CN114075521A - Method for screening valine production strain with phage resistance and application thereof - Google Patents
Method for screening valine production strain with phage resistance and application thereof 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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- 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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- 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
- C12P13/04—Alpha- or beta- amino acids
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
Abstract
The present invention relates to a method for screening a valine-producing strain having phage-resistant ability, comprising the steps of; s1: separating out a phage solution from the valine producer fermentation liquor infected by the phage; s2: purifying and culturing the phage solution prepared in the step S1 to obtain a single colony; s3: and (4) sequentially inoculating the single colony obtained in the step (S2) and the valine producing bacteria into a fermentation culture medium for fermentation culture, and screening to obtain the valine producing strain with the phage resistance. The method has the advantages of simple operation, high screening efficiency, suitability for industrial production and the like.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for screening a valine production strain with phage resistance and application thereof.
Background
Valine is an essential amino acid and a nutrient substance for organisms, is widely applied to the fields of foods, medicines, cosmetics, feeds and the like, is widely used as a food additive, a feed additive, a nutritional supplement, a flavoring agent, a cosmetic additive, a preparation precursor of medicines (such as antibiotics, herbicides and the like) and the like, and has great market demand.
The valine produced by the microbial fermentation method has the advantages of bio-based raw materials, high efficiency conversion, mild reaction, environmental protection, economy and the like, and is widely applied to the industry. However, the microorganism is easily infected by bacteriophage to affect the fermentation efficiency and the purity of the fermentation product, which leads to low fermentation yield, prolonged fermentation period, reduced product accumulation, increased production of miscellaneous acids, increased difficulty in purification of the fermentation product and recovery of three wastes, and even fermentation failure, resulting in difficult guarantee of fermentation quality, low utilization rate of fermentation resources, high fermentation cost, and the like.
The existing phage pollution resistance measures comprise infection source control, strain rotation, drug inhibitor application and the like, but the technical problems of microbial strain infection, poor fermentation and the like cannot be effectively solved by the measures. Therefore, how to efficiently screen a valine fermentation strain having phage resistance becomes a technical problem which needs to be solved in the field.
The disclosure of Chinese patent applications (CN202010401422.5, CN202010466347.0, CN202010460035.9 and CN202010522549.2) is an essential component of the invention.
Disclosure of Invention
The invention aims to provide a method for screening a valine fermentation strain with phage resistance, which comprises the following steps:
s1: separating out a phage solution from the valine producer fermentation liquor infected by the phage;
s2: performing mixed culture on the valine producing strain and the phage solution prepared in the step S1, and performing separation and purification treatment on the culture solution to obtain a single colony;
s3: and (4) sequentially inoculating the single colony obtained in the step (S2) and the valine producing bacteria into a fermentation culture medium for fermentation culture, and screening to obtain the valine producing strain with the phage resistance.
In a preferred technical scheme of the invention, the valine-producing bacteria are:
wild type brevibacterium flavum;
or, wild-type E.coli;
or, the strain is obtained by taking wild type Brevibacterium flavum or wild type Escherichia coli as starting bacteria and performing natural mutation and/or physicochemical mutagenesis and/or gene recombination.
In the preferred technical scheme of the invention, the valine-producing strain is Brevibacterium flavum (preservation number: CCTCC NO: M2019496), and is submitted to preservation in 2019, 7 and 1, wherein the preservation unit is as follows: china center for type culture Collection, collection address: eight-path Lojia mountain in Wuchang region of Wuhan city, Hubei province.
In a preferred technical scheme of the invention, the valine-producing strain is any one of an Sval031 strain, an Sval049 strain and an Sval065 strain;
wherein the Sval031 strain (preservation number: CGMCC No.19456) is submitted to the preservation 3/6 in 2020, and is classified and named as follows: escherichia coli (Escherichia coli), deposit unit: china general microbiological culture Collection center, preservation Address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North;
wherein the Sval049 strain (preservation number: CGMCC No.19457) is submitted to the preservation 3/6 in 2020, and is classified and named as follows: escherichia coli (Escherichia coli), deposit unit: china general microbiological culture Collection center, preservation Address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North;
wherein the Sval065 strain (preservation number: CGMCC No.19458) is submitted to the preservation 3/6 in 2020, and is classified and named as follows: escherichia coli (Escherichia coli), deposit unit: china general microbiological culture Collection center, preservation Address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
In a preferred embodiment of the present invention, the separation method in step S1 is a double-plate method.
