CN109652475B - Fermentation method for increasing L-isoleucine yield - Google Patents
Fermentation method for increasing L-isoleucine yield Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
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- C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
Abstract
The invention provides a fermentation method for improving the yield of L-isoleucine, which comprises the following steps: (1) inoculating lactobacillus fermentation Brevibacterium (Brevibacterium lactofermentum) into a seed culture medium for propagation to obtain a seed solution 1; (2) inoculating Lactobacillus rhamnosus (Lactobacillus rhamnosus GG) into a seed culture medium for propagation to obtain a seed solution 2; (3) inoculating the seed liquid 2 into a fermentation culture medium, then continuously inoculating the seed liquid 1, and obtaining fermentation liquid through fermentation culture. The method obviously improves the saccharic acid conversion rate and the L-isoleucine yield by jointly using the lactose fermentation brevibacterium seed liquid and the lactobacillus rhamnosus seed liquid, and improves the saccharic acid conversion rate and the L-isoleucine yield again by further controlling the dosage ratio of the lactose fermentation brevibacterium and the lactobacillus rhamnosus in the fermentation process.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for improving the yield of L-isoleucine.
Background
L-Isoleucine (L-Isoleucine) is chemically named as L-a-amino- β -methylvaleric acid, is one of eight essential amino acids of human body, is one of three branched chain amino acids, is a raw material for synthesizing human hormone and enzymes, has the effects of promoting protein synthesis and inhibiting protein decomposition, plays an important role in human life activities, and is widely used for lactation promotion of dairy cows, preparation of feed additives and production of functional beverages abroad.
The production methods of L-isoleucine include three methods, i.e., extraction method, chemical synthesis method and fermentation method, and because it is difficult to separate L-isoleucine produced by the extraction method and chemical synthesis method from its isomer, industrial production has not been achieved. Currently, only fermentation methods are implemented in industrial production.
The fermentation method is a process of utilizing the metabolism of microorganisms, biosynthesizing and excessively accumulating L-isoleucine, and comprises two types of fermentation methods of adding precursors and direct fermentation methods. Because precursor substances are rare or expensive, the method for producing L-isoleucine by adding precursor fermentation is rarely adopted at present. The direct fermentation method is to accumulate certain amino acid in excess amount by means of the ability of microbe to synthesize the required amino acid. The method has the advantages of low raw material cost, wide sources, high product purity, mild reaction conditions and the like, and has the potential of industrial production.
The existing L-isoleucine producing bacteria mainly comprise corynebacterium glutamicum, brevibacterium flavum, escherichia coli, lactobacillus fermentum, corynebacterium crenatum and corynebacterium viscosus, wherein except the corynebacterium glutamicum and the brevibacterium flavum, the acid production capacity of the rest bacteria is quite low, and only one third to one half of that of the corynebacterium glutamicum and the brevibacterium flavum.
In order to improve the acid production level of corynebacterium glutamicum, brevibacterium flavum, brevibacterium lactofermentum, corynebacterium crenatum, and the like, many scholars have tried to obtain corynebacterium glutamicum, brevibacterium flavum, brevibacterium lactofermentum, corynebacterium mucosae, and the like with improved acid production level by mutating strains and constructing genetically engineered bacteria from the perspective of strains, chinese patent publication No. CN1834228 discloses a brevibacterium flavum mutant strain and application thereof in the production of L-isoleucine by fermentation, a brevibacterium flavum 41 (deposited in the china industrial microbial strain collection management center and deposited under the accession number of cic 10135) of corynebacterium sp is taken as a starting strain, and is directionally bred into an L-isoleucine producing strain TC21 by diethyl sulfate (DES) and ultraviolet mutagenesis treatment, which is genetically marked as methionine deficiency, ethionine, α -aminobutyric acid, S-2-aminoethyl-L-cysteine resistance (Met- + Ethr + ABr + aeccr), so that the L-isoleucine-synthesizing strains have L-amino acid resistance (Met- + α -ABr-amino acid, S-L-cysteine resistance (Met- + 104878051A) and L-isoleucine-synthesizing strains, and the like, thus, a new strain fermentation promoting fermentation method for improving the production of strains and improving the production of lactobacillus by fermentation and improving the metabolic regulation of strains has been found in domestic fermentation and improving the production of strains.
