CN116622520A - Strain for high-yield L-malic acid and application thereof - Google Patents
Strain for high-yield L-malic acid and application thereof Download PDFInfo
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- CN116622520A CN116622520A CN202310506071.8A CN202310506071A CN116622520A CN 116622520 A CN116622520 A CN 116622520A CN 202310506071 A CN202310506071 A CN 202310506071A CN 116622520 A CN116622520 A CN 116622520A
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- malic acid
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- aspergillus niger
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- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 title claims abstract description 120
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 title claims abstract description 75
- 235000011090 malic acid Nutrition 0.000 title claims abstract description 69
- 229940116298 l- malic acid Drugs 0.000 title claims abstract description 58
- 238000000855 fermentation Methods 0.000 claims abstract description 54
- 230000004151 fermentation Effects 0.000 claims abstract description 54
- 241000228245 Aspergillus niger Species 0.000 claims abstract description 26
- 238000004519 manufacturing process Methods 0.000 claims abstract description 20
- 238000000034 method Methods 0.000 claims description 21
- 239000002609 medium Substances 0.000 claims description 19
- 239000001963 growth medium Substances 0.000 claims description 17
- 239000000725 suspension Substances 0.000 claims description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 238000011218 seed culture Methods 0.000 claims description 12
- 238000012258 culturing Methods 0.000 claims description 11
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 8
- 239000008103 glucose Substances 0.000 claims description 8
- 238000004321 preservation Methods 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 239000012137 tryptone Substances 0.000 claims description 6
- 238000009629 microbiological culture Methods 0.000 claims description 3
- 238000012262 fermentative production Methods 0.000 claims description 2
- 238000002703 mutagenesis Methods 0.000 abstract description 22
- 231100000350 mutagenesis Toxicity 0.000 abstract description 22
- YPHMISFOHDHNIV-FSZOTQKASA-N cycloheximide Chemical compound C1[C@@H](C)C[C@H](C)C(=O)[C@@H]1[C@H](O)CC1CC(=O)NC(=O)C1 YPHMISFOHDHNIV-FSZOTQKASA-N 0.000 abstract description 18
- 238000012216 screening Methods 0.000 abstract description 16
- 235000013305 food Nutrition 0.000 abstract description 10
- 230000000813 microbial effect Effects 0.000 abstract description 4
- 239000002253 acid Substances 0.000 description 14
- 239000006228 supernatant Substances 0.000 description 10
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 9
- 241000233866 Fungi Species 0.000 description 8
- 238000004128 high performance liquid chromatography Methods 0.000 description 7
- 238000011081 inoculation Methods 0.000 description 7
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 239000002244 precipitate Substances 0.000 description 6
- BJEPYKJPYRNKOW-UWTATZPHSA-N (R)-malic acid Chemical compound OC(=O)[C@H](O)CC(O)=O BJEPYKJPYRNKOW-UWTATZPHSA-N 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 239000011324 bead Substances 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 239000002054 inoculum Substances 0.000 description 4
- 229940099690 malic acid Drugs 0.000 description 4
- 239000001630 malic acid Substances 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 235000010633 broth Nutrition 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
- 150000007524 organic acids Chemical class 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- -1 compound amino acid Chemical class 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 235000006408 oxalic acid Nutrition 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- STGNLGBPLOVYMA-MAZDBSFSSA-N (E)-but-2-enedioic acid Chemical compound OC(=O)\C=C\C(O)=O.OC(=O)\C=C\C(O)=O STGNLGBPLOVYMA-MAZDBSFSSA-N 0.000 description 1
- STGNLGBPLOVYMA-TZKOHIRVSA-N (z)-but-2-enedioic acid Chemical compound OC(=O)\C=C/C(O)=O.OC(=O)\C=C/C(O)=O STGNLGBPLOVYMA-TZKOHIRVSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 208000037157 Azotemia Diseases 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 241001391944 Commicarpus scandens Species 0.000 description 1
- 241000186145 Corynebacterium ammoniagenes Species 0.000 description 1
- 241000319304 [Brevibacterium] flavum Species 0.000 description 1
- CKUAXEQHGKSLHN-UHFFFAOYSA-N [C].[N] Chemical compound [C].[N] CKUAXEQHGKSLHN-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000002053 acidogenic effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004820 blood count Methods 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 108010079058 casein hydrolysate Proteins 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000001530 fumaric acid Substances 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 238000012803 optimization experiment Methods 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- UQDJGEHQDNVPGU-UHFFFAOYSA-N serine phosphoethanolamine Chemical compound [NH3+]CCOP([O-])(=O)OCC([NH3+])C([O-])=O UQDJGEHQDNVPGU-UHFFFAOYSA-N 0.000 description 1
- 238000004513 sizing Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
- 208000009852 uremia Diseases 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
- C12N1/14—Fungi; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
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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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/44—Polycarboxylic acids
- C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
- C12R2001/685—Aspergillus niger
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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Abstract
The application relates to the technical field of microbial fermentation engineering, in particular to a strain for high-yield L-malic acid and application thereof. The strain is an aspergillus niger strain, is obtained by ultraviolet mutagenesis and high-concentration cycloheximide iterative mutagenesis screening, has short fermentation period and high L-malic acid yield, can meet the strict production strain requirements in the food field, and provides an excellent strain for L-malic acid production. Provides a new way for the production of the L-malic acid, greatly shortens the fermentation period and improves the yield of the L-malic acid.