In a preferred embodiment of the present invention, step S1 includes the following steps:
(1-1) taking the valine producer fermentation liquor infected by the phage, centrifuging for 10-30 minutes at 5000-;
(1-2) transferring the supernatant and the fermentation seed liquid into an LB semisolid culture medium according to the inoculation amount of 1-5%, uniformly mixing, pouring the obtained mixed solution onto the LB solid culture medium, and culturing for 20-40 hours at the temperature of 30-40 ℃ to obtain the plaques;
(1-3) washing the LB solid medium plate with sterilized normal saline, collecting the effluent liquid, filtering, and collecting the filtrate, wherein the obtained filtrate is the phage solution.
In a preferred embodiment of the present invention, the supernatant obtained in step (1-1) is filtered before being added to the LB semi-solid medium.
In a preferred embodiment of the present invention, the filtration mode in the step (1-1) and the step (1-3) is membrane filtration, and is preferably either ultrafiltration membrane filtration or microfiltration membrane filtration.
In a preferred technical scheme of the invention, the microfiltration membrane is a water-based microfiltration membrane, and the pore diameter of the microfiltration membrane is 0.22 um.
In a preferred embodiment of the present invention, the purification and culture method in step S2 is any one of a plate drawing method and a dilution coating method.
In a preferred embodiment of the present invention, step S2 includes the following steps:
(2-1) sequentially transferring the fermentation seed solution into an LB liquid culture medium according to the inoculum size of 1-5% and the phage solution according to the inoculum size of 0.1-1%, culturing for 18-24 hours at 30-40 ℃, centrifuging for 10-30 minutes at 5000-10000rpm, and collecting bottom filter residue;
(2-2) transferring the bottom filter residue collected in the step (2-1) and the phage solution prepared in the step (1-3) into an LB liquid culture medium according to the inoculation amount of 0.1-1%, culturing for 12-36 hours at 30-40 ℃, centrifuging for 10-30 minutes at 5000-10000rpm, and collecting the bottom filter residue;
(2-3) transferring the bottom filter residue collected in the step (2-2) and the phage solution prepared in the step (1-3) into an LB liquid culture medium according to the inoculation amount of 0.1-1%, culturing for 12-36 hours at the temperature of 30-40 ℃, and collecting a culture solution;
(2-4) carrying out gradient dilution on the culture solution collected in the step (2-3), coating the obtained diluted bacterial solution on an LB solid culture medium, and culturing for 20-40 hours at the temperature of 30-40 ℃ to obtain a single colony.
In the preferred technical scheme of the invention, the repeated operation times of the step (2-2) are more than or equal to 2, and the preferred repeated operation times are 2-9.
In a preferred embodiment of the present invention, the dilution degree of the gradient dilution in the step (2-4) is selected from 10-3、10-4、10-5、10-6、10-7、10-8Any one or combination thereof.
In a preferred embodiment of the present invention, step S3 includes the following steps:
(3-1) inoculating the single colony prepared in the step (2-4) into an LB liquid culture medium, and culturing for 15-25h at the temperature of 30-40 ℃ to obtain a culture solution with OD being more than or equal to 2, namely a single colony seed solution;
(3-2) inoculating the single colony seed culture solution prepared in the step (3-1) into a fermentation culture medium according to the inoculation amount of 5-10% for culturing for 5-90 hours until the OD value of the fermentation liquid does not decrease, and separating out a strain from the culture solution, namely the valine producing strain with the phage resistance.
In a preferred technical scheme of the invention, the fermentation seed liquid is prepared according to the following steps: inoculating valine producing bacteria into LB liquid culture medium according to the inoculation amount of 5-10 per mill, culturing at 30-40 deg.C and 150-.
In the preferable technical scheme of the invention, the LB solid culture medium comprises 5g/L of yeast powder, 10g/L of peptone, 10g/L of sodium chloride and 20g/L of agar powder.
In the preferable technical scheme of the invention, the LB liquid culture medium comprises 5-10g/L of yeast powder, 10-20g/L of peptone and 10-20g/L of sodium chloride, and preferably comprises 5g/L of yeast powder, 10g/L of peptone and 10g/L of sodium chloride.
In the preferable technical scheme of the invention, the LB semisolid culture medium comprises 5g/L of yeast powder, 10g/L of peptone, 10g/L of sodium chloride and 9g/L of agar powder.
In the preferable technical scheme of the invention, the fermentation medium comprises 10-20g/L potassium dihydrogen phosphate, 1-5g/L yeast powder, 1-10g/L peptone and 10-40g/L glucose, preferably 20g/L potassium dihydrogen phosphate, 2g/L yeast powder, 5g/L peptone and 30g/L glucose.
In a preferred technical scheme of the invention, the fermentation medium is prepared by tap water.