Therefore, a high-efficiency fermentation production method with high L-isoleucine yield and high saccharic acid conversion rate is needed at present.
Disclosure of Invention
The invention provides a fermentation method for improving the yield of L-isoleucine, which does not relate to variation of microorganisms, ensures higher acid yield and sugar-acid conversion rate and has good stability.
Description of the drawings: 1. the inoculation amount is the ratio of the volume of the seed solution transferred to the volume of the culture solution after inoculation; 2. the total inoculum size is the ratio of the sum of the volumes of the two seed solutions transferred to the volume of the culture solution after inoculation.
The invention provides a fermentation method for improving the yield of L-isoleucine, which comprises the following steps:
(1) inoculating lactobacillus fermentation Brevibacterium (Brevibacterium lactofermentum) into a seed culture medium for propagation to obtain a seed solution 1;
(2) inoculating Lactobacillus rhamnosus (Lactobacillus rhamnosus GG) into a seed culture medium for propagation to obtain a seed solution 2;
(3) inoculating the seed liquid 2 into a fermentation culture medium, then continuously inoculating the seed liquid 1, and obtaining fermentation liquid through fermentation culture.
The seed culture medium in the step (1) comprises: 95-98g/L of glucose, 7-10g/L of ammonium sulfate, 95-99g/L of peptone, 4-8g/L of ammonium nitrate, 3-4g/L of monopotassium phosphate and 1-2g/L of magnesium sulfate.
The seed culture medium in the step (2) comprises: 20-50g/L glucose, 2-8g/L yeast extract, 15-25g/L soyapeptone, 1-5g/L potassium dihydrogen phosphate, 0.5 magnesium sulfate-
1.2g/L and 0.4-1.0g/L of sodium chloride.
The culture expanding temperature in the step (1) is 30-32 ℃, the time is 16-18h, and the rotating speed is 210-290 rpm.
The culture expanding temperature in the step (2) is 30-32 ℃, the time is 12-15h, and the rotating speed is 100-130 rpm.
The fermentation medium in the step (3) comprises: 20-25g/L of glucose, 10-38g/L of ammonium sulfate, 3.0-4.8g/L of beef extract, 2-18g/L of monopotassium phosphate and 2-8g/L of magnesium sulfate.
The temperature of the fermentation culture in the step (3) is 28-35 ℃, the time is 65-78h, and the rotation speed is 120-135 rpm.
The pH value of the seed culture medium in the step (1) is 6.2-6.7, and the pH value of the seed culture medium in the step (2) is 7.0-7.2.
The inoculation amount in the step (1) is 0.1g/L calculated by lactose fermentation brevibacterium strain powder, and the inoculation amount in the step (2) is 0.05g/L calculated by rhamnose lactobacillus strain powder.
The volume ratio of the seed liquid 1 to the seed liquid 2 inoculated in the fermentation medium in the step (3) is 5-15: 1.
Preferably, the volume ratio of the seed liquid 1 to the seed liquid 2 inoculated in the fermentation medium in the step (3) is 5-10: 1.
The total inoculation amount in the step (3) is 5-8%.
In the step (3), the seed solution 1 is inoculated 10-40min after the seed solution 2 is inoculated.
Compared with the prior art, the invention has the beneficial effects that:
(1) the method obviously improves the saccharic acid conversion rate and the L-isoleucine yield by jointly using the lactose fermentation brevibacterium seed liquid and the lactobacillus rhamnosus seed liquid, and improves the saccharic acid conversion rate and the L-isoleucine yield again by further controlling the dosage ratio of the lactose fermentation brevibacterium and the lactobacillus rhamnosus in the fermentation process.
(2) According to the invention, the inoculation sequence of the lactose fermentation bacillus brevis seed liquid and the rhamnose lactobacillus seed liquid in the fermentation culture medium is controlled, so that the saccharic acid conversion rate is obviously improved, and the effective utilization rate of raw materials is improved.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The examples do not show specific techniques or conditions, and are carried out by means of a conventional technique in the art.