Description
Technical Field
The application relates to the technical field of microbial fermentation engineering, in particular to a strain for high-yield L-malic acid and application thereof.
Background
Malic acid, also known as 2-hydroxysuccinic acid, is mainly used in the industries of food, medicine, chemical industry and the like. Malic acid is classified into three structures of L-malic acid (L-form), D-malic acid (D-form) and racemic malic acid (DL-form). Most of the malic acids currently sold in the market are DL-malic acid and L-malic acid. Compared with DL-type malic acid, L-malic acid has the advantage of easy absorption, is a main trend of the development of the L-malic acid industry, and is a technical attack trend for the next years. The raw materials used for synthesizing DL-malic acid or L-malic acid by a chemical synthesis method and an enzyme conversion method are maleic acid (maleic acid) or fumaric acid (fumaric acid) which are derived from petroleum-based chemicals and synthesized by benzene respectively, and the method has a great challenge to the serious energy problem. The fumaric acid enzyme commonly used in the enzyme conversion method is mostly from Brevibacterium ammoniagenes, brevibacterium flavum and the like, and is the main method for producing the L-malic acid at present. However, the method has the defects of high substrate cost, low production efficiency, complex downstream separation and purification process and the like, and limits the wide application of the method in L-malic acid production. Compared with the two, the microbial fermentation method utilizes the advantages that the microbes can efficiently utilize various carbon sources of renewable biomass, greatly reduces the production cost, is more environment-friendly, and is a field greatly supported by China.
In the food industry, L-malic acid is mainly used as food acidulant, and compared with citric acid, the L-malic acid has the characteristics of soft taste, large acidity, long residence time and the like, does not damage teeth of the oral cavity and accumulate fat, and becomes a low-calorie safe food acidulant recognized by the international food industry. In the pharmaceutical industry, L-malic acid is used for treating various diseases such as liver diseases, anemia, uremia and the like. And because L-malic acid is favorable for the absorption of amino acid metabolically, the L-malic acid is often matched into compound amino acid injection. The range of application of L-malic acid, a natural product produced by fermentation, in the fields of food and medicine is expanding, and in recent years, the application of L-malic acid in the food industry has gradually replaced citric acid. With the growing demand for health foods, L-malic acid has a great market potential in the future.
Aspergillus niger has been an important cell factory for over 100 years with inexpensive carbon sources for fermentative production of organic acids. Furthermore, if the microbial fermentation is used in the food field, it is necessary that the strain for producing L-malic acid is a non-genetically engineered strain. Therefore, the Aspergillus niger for producing the L-malic acid by fermentation through an iterative mutagenesis method has important value and significance.
Disclosure of Invention
Aiming at the problems in the prior art, the application provides a strain for high-yield L-malic acid and application thereof. The strain is an aspergillus niger strain, is obtained by ultraviolet mutagenesis and high-concentration cycloheximide iterative mutagenesis screening, has short fermentation period and high L-malic acid yield, can meet the strict production strain requirements in the food field, and provides an excellent strain for L-malic acid production. Provides a new way for the production of the L-malic acid, greatly shortens the fermentation period and improves the yield of the L-malic acid.