Another object of the present invention is to provide a valine-producing strain having phage-resistant ability, which is prepared by the steps of:
s1: separating out a phage solution from the valine producer fermentation liquor infected by the phage;
s2: performing mixed culture on the valine producing strain and the phage solution prepared in the step S1, and performing separation and purification treatment on the culture solution to obtain a single colony;
s3: and (4) sequentially inoculating the single colony obtained in the step (S2) and the valine producing bacteria into a fermentation culture medium for fermentation culture, and screening to obtain the valine producing strain with the phage resistance.
In a preferred technical scheme of the invention, the valine-producing bacteria are:
wild type brevibacterium flavum;
or, wild-type E.coli;
or, the strain is obtained by taking wild type Brevibacterium flavum or wild type Escherichia coli as starting bacteria and performing natural mutation and/or physicochemical mutagenesis and/or gene recombination.
In the preferred technical scheme of the invention, the valine-producing strain is Brevibacterium flavum (preservation number: CCTCC NO: M2019496), and is submitted to preservation in 2019, 7 and 1, wherein the preservation unit is as follows: china center for type culture Collection, collection address: eight-path Lojia mountain in Wuchang region of Wuhan city, Hubei province.
In a preferred technical scheme of the invention, the valine-producing strain is any one of an Sval031 strain, an Sval049 strain and an Sval065 strain;
wherein the Sval031 strain (preservation number: CGMCC No.19456) is submitted to the preservation 3/6 in 2020, and is classified and named as follows: escherichia coli (Escherichia coli), deposit unit: china general microbiological culture Collection center, preservation Address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North;
wherein the Sval049 strain (preservation number: CGMCC No.19457) is submitted to the preservation 3/6 in 2020, and is classified and named as follows: escherichia coli (Escherichia coli), deposit unit: china general microbiological culture Collection center, preservation Address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North;
wherein the Sval065 strain (preservation number: CGMCC No.19458) is submitted to the preservation 3/6 in 2020, and is classified and named as follows: escherichia coli (Escherichia coli), deposit unit: china general microbiological culture Collection center, preservation Address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
In a preferred embodiment of the present invention, the separation method in step S1 is a double-plate method.
In a preferred embodiment of the present invention, step S1 includes the following steps:
(1-1) taking the valine producer fermentation liquor infected by the phage, centrifuging for 10-30 minutes at 5000-;
(1-2) transferring the supernatant and the fermentation seed liquid into an LB semisolid culture medium according to the inoculation amount of 1-5%, uniformly mixing, pouring the obtained mixed solution onto the LB solid culture medium, and culturing for 20-40 hours at the temperature of 30-40 ℃ to obtain the plaques;
(1-3) washing the LB solid medium plate with sterilized normal saline, collecting the effluent liquid, filtering, and collecting the filtrate, wherein the obtained filtrate is the phage solution.
In a preferred embodiment of the present invention, the supernatant obtained in step (1-1) is filtered before being added to the LB semi-solid medium.
In a preferred technical scheme of the invention, the filtration mode is membrane filtration, and is preferably any one of ultrafiltration membrane filtration and microfiltration membrane filtration.
In a preferred technical scheme of the invention, the microfiltration membrane is a water-based microfiltration membrane, and the pore diameter of the microfiltration membrane is 0.22 um.
In a preferred embodiment of the present invention, the purification and culture method in step S2 is any one of a plate drawing method and a dilution coating method.
In a preferred embodiment of the present invention, step S2 includes the following steps:
(2-1) sequentially transferring the fermentation seed solution into an LB liquid culture medium according to the inoculum size of 1-5% and the phage solution according to the inoculum size of 0.1-1%, culturing for 18-24 hours at 30-40 ℃, centrifuging for 10-30 minutes at 5000-10000rpm, and collecting bottom filter residue;
(2-2) transferring the bottom filter residue collected in the step (2-1) and the phage solution prepared in the step (1-3) into an LB liquid culture medium according to the inoculation amount of 0.1-1%, culturing for 12-36 hours at 30-40 ℃, centrifuging for 10-30 minutes at 5000-10000rpm, and collecting the bottom filter residue;
(2-3) transferring the bottom filter residue collected in the step (2-2) and the phage solution prepared in the step (1-3) into an LB liquid culture medium according to the inoculation amount of 0.1-1%, culturing for 12-36 hours at the temperature of 30-40 ℃, and collecting a culture solution;
(2-4) carrying out gradient dilution on the culture solution collected in the step (2-3), coating the obtained diluted bacterial solution on an LB solid culture medium, and culturing for 20-40 hours at the temperature of 30-40 ℃ to obtain a single colony.
In the preferred technical scheme of the invention, the repeated operation times of the step (2-2) are more than or equal to 2, and the preferred repeated operation times are 2-9.