EXAMPLE 1 fermentation method for increasing L-isoleucine production
(1) Inoculating lactobacillus fermentation Brevibacterium (Brevibacterium lactofermentum) into a seed culture medium for propagation to obtain a seed solution 1, wherein the seed culture medium comprises: 95g/L glucose, 10g/L ammonium sulfate, 95g/L peptone, 8g/L ammonium nitrate, 3g/L potassium dihydrogen phosphate and 2g/L magnesium sulfate, wherein the culture expanding temperature is 30 ℃, the time is 18h, the rotating speed is 210rpm, the pH of a seed culture medium is 6.7, and the inoculation amount is 0.1g/L calculated by the Brevibacterium lactofermentum powder;
(2) inoculating Lactobacillus rhamnosus (Lactobacillus rhamnosus GG) into a seed culture medium for propagation to obtain a seed liquid 2, wherein the seed culture medium comprises: 50g/L of glucose, 2g/L of yeast extract, 25g/L of soybean peptone, 1g/L of potassium dihydrogen phosphate, 1.2g/L of magnesium sulfate and 0.4g/L of sodium chloride, wherein the culture expanding temperature is 32 ℃, the time is 12h, the rotating speed is 130rpm, the pH value of a seed culture medium is 7.2, and the inoculation amount is 0.05g/L calculated by lactobacillus rhamnosus powder;
(3) inoculating the seed liquid 2 into a fermentation culture medium, inoculating the seed liquid 1 after 10min, and performing fermentation culture to obtain a fermentation liquid, wherein the fermentation culture medium comprises: 25g/L of glucose, 10g/L of ammonium sulfate, 4.8g/L of beef extract, 2g/L of monopotassium phosphate and 8g/L of magnesium sulfate, wherein the temperature of fermentation culture is 28 ℃, the time is 78 hours, the rotating speed is 120rpm, the volume ratio of the seed solution 1 to the seed solution 2 inoculated in the fermentation culture medium is 15:1, and the total inoculation amount is 5%.
Example 2 fermentation method for increasing L-isoleucine production
(1) Inoculating lactobacillus fermentation Brevibacterium (Brevibacterium lactofermentum) into a seed culture medium for propagation to obtain a seed solution 1, wherein the seed culture medium comprises: 98g/L glucose, 7g/L ammonium sulfate, 99g/L peptone, 4g/L ammonium nitrate, 4g/L potassium dihydrogen phosphate and 1g/L magnesium sulfate, wherein the culture expanding temperature is 32 ℃, the time is 16h, the rotating speed is 290rpm, the pH of a seed culture medium is 6.2, and the inoculation amount is 0.1g/L calculated by the Brevibacterium lactofermentum powder;
(2) inoculating Lactobacillus rhamnosus (Lactobacillus rhamnosus GG) into a seed culture medium for propagation to obtain a seed liquid 2, wherein the seed culture medium comprises: 20g/L of glucose, 8g/L of yeast extract, 15g/L of soybean peptone, 5g/L of potassium dihydrogen phosphate, 0.5g/L of magnesium sulfate and 1.0g/L of sodium chloride, wherein the culture expanding temperature is 30 ℃, the time is 15h, the rotating speed is 100rpm, the pH value of a seed culture medium is 7.0, and the inoculation amount is 0.05g/L calculated by lactobacillus rhamnosus powder;
(3) inoculating the seed liquid 2 into a fermentation culture medium, inoculating the seed liquid 1 after 40min, and performing fermentation culture to obtain a fermentation liquid, wherein the fermentation culture medium comprises: 20g/L of glucose, 38g/L of ammonium sulfate, 3.0g/L of beef extract, 18g/L of monopotassium phosphate and 2g/L of magnesium sulfate, wherein the fermentation culture temperature is 35 ℃, the time is 65 hours, the rotating speed is 135rpm, the volume ratio of the seed solution 1 to the seed solution 2 inoculated in the fermentation culture medium is 5:1, and the total inoculation amount is 8%.