In a first aspect, the application provides a strain with high yield of L-malic acid, which is obtained by ultraviolet mutagenesis and high-concentration ketone iterative mutagenesis screening of Aspergillus niger (Aspergillus niger), and is preserved in China general microbiological culture collection center (CGMCC) with a preservation number of 40550 in 3-month 31 of 2023.
The aspergillus niger strain is obtained through ultraviolet mutagenesis and high-concentration cycloheximide mutagenesis, and the specific mutagenesis process comprises the following steps: taking an aspergillus niger strain CGMCC NO.10142 as an original strain, carrying out ultraviolet mutagenesis on spore suspension, and primarily screening to obtain a positive mutant; the spore suspension of the positive mutant is respectively subjected to mutagenesis culture for 24 hours in culture mediums containing cycloheximide with different concentrations, and is respectively diluted and coated on a primary screening culture medium plate to carry out the screening of the positive mutant, so that the Aspergillus niger strain is finally obtained.
In a second aspect, the application provides application of the strain for producing L-malic acid in fermentation production.
In a third aspect, the present application provides a method for producing L-malic acid by fermentation of the strain for producing L-malic acid in high yield, comprising:
slant culturing Aspergillus niger strain to obtain fresh spore and sterile ddH 2 O is washed, filtered, collected and dispersed to obtain spore suspension, and inoculated into seed culture medium to make initial spore quantity reach 1X 10 6 －1×10 7 Culturing at 35-37deg.C and 180-220rpm for 22-24 hr; and (3) supplementing a fermentation culture medium, continuously culturing for 44-144h, and stopping culturing when the L-malic acid generation rate reaches the highest or begins to gradually decrease.
Preferably, the volume of the additional fermentation medium is 2-3 times that of the seed medium.
Preferably, the spore suspension is inoculated to achieve an initial spore count of 1X 10 in the seed medium 7 And each mL.
Preferably, the seed medium C: n=45-55, most preferably C: n=45.
More preferably, the seed culture medium comprises the following components: glucose 80g/L, (NH) 4 ) 2 SO 4 2.5g/L, tryptone 2g/L, KH 2 PO 4 0.65g/L，MgSO 4 ·7H 2 O 0.4g/L，FeSO 4 ·7H 2 O 0.6g/L，NaCl 0.05g/L，CaCl 2 0.13g/L, and the pH was adjusted to 4.0.
More preferably, the fermentation medium comprises the following components: glucose 160g/L, (NH) 4 ) 2 SO 4 2.5g/L, tryptone 2g/L, KH 2 PO 4 0.65g/L，MgSO 4 ·7H 2 O 0.4g/L，FeSO 4 ·7H 2 O 0.6g/L，NaCl 0.05g/L，CaCl 2 0.13g/L，8％ CaCO 3 。
The application has the following beneficial effects.
According to the application, an Aspergillus niger strain with high L-malic acid yield is screened by ultraviolet mutagenesis and high-concentration cycloheximide iterative mutagenesis methods. After fermentation conditions are optimized, fermentation is carried out for about 144 hours through test, fermentation supernatant fluid and sediment are centrifugally separated, and 10mM H is respectively used for fermentation 2 SO 4 Dilution by 20-fold HPLCAnd separating, wherein the acid mainly generated by the supernatant of the fermentation liquor is L-malic acid, the yield can reach 72.9g/L or even higher, and the purity can reach 93.4% or higher. Therefore, the strain and the method for producing the L-malic acid by fermentation of the strain are very suitable for industrial application, provide a better fermentation production path for the L-malic acid and promote the development of industry.
Preservation information
Preservation time: 2023, 3 and 31;
preservation unit name: china general microbiological culture Collection center (China Committee for culture Collection);
preservation number: CGMCC No.40550;
deposit unit address: the institute of microorganisms of national academy of sciences of China, national institute of sciences, no.1, no. 3, north Chen West Lu, the Korean region of Beijing;
classification naming: aspergillus niger (Aspergillus niger).