In a preferred embodiment of the present invention, the dilution degree of the gradient dilution in the step (2-4) is selected from 10-3、10-4、10-5、10-6、10-7、10-8Any one or combination thereof.
In a preferred embodiment of the present invention, step S3 includes the following steps:
(3-1) inoculating the single colony prepared in the step (2-4) into an LB liquid culture medium, and culturing for 15-25h at the temperature of 30-40 ℃ to obtain a culture solution with OD being more than or equal to 2, namely a single colony seed solution;
(3-2) inoculating the single colony seed culture solution prepared in the step (3-1) into a fermentation culture medium according to the inoculation amount of 5-10% for culturing for 5-90 hours until the OD value of the fermentation liquid does not decrease, and separating out a strain from the culture solution, namely the valine producing strain with the phage resistance.
In a preferred technical scheme of the invention, the fermentation seed liquid is prepared according to the following steps: inoculating valine producing bacteria into LB liquid culture medium according to the inoculation amount of 5-10 per mill, culturing at 30-40 deg.C and 150-.
In the preferable technical scheme of the invention, the LB solid culture medium comprises 5g/L of yeast powder, 10g/L of peptone, 10g/L of sodium chloride and 20g/L of agar powder.
In the preferable technical scheme of the invention, the LB liquid culture medium comprises 5-10g/L of yeast powder, 10-20g/L of peptone and 10-20g/L of sodium chloride, and preferably comprises 5g/L of yeast powder, 10g/L of peptone and 10g/L of sodium chloride.
In the preferable technical scheme of the invention, the LB semisolid culture medium comprises 5g/L of yeast powder, 10g/L of peptone, 10g/L of sodium chloride and 9g/L of agar powder.
In the preferable technical scheme of the invention, the fermentation medium comprises 10-20g/L potassium dihydrogen phosphate, 1-5g/L yeast powder, 1-10g/L peptone and 10-40g/L glucose, preferably 20g/L potassium dihydrogen phosphate, 2g/L yeast powder, 5g/L peptone and 30g/L glucose.
In a preferred technical scheme of the invention, the fermentation medium is prepared by tap water.
Another object of the present invention is to provide a method for efficiently producing valine, comprising the steps of: inoculating the valine producing strain seed culture solution into a fermentation culture medium according to the inoculation amount of 5-10%, and performing fermentation culture at 30-40 deg.C for 12-36h to obtain valine solution.
In the preferable technical scheme of the invention, the preparation of the valine producer strain culture solution comprises the following steps of inoculating the valine producer strain with the bacteriophage resistance, which is obtained by screening in the step (3-2), into an LB liquid culture medium for culturing for 10-60 hours according to the inoculation amount of 5-10 per mill until the OD value of the culture solution is more than or equal to 2, and collecting the culture solution to obtain the valine producer strain culture solution.
In the preferable technical scheme of the invention, the fermentation medium comprises 10-20g/L potassium dihydrogen phosphate, 1-5g/L yeast powder, 1-10g/L peptone and 10-40g/L glucose, preferably 20g/L potassium dihydrogen phosphate, 2g/L yeast powder, 5g/L peptone and 30g/L glucose.
In a preferred technical scheme of the invention, the fermentation medium is prepared by tap water.
In the preferable technical scheme of the invention, the content of the heteropolyacid in the valine solution is less than or equal to 1g/L, and the saccharic acid conversion rate is more than or equal to 50%.
In the preferable technical scheme of the invention, the valine is prepared by separating and purifying after pretreatment of the valine solution.
In a preferred embodiment of the present invention, the pretreatment includes any one or a combination of sterilization, desalting, removing heteropolyacid, and concentration.
In a preferred embodiment of the present invention, the separation is selected from any one of filtration, centrifugation, and membrane treatment, or a combination thereof.
In the preferred technical scheme of the invention, the purification frequency of the valine is more than or equal to 1 time, and preferably 2-4 times.
In a preferred embodiment of the present invention, the drying is selected from any one of vacuum drying, reduced pressure drying, atmospheric drying, spray drying, and boiling drying, or a combination thereof.
In the preferred technical scheme of the invention, the drying temperature is 25-80 ℃, preferably 35-70 ℃, and more preferably 40-60 ℃.
Another object of the present invention is to provide the use of the valine producing strain selected according to the present invention having phage resistance for the fermentative production of valine.
In a preferred embodiment of the present invention, the valine is prepared by the following steps: inoculating the valine producing strain seed culture solution into a fermentation culture medium according to the inoculation amount of 5-10%, and performing fermentation culture at 30-40 deg.C for 12-36h to obtain valine solution.