EXAMPLE 3 fermentation method for increasing L-isoleucine production
(1) Inoculating lactobacillus fermentation Brevibacterium (Brevibacterium lactofermentum) into a seed culture medium for propagation to obtain a seed solution 1, wherein the seed culture medium comprises: 96g/L glucose, 9g/L ammonium sulfate, 97g/L peptone, 6g/L ammonium nitrate, 3.5g/L potassium dihydrogen phosphate and 1.2g/L magnesium sulfate, wherein the culture expanding temperature is 31 ℃, the time is 17h, the rotating speed is 250rpm, the pH of a seed culture medium is 6.3, and the inoculation amount is 0.1g/L based on the lactobacillus fermentation brevibacterium strain powder;
(2) inoculating Lactobacillus rhamnosus (Lactobacillus rhamnosus GG) into a seed culture medium for propagation to obtain a seed liquid 2, wherein the seed culture medium comprises: 30g/L of glucose, 5g/L of yeast extract, 20g/L of soybean peptone, 3g/L of potassium dihydrogen phosphate, 0.8g/L of magnesium sulfate and 0.6g/L of sodium chloride, wherein the culture expanding temperature is 31 ℃, the time is 14h, the rotating speed is 120rpm, the pH value of a seed culture medium is 7.1, and the inoculation amount is 0.05g/L calculated by lactobacillus rhamnosus powder;
(3) inoculating the seed liquid 2 into a fermentation culture medium, inoculating the seed liquid 1 after 20min, and performing fermentation culture to obtain a fermentation liquid, wherein the fermentation culture medium comprises: 23g/L of glucose, 20g/L of ammonium sulfate, 4.0g/L of beef extract, 15g/L of monopotassium phosphate and 6g/L of magnesium sulfate, wherein the temperature of fermentation culture is 30 ℃, the time is 72h, the rotating speed is 130rpm, the volume ratio of the seed solution 1 to the seed solution 2 inoculated in the fermentation culture medium is 8:1, and the total inoculation amount is 6%.
Example 4 fermentation method for increasing L-isoleucine production
The only difference from example 3 is that the volume ratio of seed solution 1 and seed solution 2 inoculated in the fermentation medium in step (3) was 10: 1.
EXAMPLE 5 fermentation method for increasing L-isoleucine production
The only difference from example 3 is that the volume ratio of seed solution 1 and seed solution 2 inoculated in the fermentation medium in step (3) was 15: 1.
Comparative example 1 fermentation method for producing L-isoleucine
The only difference from example 3 is that the addition of seed liquid 2 is omitted in step (3).
Comparative example 2 fermentation method for producing L-isoleucine
The only difference from example 3 is the exchange of the order of inoculation of seed liquor 1 and seed liquor 2 in step (3).
Comparative example 3A method for increasing the yield of L-isoleucine
According to the method of example 1 in Chinese patent publication CN108893502A, the A formula in the compound nitrogen source formula in Table 1 is adopted for producing L-isoleucine.
The L-isoleucine yield detection method (paper chromatography) comprises sampling ① sample with 1 μ L sample by use of microsyringe, and performing paper chromatography with n-butanol and glacial acetic acid as developing agentWater 5:2:1, ② unfolding for more than or equal to 3 hours, drying the chromatographic filter paper after the unfolding, ③ ninhydrin color development, namely soaking the dried chromatographic filter paper in a ceramic tray containing 2% ninhydrin color development reagent, drying the paper in the air, then drying the paper in an oven at 105 ℃ for 5 minutes to form blue-purple spots, cutting the color development spots according to areas with consistent sizes, eluting the spots, ④ eluting, namely putting the cut spots in a test tube with a plug, and eluting the test tube with an eluent [ 75% (volume percentage concentration) ethanol: 0.2% (mass percentage concentration) CuSO4·5H2O＝39:1]Eluting with 5ml of the above solutions, measuring the absorbance at ⑤ nm, and finding out the corresponding content on the standard curve according to the absorbance;
⑥ drawing standard curve, selecting corresponding 5 concentration gradient standard samples according to the actual acid production rate of the fermentation liquid, spotting, developing, eluting the same sample liquid, measuring the absorbance at 520nm, and drawing the standard curve with the standard sample as abscissa and the absorbance as ordinate.