Drawings
FIG. 1 is a graph showing the comparison of the acid-producing transparent circles of the Aspergillus niger strains of the present application and the starting strain plates of example 1;
FIG. 2 is a graph showing comparison of a flat-plate acidogenic HPLC analysis of the Aspergillus niger strain of the present application with its starting strain in example 1;
FIG. 3 is a view showing the morphology of the fungus under a microscope during fermentation at different rotational speeds in a rotational speed optimization experiment in example 2;
FIG. 4 is a graph showing the comparison of seed inoculum size and acid yield over 72h of fermentation with different carbon nitrogen ratios in example 2;
FIG. 5 is a graph of HPLC analysis of 96h and 120h fermentation broths and DL-malic acid standard in example 3;
FIG. 6 is a diagram of HPLC analysis of fermentation supernatant and precipitate at various fermentation times in example 3.
Detailed Description
The above-described aspects of the present application will be described in further detail by way of the following embodiments, but it should not be construed that the scope of the above-described subject matter of the present application is limited to the following examples. All techniques realized based on the above description of the present application are within the scope of the present application, and the following examples are accomplished by conventional techniques unless otherwise specified. The experimental methods used in the following preparation examples and examples are conventional methods unless otherwise specified; materials, reagents and the like used in the following preparation examples and examples are commercially available unless otherwise specified.
The "seed inoculum size" described in the examples below refers to the initial number of spores after inoculation into the medium.
The seed culture medium formula is as follows: glucose 80g/L, (NH) 4 ) 2 SO 4 2.5g/L, tryptone 2g/L, KH 2 PO 4 0.65g/L，MgSO 4 ·7H 2 O 0.4g/L，FeSO 4 ·7H 2 O 0.6g/L，NaCl 0.05g/L，CaCl 2 0.13g/L, and the pH was adjusted to 4.0.
The following fermentation medium formula is: glucose 160g/L, (NH) 4 ) 2 SO 4 2.5g/L, tryptone 2g/L, KH 2 PO 4 0.65g/L，MgSO 4 ·7H 2 O 0.4g/L，FeSO 4 ·7H 2 O 0.6g/L，NaCl 0.05g/L，CaCl 2 0.13g/L，8％CaCO 3 。
Example 1
Screening high-yield L-malic acid industrial Aspergillus niger strains by ultraviolet mutagenesis and high-concentration cycloheximide iterative mutagenesis:
(1) Method for primary screening of ultraviolet mutagenesis and positive mutant
The Aspergillus niger strain CGMCC NO.10142 is taken as an original strain, fresh spores grow on an inclined plane for 6 days, and the spores are sterilized with ddH 2 O is washed and filtered by Miracloth, and is scattered by small glass beads to obtain spore suspension, and the spore suspension is respectively incubated for 0.5, 1, 2, 3, 4 and 5 hours at 30 ℃ and 180r/min for water absorption expansion, counted by a blood cell counting plate and diluted to the concentration of 10 7 And each mL. Taking 100 mu L of spore suspension after incubation, carrying out ultraviolet irradiation in a mutagenesis cuvette, carrying out irradiation for 1-15min at intervals of 1min, respectively diluting and coating on a primary screening culture medium plate, and carrying out ultraviolet mutagenesis lethality analysis and screening of positive mutants.
Primary screening of the culture medium: 50 XSP+N solution 20mL/L, glucose 10g/L,1M MgSO 4 2mL/L, culture1mL of basic trace elements, 1g/L of casein hydrolysate, 5g/L of yeast extract and 20g/L of agar powder.
50×asp+n solution: KH (KH) 2 PO 4 74.8g，NaNO 3 297.5g of KCl 26.1g is dissolved in an appropriate amount of distilled water, the pH is adjusted to 5.5 with KOH, and the volume is set to 1000mL.
Medium trace elements: h 3 BO 3 1.1g，MnCl 2 ·4H 2 O 0.5g，CoCl 2 ·4H 2 O 0.17g，FeSO 4 ·H 2 O 0.5g，CuSO 4 ·5H 2 O 0.16g，ZnSO 4 ·H 2 O 2.1g，Na 2 MoO 4 ·2H 2 O0.15 g, EDTA5.1g, to a volume of 1L.