In the preferable technical scheme of the invention, the preparation of the valine producer strain culture solution comprises the following steps of inoculating the valine producer strain with the bacteriophage resistance, which is obtained by screening in the step (3-2), into an LB liquid culture medium for culturing for 10-60 hours according to the inoculation amount of 5-10 per mill until the OD value of the culture solution is more than or equal to 2, and collecting the culture solution to obtain the valine producer strain culture solution.
In the preferable technical scheme of the invention, the fermentation medium comprises 10-20g/L potassium dihydrogen phosphate, 1-5g/L yeast powder, 1-10g/L peptone and 10-40g/L glucose, preferably 20g/L potassium dihydrogen phosphate, 2g/L yeast powder, 5g/L peptone and 30g/L glucose.
In a preferred technical scheme of the invention, the fermentation medium is prepared by tap water.
In the preferable technical scheme of the invention, the content of the heteropolyacid in the valine solution is less than or equal to 1g/L, and the saccharic acid conversion rate is more than or equal to 50%.
In the preferable technical scheme of the invention, the valine is prepared by separating and purifying after pretreatment of the valine solution.
In a preferred embodiment of the present invention, the pretreatment includes any one or a combination of sterilization, desalting, removing heteropolyacid, and concentration.
In the preferred technical scheme of the invention, the purification frequency of the valine is more than or equal to 1 time, and preferably 2-4 times.
Unless otherwise indicated, when the present invention relates to percentages between liquids, said percentages are volume/volume percentages; the invention relates to the percentage between liquid and solid, said percentage being volume/weight percentage; the invention relates to the percentages between solid and liquid, said percentages being weight/volume percentages; the balance being weight/weight percent.
Unless otherwise indicated, the following detection methods were employed in the present invention:
OD value
Instruments and reagents: ultraviolet-visible spectrophotometer, glass cuvette and pure water
The experimental steps are as follows: starting up the spectrophotometer for 15 minutes in advance before use, preheating, adjusting the wavelength to 600nm, adding pure water into the cuvette, putting the cuvette into the spectrophotometer for zero calibration, taking out the cuvette, pouring the pure water, adding a sample to be detected, putting the sample into the spectrophotometer, and reading the absorbance value to obtain the OD value of the sample.
2. Amount of mixed acid and amount of acid produced
Instruments and reagents: shimadzu LC-16 liquid chromatograph, C18 chromatograph group, derivatization agent is 5% o-phthalaldehyde ethanol water solution, 30% methanol mobile phase, valine standard is sigma analysis standard, and heteropolyacid is other commercially available amino acid with content of 99%.
The experimental steps are as follows: diluting the fermentation liquor by 100 times, filtering, adding a derivatization agent for derivatization for 2 minutes, sampling after derivatization, wherein the wavelength is 234nm, the sampling amount is 10ul, and calculating the concentration of amino acid in the sample according to the peak area of a standard sample.
Amount of heteropolyacid ═ area of heteropolyacid standard peak/area of heteropolyacid peak ═ concentration of heteropolyacid standard.
The amount of acid produced is valine standard peak area/valine peak area valine standard concentration.
3. Residual sugar content
The glucose content of the fermentation product was measured using the SBA-40D biosensor from the institute of biology, Shandong academy of sciences.
4. Conversion rate of sugar and acid
The conversion of sugar acid was calculated as acid yield/(mass of total glucose in the fermentation medium 0.9).
Compared with the prior art, the invention has the following beneficial technical effects:
1. the valine producing strain with stronger resistance to the phage, which is screened by the invention, has the advantages of phage infection resistance, high conversion rate of the saccharic acid, low content of the heteroacid, low content of the residual sugar and the like, remarkably improves the yield and the quality of the valine, reduces the generation of side reactions and byproducts, optimizes the separation and purification of the valine, and remarkably improves the stability of the fermentation process and the controllability of the fermentation process.
2. The screening method has the advantages of simple and convenient operation, high screening efficiency, better cost, suitability for industrial production and the like.
Drawings
FIG. 1 comparison of OD values during fermentation in examples 2 to 4 and comparative examples 1 to 2;
FIG. 2 comparison of OD values after 48h fermentation for examples 2 to 4 and comparative examples 1 to 2;
FIG. 3 comparison of the total heteropolyacid content after 48h fermentation for examples 2-4 and comparative examples 1-2;
FIG. 4 comparison of residual sugar content after 48h fermentation for examples 2-4 and comparative examples 1-2;
FIG. 5 comparison of the yields of examples 2 to 4 and comparative examples 1 to 2 after 48h of fermentation;
FIG. 6 is a schematic process flow diagram of the present invention.
Detailed Description
The present invention will be further described with reference to the following examples.
In the specific embodiment (examples or comparative examples), Sval065 strain (accession number: CGMCC NO.19458) is exemplified.
