-7.2 by using ammonia water; culturing at 28 + -1 deg.C and 180r/min for 24 hr to obtain seed solution.
Taking the obtained seed liquid, and carrying out fermentation culture under the following fermentation culture medium and culture conditions to produce the L-valine:
fermentation medium: 200g/L glucose, 15g/L diammonium hydrogen phosphate, 15g/L potassium dihydrogen phosphate, 3g/L ammonium sulfate, 0.3g/L magnesium sulfate heptahydrate, 0.05g/L ferrous sulfate heptahydrate, 160 mu g/L vitamin B and the pH value is continuously adjusted to 6.8 by ammonia water;
fermentation culture conditions: the culture conditions of 0 to 9 hours are: the temperature is 31 ℃, the tank pressure is 0.16-0.20 Mpa, the air volume is 0.3v/vm, and the rotating speed is 150 r/min; the culture conditions of 9 to 46 hours are as follows: the temperature is 30 ℃, the air is isolated, and the anaerobic reaction is carried out, and the rotating speed is 100 r/min.
The yield of the obtained fermentation liquor is 94.52g/L through HPLC detection, and the gluconic acid conversion rate is 48.12%.
Claims (10)
1. An L-valine-producing Brevibacterium flavum (Brevibacterium flavum), characterized in that: the Brevibacterium flavum is preserved in China center for type culture Collection in 2019, 07 and 01, with the preservation number of CCTCC M2019496 and the preservation address of eight-way Lopa nationality mountain in Wuchang district, Wuhan City, Hubei province.
2. The Brevibacterium flavum producing L-valine according to claim 1, characterized in that: the strain is a Brevibacterium flavum subjected to anaerobic acclimation, and the acclimation process comprises the following steps:
(1) inoculating and culturing L-valine-producing brevibacterium flavum as a starting strain to obtain a seed solution, carrying out anaerobic fermentation on the seed solution in a fermentation culture medium with the glucose concentration of A, stopping fermentation to obtain a fermentation broth after the glucose concentration in the fermentation culture medium is reduced to the concentration of B, carrying out anaerobic fermentation on the fermentation broth serving as the seed solution for the second time, wherein the culture medium and the fermentation conditions are the same as those of the previous time, stopping fermentation to obtain the fermentation broth after the glucose concentration in the culture medium is reduced to the concentration of B in the second time of fermentation, repeating the fermentation culture process by using the fermentation broth obtained in the second time as the seed solution, recording the acid production rate in each fermentation culture process, and the concentration of B is 40-60% of the concentration of A;
(2) when the acid production rate and the sugar consumption rate of the Nth fermentation are obviously higher than those of the previous fermentation, the glucose concentration of the culture medium is increased during the (N + 1) th fermentation, and other fermentation conditions are the same as those of the previous fermentation; continuously monitoring the acid production rate in the fermentation process, and repeating the process of increasing the glucose concentration of the culture medium, wherein the glucose concentration increase value is 8-10% of the concentration A each time;
(3) and when the glucose concentration of the fermentation medium is increased to 30-50 times of the concentration A, completing anaerobic acclimation to obtain the anaerobically acclimated brevibacterium flavum.
3. The Brevibacterium flavum producing L-valine according to claim 2, characterized in that: the glucose concentration A is 2-10 g/L.
4. The Brevibacterium flavum producing L-valine according to claim 3, wherein: the culture medium further comprises: 1-20 g/L diammonium hydrogen phosphate, 1-20 g/L potassium dihydrogen phosphate, 1-5 g/L ammonium sulfate, 0.2-2 g/L magnesium sulfate heptahydrate, 0.01-0.05 g/L ferrous sulfate heptahydrate, 1-500 mu g/L biotin and vitamin B11-500 mu g/L, and continuously adjusting the pH value to 6.5-7.8 by using ammonia water;
fermentation conditions are as follows: the temperature is 25-35 ℃, and the rotating speed is 50-200 r/min.
5. A process for producing L-valine by using Brevibacterium flavum according to any one of claims 1 to 4, which comprises: and (2) performing activation culture and seed liquid culture on the anaerobically domesticated brevibacterium flavum strain, and inoculating the obtained seed liquid into a fermentation culture medium for fermentation culture, wherein the fermentation culture process comprises aerobic fermentation and then anaerobic fermentation.
6. The process for producing L-valine according to claim 5, wherein: the fermentation conditions of the aerobic fermentation are as follows: the temperature is 25-35 ℃, the rotating speed is 50-200 r/min, the pressure is maintained by introducing sterile air into the tank to be 0.12-0.2 Mpa, the air volume is 0.1-1 v/vm, and the fermentation time is 8-12 h.
7. The process for producing L-valine according to claim 5, wherein: the fermentation conditions of the anaerobic fermentation are as follows: the temperature is 25-35 ℃, the rotating speed is 50-200 r/min, the fermentation tank is sealed to isolate air, and the fermentation time is 28-38 h.
8. The process for producing L-valine according to claim 4, wherein: the fermentation medium is as follows: the glucose concentration A is 100-200 g/L.
9. The process for producing L-valine according to claim 5, wherein: the activation culture is cultured in an activation slant culture medium to a logarithmic phase; the activated slant culture medium comprises: 6.0-15.0 g/L of peptone, 3.0-7.0 g/L of sodium chloride, 3.0-10.0 g/L of yeast extract or yeast powder, 3.0-10.0 g/L of beef extract, 15-20.0 g/L of agar and adjusting the pH value to 7.0-7.2.
10. The process for producing L-valine according to claim 5, wherein: the seed liquid culture is to culture 3-5 bacterial colonies in a seed culture medium after activating and culturing the bacterial strains to obtain a seed liquid; the seed culture medium is as follows: 10-30 g/L of glucose, 5-20 g/L of diammonium hydrogen phosphate, 5-20 g/L of potassium dihydrogen phosphate, 0.2-0.9 g/L of magnesium sulfate heptahydrate, 0.01-0.05 g/L of ferrous sulfate heptahydrate, 150-500 mu g/L of vitamin B, and continuously adjusting the pH value to 6.5-7.8 by using ammonia water.
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