(2) Preliminary screening method for high-concentration cycloheximide mutagenesis and positive mutant
The positive mutant screened by ultraviolet mutagenesis grows fresh spores for 6 days on an inclined plane, and the spores are sterile ddH 2 O is washed and is filtered and collected by Miracloth, and is scattered by small glass beads to obtain spore suspension which is diluted to the concentration of 10 8 Performing mutagenesis culture on 1mL of spore suspension in a culture medium containing 2-10 mu g/mL of cycloheximide for 24 hours, respectively diluting and coating the culture medium on a preliminary screening culture medium plate, screening positive mutants, performing passage on the screened positive mutants (namely, the aspergillus niger strain), dibbling the spore suspension on a preliminary screening plate added with 6 mu g/mL of cycloheximide, performing acid-producing transparent ring analysis (shown in figure 1), taking a starting strain as a control strain, forming no transparent ring on the control strain, forming obvious transparent rings on the obtained positive mutant strain, and taking 1cm of acid-producing transparent ring formed on the plate when the strain grows to the 4 th day and the 8 th day on the plate 2 Dissolved in 10mM H 2 SO 4 In (c) for HPLC acidogenesis analysis (as shown in FIG. 2), the results showed that the positive mutant strain produced a large amount of L-malic acid in the transparent circle at day 8.
Example 2
The fermentation culture conditions of the high-yield L-malic acid Aspergillus niger strain are optimized:
fresh spores of the positive mutant strain obtained by screening in example 1 were grown obliquely for 6 days, and were sterilized with ddH 2 O cleaning andcollected by Miracloth filtration, broken up by small glass beads to obtain a spore suspension, which was inoculated into seed medium.
Other condition settings:
(1) And (3) optimizing the rotating speed: setting 5 groups of experiments, setting the rotation speed of each group of seed culture process to 300rpm, 280rpm, 250rpm, 220rpm, 200rpm and 180rpm respectively, and the seed inoculation amount is 1 multiplied by 10 7 The culture temperature is 37 ℃ and the culture of the seed culture medium is 30mL, and the culture time is 24h. As shown in FIG. 3 (observation under a 40-fold microscope), the rotation speed higher than 250rpm can lead to the abnormal formation of the fungus balls and the easy breakage of hyphae, and the fungus balls can be formed normally at the rotation speed of 180-220rpm, and the shape and the size of the fungus balls can reach the optimal state at the rotation speed of 220rpm.
(2) And (3) inoculation amount optimization: setting 3 groups of experiments, wherein seed inoculum size of each group of seed culture medium is respectively 1 multiplied by 10 8 、1×10 7 、1×10 6 Three groups of fermentation media with different concentrations per mL are cultured for 24 hours at the culture temperature of 37 ℃ and the seed culture medium of 30mL at the rotation speed of 200r/min, 60mL of fermentation media are added, and the culture is continued for 72 hours, so that the acid production condition is analyzed. The acid production result of 72h is shown in FIG. 4A, and the seed inoculation amount is 1×10 6 -1×10 7 The seed inoculation amount is 1X 10, and the optimal seed inoculation amount is in a proper range of one/mL 7 The acid yield is up to 18.3g/L per mL.
(3) C, N optimizing: set up 4 experiments, carbon source in each group of seed media: the nitrogen sources (namely C: N) are respectively 60, 55, 50 and 45, and the specific operations are as follows: the main nitrogen source in the seed medium is (NH) 4 ) 2 SO 4 The content is 2.5g/L, and the glucose concentration is adjusted to 79.5g/L, 72.9g/L, 66.3g/L and 59.6g/L respectively, and other conditions are that: the spore inoculation amount is 1 multiplied by 10 7 30mL of seed culture medium, the rotation speed is 200r/min, the culture temperature is 37 ℃, the culture is carried out for 24 hours, 60mL of fermentation culture medium is added, the culture is continued for 72 hours, and the acid production condition is analyzed. The result of 72h acid production is shown in fig. 4B, preferably C: n=45-55, most preferably C: n=45, and the acid yield is up to 22.6g/L.
Finally, the optimal seed culture conditions are determined: seed inoculum size 1X 10 7 And (3) per mL, C: N=45, and the rotating speed is 220rpm.
The Aspergillus niger cannot form the change from forming the fungus ball to forming the normal fungus ball in the fermentation process, the fungus ball is irregularly formed before optimization, the edge hypha is overlong, the regular fungus ball is formed after optimization, and the edge hypha is relatively short and small and branches are more; under the condition of high rotating speed of 300rpm, the edge hyphae are easy to break, after the rotating speed is reduced, the hyphae can be well protected, but the generation of malic acid can be influenced after the rotating speed is reduced.