Composition of LB liquid medium: 5g/L of yeast powder, 10g/L of peptone and 10g/L of sodium chloride, and the balance of water.
Composition of LB solid medium: 5g/L of yeast powder, 10g/L of peptone, 10g/L of sodium chloride, 20g/L of agar powder and the balance of water.
Composition of LB semisolid medium: 5g/L of yeast powder, 10g/L of peptone, 10g/L of sodium chloride, 9g/L of agar powder and the balance of water.
Composition of the fermentation medium: 20g/L potassium dihydrogen phosphate, 2g/L yeast powder, 5g/L peptone and 30g/L glucose, and the balance of water.
The preparation of the fermentation seed liquid comprises the following steps: inoculating Sval065 strain into LB liquid culture medium at 5 ‰ inoculum size, culturing at 37 deg.C and 220rpm for 24 hr until OD value of the culture solution is 2, and collecting the culture solution to obtain fermented seed solution.
Example 1Screening of valine-producing Strain having phage-resistant ability
S1: isolation of bacteriophages
(1-1) taking Sval065 strain fermentation liquor infected by phage in a workshop of Anhui Hua Heng Biotech Co., Ltd, centrifuging at 6500rpm for 10 minutes, filtering the obtained supernatant with 0.22um water system filter membrane, and collecting filtrate;
(1-2) transferring 500uL of filtrate and 4ml of fermentation seed liquid into 200ml of LB semisolid culture medium, then pouring the obtained mixed solution onto the LB solid culture medium, and culturing at 37 ℃ for 24 hours to obtain plaques;
(1-3) flushing the culture medium plate with 0.9% sterilized normal saline, collecting effluent, and filtering with 0.22um water system filter membrane to obtain filtrate as bacteriophage solution.
S2: isolation and purification of phages
(2-1) transferring 8ml of fermentation seed solution and 1ml of phage solution into 200ml of LB liquid culture medium, culturing at 37 ℃ for 18 hours, centrifuging at 10000rpm for 10 minutes, removing supernatant, and collecting bottom filter residue;
(2-2) inoculating 1ml of filter residue and 1ml of phage solution into 200ml of LB liquid culture medium, culturing at 37 ℃ for 24 hours, centrifuging at 10000rpm for 10 minutes, removing supernatant, and collecting bottom filter residue;
(2-3) repeating the operation of step (2-2) 9 times;
(2-4) inoculating 1ml of filter residue and 1ml of phage solution into 200ml of LB liquid culture medium, culturing at 37 ℃ for 24 hours, and collecting the culture solution;
(2-5) the culture solution collected in the step (2-4) was diluted in a gradient manner, and 0.5ml of each diluted bacterial solution was applied to LB solid medium in this order and cultured at 37 ℃ for 24 hours to obtain 40 single colonies.
Wherein, the gradient dilution comprises the following steps:
(a) taking 10 test tubes of 100ml, filling 10ml of 0.9% sterilized normal saline into each test tube, plugging the opening of the test tube tightly by a cotton plug to ensure the sealing, then placing the test tubes into a sterilizing pot to sterilize for 30 minutes at 121 ℃, and cooling for standby application, wherein the test tubes are numbered 1-10;
(b) on aseptic superclean bench, get 1ml supernatant with aseptic rifle head and add into No.1 test tube, after the misce bene, get 1ml to No. 2 test tubes from No.1 test tube, get 1ml to No. 3 test tubes after No. 2 test tube misce benes to this analogizes to No. 10 test tubes.
S3: screening of valine-producing strains having phage-resistant ability:
(3-1) selecting each single colony from the LB solid culture medium obtained in the step (2-5), sequentially transferring the single colony into each LB liquid culture medium, culturing at 37 ℃ for 20 hours, detecting the absorbance of the culture solution, and collecting the culture solution when the OD is 2, namely the single colony seed solution, wherein the serial number of the culture solution is 1-40;
(3-2) sequentially inoculating No. 1-40 single colony seed solution (20ml) and 1ml phage solution into 40 fermentation culture media (200ml), and culturing at 37 ℃ for 24 hours; sampling every 4 hours during the culture process, detecting the absorbance of the culture solution, and recording the change of the OD value; and detecting the product concentration in a liquid phase after the culture is finished, and calculating the acid yield.