TABLE 1
| Experiment number | L-isoleucine yield/(g/L) | Sugar acid conversion/% |
| Example 1 | 21 | 23 |
| Example 2 | 22 | 25 |
| Example 3 | 25.5 | 26 |
| Example 4 | 25.3 | 24 |
| Example 5 | 24.9 | 23 |
| Comparative example 1 | 18.4 | 17.9 |
| Comparative example 2 | 20.4 | 18.5 |
| Comparative example 3 | 18.4 | 17.8 |
The experimental data in Table 1 show that the fermentation method of the invention can be used for producing L-isoleucine, the yield of the L-isoleucine is up to 25.5g/L, and the saccharic acid conversion rate is up to 26%. Comparing the experimental data of examples 3-4 in table 1, it can be found that the inoculation amount of the seed liquid 1 and the seed liquid 2 in the fermentation culture can affect the yield of the L-isoleucine and the sugar-acid conversion rate, and when the ratio of the inoculation amount of the seed liquid 1 and the inoculation amount of the seed liquid 2 in the fermentation culture medium is 10:1 and 8:1, higher yield of the L-isoleucine and higher sugar-acid conversion rate can be obtained. Further comparing the experimental data of example 3 and comparative examples 1-2 in table 1, it can be found that the inoculation of the seed liquid of lactobacillus rhamnosus in the fermentation culture during the production of L-isoleucine now contributes to the improvement of the yield and sugar-acid conversion rate of L-isoleucine.
Claims (10)
1. A fermentation method for improving the yield of L-isoleucine is characterized in that: the method comprises the following steps: (1) inoculating lactobacillus fermentation brevibacterium into a seed culture medium for propagation culture to obtain a seed solution 1; (2) inoculating lactobacillus rhamnosus into a seed culture medium for propagation to obtain a seed solution 2; (3) inoculating the seed liquid 2 into a fermentation culture medium, then continuously inoculating the seed liquid 1, and obtaining fermentation liquid through fermentation culture.
2. The fermentation process of claim 1, wherein: the seed culture medium in the step (1) comprises: 95-98g/L of glucose, 7-10g/L of ammonium sulfate, 95-99g/L of peptone, 4-8g/L of ammonium nitrate, 3-4g/L of monopotassium phosphate and 1-2g/L of magnesium sulfate.
3. The fermentation process of claim 1, wherein: the seed culture medium in the step (2) comprises: 20-50g/L of glucose, 2-8g/L of yeast extract, 15-25g/L of soybean peptone, 1-5g/L of monopotassium phosphate, 0.5-1.2g/L of magnesium sulfate and 0.4-1.0g/L of sodium chloride.
4. The fermentation process of claim 1, wherein: the culture expanding temperature in the step (1) is 30-32 ℃, the time is 16-18h, and the rotating speed is 210-290 rpm.
5. The fermentation process of claim 1, wherein: the culture expanding temperature in the step (2) is 30-32 ℃, the time is 12-15h, and the rotating speed is 100-130 rpm.
6. The fermentation process of claim 1, wherein: the fermentation medium in the step (3) comprises: 20-25g/L of glucose, 10-38g/L of ammonium sulfate, 3.0-4.8g/L of beef extract, 2-18g/L of monopotassium phosphate and 2-8g/L of magnesium sulfate.
7. The fermentation process of claim 1, wherein: the temperature of the fermentation culture in the step (3) is 28-35 ℃, the time is 65-78h, and the rotation speed is 120-135 rpm.
8. The fermentation process of claim 1, wherein: the volume ratio of the seed liquid 1 to the seed liquid 2 inoculated in the fermentation medium in the step (3) is 5-15: 1.
9. The fermentation process of claim 1, wherein: the total inoculation amount in the step (3) is 5-8%.
10. The fermentation process of claim 1, wherein: in the step (3), the seed solution 1 is inoculated 10-40min after the seed solution 2 is inoculated.
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| Combined dissolved oxygen and pH control strategy to improve;Zhijian Peng等;《Bioprocess Biosyst Eng》;20090516;第33卷(第3期);339-345 * |
| L-异亮氨酸互生菌发酵条件的研究;浦军平 等;《大连轻工业学院学报》;19980930;第17卷(第3期);68-70 * |
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