Example 3
High-yield L-malic acid Aspergillus niger strain is fermented to produce L-malic acid:
based on optimized seed culture conditions, the Aspergillus niger strain of the application grows fresh spores for 6 days on an inclined plane, and is sterile in ddH 2 O is washed and filtered and collected by Miracloth, and is scattered by small glass beads to obtain spore suspension, and the spore suspension is respectively inoculated into 5 groups of 30ml seed culture media to lead the spore number to reach 10 7 Culturing at 37deg.C and 220rpm for 24 hr, adding 60ml fermentation medium, continuously culturing, stopping culturing at 44 hr, 72 hr, 96 hr, 120 hr and 144 hr for acid production analysis, re-sizing the fermentation liquid to 90ml, centrifuging to separate fermentation supernatant and precipitate, and subjecting to 10mM H 2 SO 4 HPLC analysis was performed at 20-fold dilution as shown in fig. 6.
HPLC analysis of organic acid components of fermentation broth:
the analysis method comprises the following steps: centrifuging the fermentation broth to separate fermentation supernatant and precipitate, and subjecting the supernatant and precipitate to 10mM H respectively 2 SO 4 Diluting for 20 times, filtering with membrane with diameter of 0.22 μm, detecting, separating with Aminex organic acid analysis column (300 mm×7.8mm), and adjusting flow rate to 0.6mL/min with 5mM dilute sulfuric acid as mobile phase, wherein organic acid can be separated and accurately quantified at 210nm of ultraviolet detection wavelength. The DL-malic acid standard substances with different concentrations of 2mg/L, 1mg/L, 0.8mg/L, 0.6mg/L, 0.4mg/L, 0.2mg/L, 0.1mg/L and the like are selected for standard curve drawing; and (3) carrying out peak diagram overlapping comparison (shown in figure 5) on fermentation liquor obtained by fermenting for 96 hours and 144 hours and a standard DL-malic acid HPLC analysis result, wherein the L-malic acid in the supernatant of the fermentation liquor is determined to be the most main product, and a small amount of D-malic acid is obtained, and the specific data are as follows:
in the whole fermentation process, the main acid generated in the fermentation supernatants of 44h, 72h, 96h, 120h and 144h is L-malic acid (the detection content is 16.9g/L, 30.4g/L, 39.9g/L, 66.3g/L and 72.9g/L respectively), the main byproducts in the fermentation supernatants of 44h and 72h are oxalic acid (the detection content is 1.63g/L and 1.35g/L respectively) and D-malic acid (the detection content is 1.59g/L and 2.26g/L respectively), the main byproducts of 96h, 120h and 144h are D-malic acid (the detection content is 3.40g/L, 6.14g/L and 5.13g/L respectively), and the purity of the L-malic acid is about 84% -93.4%.
The primary acids produced in 44h, 72h, 96h, 120h and 144h fermentation precipitates were L-malic acid (detected at levels of 6.7g/L, 11.2g/L, 10.3g/L, 17.7g/L and 15.4g/L, respectively), followed by citric acid (detected at levels of 1.69g/L, 4.30g/L, 4.96g/L, 10.05g/L and 8.49g/L, respectively), and small amounts of D-malic acid and oxalic acid at concentrations less than 1g/L, with the L-malic acid being more pure than the supernatant, and approximately 56.42% -69.5% in the precipitates.
Claims (9)
1. A strain for high-yield L-malic acid is characterized in that the strain is Aspergillus niger (Aspergillus niger) which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 40550 in the 3 rd month 31 th year 2023.
2. Use of the strain of claim 1 for the fermentative production of L-malic acid.
3. The fermentation method of the L-malic acid is characterized by comprising the following specific steps:
slant culturing Aspergillus niger strain to obtain fresh spore and sterile ddH 2 O is washed, filtered, collected and dispersed to obtain spore suspension, and inoculated into seed culture medium to make initial spore quantity reach 1X 10 6 －1×10 7 Culturing at 35-37deg.C and 180-220rpm for 22-24 hr, adding fermentation medium, continuously culturing for 44-144 hr, and stopping culturing when L-malic acid production rate reaches maximum or begins to gradually decrease, wherein the Aspergillus niger strain is the strain of claim 1。
4. The method of fermentation culture of L-malic acid according to claim 3, wherein the volume of the additional fermentation medium is 2 to 3 times that of the seed medium.