The detection method of the invention has the following detection results: in 40 fermentation culture mediums, 26 fermentation culture mediums exist, and after the thalli are cultured for 24 hours, the OD of a culture solution is reduced to be less than or equal to 1; 5 fermentation culture mediums are provided, and the saccharic acid conversion rate is less than or equal to 30 percent after the thalli are cultured for 24 hours; 6 fermentation culture mediums are provided, after the thalli are cultured for 24 hours, the residual sugar content is more than or equal to 30g/L, and the total content of the heteropolyacid is more than or equal to 3 g/L; the method comprises the following steps of providing 3 fermentation culture media, after culturing thalli for 24 hours, enabling OD of a culture solution to be more than or equal to 8, enabling a saccharic acid conversion rate to be more than or equal to 50%, enabling residual sugar content to be less than or equal to 5g/L, enabling the total content of heteropolyacid to be less than or equal to 1g/L, respectively carrying out centrifugal treatment on the culture solutions in the 3 fermentation culture media, collecting filter residues, obtaining valine production strains with bacteriophage resistance, and respectively naming the valine production strains as bacteriophage resistance strains 1, bacteriophage resistance strains 2 and bacteriophage resistance strains 3.
Example 2Production of valine
The preparation of valine comprises the following steps:
1) inoculating the anti-phage strain 1 screened out in the example 1 into an LB liquid culture medium according to the inoculation amount of 5 per mill, culturing for 24 hours under the conditions of 37 ℃ and 220rpm, and collecting a culture solution to prepare a single colony seed culture solution 1;
2) 20ml of the single colony seed culture solution 1 and 1ml of the phage solution prepared in example 1 were inoculated into 200ml of the fermentation medium, respectively, and cultured at 37 ℃ for 52 hours.
Example 3Production of valine
The preparation of valine comprises the following steps:
1) inoculating the anti-phage strain 2 screened out in the example 1 into an LB liquid culture medium according to the inoculation amount of 5 per mill, culturing for 24 hours under the conditions of 37 ℃ and 220rpm, and collecting a culture solution to obtain a single colony seed culture solution 2;
2) 20ml of the single colony seed culture solution 2 and 1ml of the phage solution prepared in example 1 were inoculated into 200ml of the fermentation medium, respectively, and cultured at 37 ℃ for 52 hours.
Example 4Production of valine
The preparation of valine comprises the following steps:
1) inoculating the anti-phage strain 3 screened out in the example 1 into an LB liquid culture medium according to the inoculation amount of 5 per mill, culturing for 24 hours under the conditions of 37 ℃ and 220rpm, and collecting a culture solution to obtain a single colony seed culture solution 3;
2) 20ml of the single colony seed culture solution 3 and 1ml of the phage solution prepared in example 1 were inoculated into 200ml of the fermentation medium, respectively, and cultured at 37 ℃ for 52 hours.
Comparative example 1Production of valine
20ml of the fermentation seed solution was inoculated into 200ml of the fermentation medium, and the mixture was cultured at 37 ℃ for 52 hours, followed by collecting the culture solution.
Comparative example 2Production of valine
20ml of the fermentation seed solution and 1ml of the phage solution prepared in example 1 were inoculated into 200ml of the fermentation medium, respectively, and cultured at 37 ℃ for 68 hours, and the culture solution was collected.
In the culture processes of examples 2-4 and comparative examples 1-2, samples were taken every 4 hours and the OD values of the culture liquids and their changes were examined, and the results are shown in FIG. 1.
After 48 hours of culture, the OD value, the total content of the heteropolyacid, the amount of residual sugar and the conversion rate of the saccharic acid in the products of examples 2 to 4 and comparative examples 1 to 2 were measured, and the results are shown in FIGS. 2 to 5.
The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined in the appended claims.
Claims (10)
1. A method for screening a valine fermentation strain having phage resistance, comprising the steps of:
s1: separating out a phage solution from the valine producer fermentation liquor infected by the phage;
s2: performing mixed culture on the valine producing strain and the phage solution prepared in the step S1, and performing separation and purification treatment on the culture solution to obtain a single colony;
s3: and (4) sequentially inoculating the single colony obtained in the step (S2) and the valine producing bacteria into a fermentation culture medium for fermentation culture, and screening to obtain the valine producing strain with the phage resistance.
2. The method for screening a valine fermenting strain having phage resistance according to claim 1, wherein said valine producing bacterium is:
wild type brevibacterium flavum;
or, wild-type E.coli;
or, the strain is obtained by taking wild type Brevibacterium flavum or wild type Escherichia coli as starting bacteria and performing natural mutation and/or physicochemical mutagenesis and/or gene recombination.
3. The method according to claim 1 or 2, wherein the valine-producing bacterium is any one selected from the group consisting of Brevibacterium flavum (accession number: CCTCC NO: M2019496), Sval031 strain (accession number: CGMCC No.19456), Sval049 strain (accession number: CGMCC No.19457), and Sval065 (accession number: CGMCC No. 19458).
4. The method according to any one of claims 1 to 3, wherein the step S1 includes the steps of:
(1-1) taking the valine producer fermentation liquor infected by the phage, centrifuging for 10-30 minutes at 5000-;
(1-2) transferring the supernatant and the fermentation seed liquid into an LB semisolid culture medium according to the inoculation amount of 1-5%, uniformly mixing, pouring the obtained mixed solution onto the LB solid culture medium, and culturing for 20-40 hours at the temperature of 30-40 ℃ to obtain the plaques;
(1-3) washing the LB solid medium plate with sterilized normal saline, collecting the effluent liquid, filtering, and collecting the filtrate, wherein the obtained filtrate is the phage solution.
5. The method according to any one of claims 1 to 4, wherein the step S2 includes the steps of:
(2-1) sequentially transferring the fermentation seed solution into an LB liquid culture medium according to the inoculum size of 1-5% and the phage solution according to the inoculum size of 0.1-1%, culturing for 18-24 hours at 30-40 ℃, centrifuging for 10-30 minutes at 5000-10000rpm, and collecting bottom filter residue;
(2-2) transferring the bottom filter residue collected in the step (2-1) and the phage solution prepared in the step (1-3) into an LB liquid culture medium according to the inoculation amount of 0.1-1%, culturing for 12-36 hours at 30-40 ℃, centrifuging for 10-30 minutes at 5000-10000rpm, and collecting the bottom filter residue;
(2-3) transferring the bottom filter residue collected in the step (2-2) and the phage solution prepared in the step (1-3) into an LB liquid culture medium according to the inoculation amount of 0.1-1%, culturing for 12-36 hours at the temperature of 30-40 ℃, and collecting a culture solution;
(2-4) carrying out gradient dilution on the culture solution collected in the step (2-3), coating the obtained diluted bacterial solution on an LB solid culture medium, and culturing for 20-40 hours at the temperature of 30-40 ℃ to obtain a single colony.
6. The method according to any one of claims 1 to 5, wherein the step S3 includes the steps of:
(3-1) inoculating the single colony prepared in the step (2-4) into an LB liquid culture medium, and culturing for 15-25h at the temperature of 30-40 ℃ to obtain a culture solution with OD being more than or equal to 2, namely a single colony seed solution;
(3-2) inoculating the single colony seed culture solution prepared in the step (3-1) into a fermentation culture medium according to the inoculation amount of 5-10% for culturing for 5-90 hours until the OD value of the fermentation liquid does not decrease, and separating out a strain from the culture solution, namely the valine producing strain with the phage resistance.
7. The method of any one of claims 1-6, wherein the fermented seed liquid is prepared by the steps of: inoculating valine producing bacteria into LB liquid culture medium according to the inoculation amount of 5-10 per mill, culturing at 30-40 deg.C and 150-.
8. A valine-producing strain having phage-resistant ability selected by the method according to any one of claims 1 to 7.
9. A method for efficiently preparing valine, comprising the following steps: inoculating a valine producing strain seed culture solution into a fermentation culture medium according to the inoculation amount of 5-10%, performing fermentation culture at 30-40 ℃ for 12-36h to obtain a valine solution, preferably inoculating a valine producing strain with phage resistance capability prepared by the screening method of any one of claims 1-7 into an LB liquid culture medium according to the inoculation amount of 5-10% for 10-60 h until the OD value of the culture solution is more than or equal to 2, and collecting the culture solution to obtain the valine producing strain seed culture solution.
10. Use of a valine-producing strain having phage-resistant ability produced by the screening method according to any one of claims 1 to 7 for producing valine.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102010847A (en) * | 2010-03-15 | 2011-04-13 | 江南大学 | Antiphagin L-phenylalanine producing strain as well as breeding method and application thereof |
| CN109370936A (en) * | 2018-10-25 | 2019-02-22 | 中国人民解放军军事科学院军事医学研究院 | Escherichia coli BL21 (the DE3)-PR of one plant of wide spectrum antiphagin and its application |
| CN110643547A (en) * | 2019-11-07 | 2020-01-03 | 巴彦淖尔华恒生物科技有限公司 | Brevibacterium flavum for producing L-valine and method for producing L-valine by using same |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102010847A (en) * | 2010-03-15 | 2011-04-13 | 江南大学 | Antiphagin L-phenylalanine producing strain as well as breeding method and application thereof |
| CN109370936A (en) * | 2018-10-25 | 2019-02-22 | 中国人民解放军军事科学院军事医学研究院 | Escherichia coli BL21 (the DE3)-PR of one plant of wide spectrum antiphagin and its application |
| CN110643547A (en) * | 2019-11-07 | 2020-01-03 | 巴彦淖尔华恒生物科技有限公司 | Brevibacterium flavum for producing L-valine and method for producing L-valine by using same |
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