5. The method for fermentation culture of L-malic acid according to claim 3, wherein the number of initial spores in the seed medium is up to 1X 10 7 And each mL.
6. The method for fermentation culture of L-malic acid according to claim 3, wherein the seed medium C is N=45-55.
7. The method of claim 6, wherein the seed medium C is n=45.
8. The method for fermentation culture of L-malic acid according to claim 3, wherein the seed medium comprises the following components: glucose 80g/L, (NH) 4 ) 2 SO 4 2.5g/L, tryptone 2g/L, KH 2 PO 4 0.65g/L，MgSO 4 ·7H 2 O 0.4g/L，FeSO 4 ·7H 2 O 0.6g/L，NaCl 0.05g/L，CaCl 2 0.13g/L, and the pH was adjusted to 4.0.
9. The method for fermentation culture of L-malic acid according to claim 3, wherein the fermentation medium comprises the following components: glucose 160g/L, (NH) 4 ) 2 SO 4 2.5g/L, tryptone 2g/L, KH 2 PO 4 0.65g/L，MgSO 4 ·7H 2 O 0.4g/L，FeSO 4 ·7H 2 O 0.6g/L，NaCl 0.05g/L，CaCl 2 0.13g/L，8％CaCO 3 。
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2009224749A1 (en) * | 2008-03-13 | 2009-09-17 | Dsm Ip Assets B.V. | Selection of organisms capable of fermenting mixed substrates |
| CN109207373A (en) * | 2018-09-21 | 2019-01-15 | 天津科技大学 | The method that one plant height produces the microbial strains and its fermentation starch saccharic production citric acid of citric acid |
| CN109207383A (en) * | 2018-08-28 | 2019-01-15 | 天津科技大学 | The aspergillus niger strain and its building and application of a kind of high yield L MALIC ACID |
| CN110684673A (en) * | 2019-11-05 | 2020-01-14 | 南京师范大学 | L-malic acid high-yield strain and application thereof |
| CN110734865A (en) * | 2019-12-02 | 2020-01-31 | 天津科技大学 | Aspergillus niger genetic engineering strain for high yield of malic acid under low pH condition and application thereof |
| CN111218408A (en) * | 2020-01-21 | 2020-06-02 | 天津科技大学 | Aspergillus niger strain for efficiently producing malic acid, construction method and application |
| CN114606142A (en) * | 2022-05-12 | 2022-06-10 | 南京昊禾生物科技有限公司 | L-malic acid producing strain with high production efficiency, method and application |
-
2023
- 2023-05-04 CN CN202310506071.8A patent/CN116622520A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2009224749A1 (en) * | 2008-03-13 | 2009-09-17 | Dsm Ip Assets B.V. | Selection of organisms capable of fermenting mixed substrates |
| CN109207383A (en) * | 2018-08-28 | 2019-01-15 | 天津科技大学 | The aspergillus niger strain and its building and application of a kind of high yield L MALIC ACID |
| CN109207373A (en) * | 2018-09-21 | 2019-01-15 | 天津科技大学 | The method that one plant height produces the microbial strains and its fermentation starch saccharic production citric acid of citric acid |
| CN110684673A (en) * | 2019-11-05 | 2020-01-14 | 南京师范大学 | L-malic acid high-yield strain and application thereof |
| CN110734865A (en) * | 2019-12-02 | 2020-01-31 | 天津科技大学 | Aspergillus niger genetic engineering strain for high yield of malic acid under low pH condition and application thereof |
| CN111218408A (en) * | 2020-01-21 | 2020-06-02 | 天津科技大学 | Aspergillus niger strain for efficiently producing malic acid, construction method and application |
| CN114606142A (en) * | 2022-05-12 | 2022-06-10 | 南京昊禾生物科技有限公司 | L-malic acid producing strain with high production efficiency, method and application |
Non-Patent Citations (1)
| Title |
|---|
| 乔长晟;郝华旋;姜少丽;敖爱华;王坤;: "紫外氦氖诱变选育高产聚苹果酸菌株", 现代食品科技, no. 11, 15 November 2011 (2011-11-15) * |
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