A strain with high L-malic acid production and its application

Abstract

The present invention relates to the technical field of microbial fermentation engineering, and in particular to a strain with high yield of L-malic acid and its application. The strain is an Aspergillus niger, which is obtained by ultraviolet mutagenesis and high-concentration cycloheximide iterative mutagenesis screening, has a short fermentation cycle, 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. It provides a new way for L-malic acid production, greatly shortens the fermentation cycle, and increases the yield of L-malic acid.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation engineering, and in particular to a strain with high L-malic acid production and application thereof. Background Art

[0002] Malic acid, also known as 2-hydroxysuccinic acid, is mainly used in food, medicine, chemical and other industries. Malic acid is divided into three structures: L-malic acid (L-type), D-malic acid (D-type) and racemic malic acid (DL-type). Most of the malic acid sold in the current market is DL-malic acid and L-malic acid. Compared with DL-malic acid, L-malic acid has the advantage of easy absorption and is the main trend of the development of the L-malic acid industry. L-malic acid will be the trend of technical breakthroughs in the next few years. The raw materials used for the synthesis of DL-malic acid or L-malic acid by chemical synthesis and enzyme conversion are maleic acid (maleic acid) or fumaric acid (fumaric acid) synthesized from petroleum-based chemicals benzene, which is a great challenge to the increasingly severe energy problem. The fumarase commonly used in the enzyme conversion method mostly comes from Brevibacterium ammoniagenes and Brevibacterium flavum, etc., and is currently the main method for the production of L-malic acid. However, this method has the disadvantages of high substrate cost, low production efficiency, and complex downstream separation and purification process, which limits its wide application in L-malic acid production. Compared with the other two, the microbial fermentation method takes advantage of the ability of microorganisms to efficiently utilize multiple carbon sources of renewable biomass, greatly reducing production costs. In addition, the microbial fermentation method is more environmentally friendly and is a field strongly supported by the country.

[0003] In the food industry, L-malic acid is mainly used as a food acidulant. Compared with citric acid, it has the characteristics of soft taste, high acidity, long retention time, etc., and does not damage oral teeth and does not accumulate fat. It has become a low-calorie food acidulant recognized by the international food industry as safe. In the pharmaceutical industry, L-malic acid is used to treat liver disease, anemia, uremia and other diseases. And because L-malic acid is beneficial to the absorption of amino acids in metabolism, it is often added to compound amino acid injections. The application scope of L-malic acid, a natural product produced by fermentation, is expanding in the fields of food and medicine. In recent years, the application of L-malic acid in the food industry has gradually replaced citric acid. With the growing demand for healthy food, L-malic acid has huge market potential in the future.

[0004] Aspergillus niger, as an important cell factory, has a history of more than 100 years of fermentation production of organic acids using cheap carbon sources. Furthermore, if the production of L-malic acid by microbial fermentation is applied to the food field, it is necessary that the production strain is a non-genetically engineered strain. Therefore, it is of great value and significance to develop Aspergillus niger for fermentation production of L-malic acid by iterative mutagenesis. Summary of the invention

[0005] The present invention aims at the problems of the prior art and provides a strain with high yield of L-malic acid and its application. The strain is an Aspergillus niger, which is obtained by ultraviolet mutagenesis and high-concentration cycloheximide iterative mutagenesis screening, has a short fermentation cycle, 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. It provides a new way for L-malic acid production, greatly shortens the fermentation cycle, and increases the L-malic acid yield.

[0006] In the first aspect, the present invention provides a strain with high L-malic acid production, which is Aspergillus niger, which is obtained by ultraviolet mutagenesis and high-concentration ketone iterative mutagenesis screening, and was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on March 31, 2023, with the deposit number CGMCC NO.40550.

[0007] The Aspergillus niger strain is obtained by ultraviolet mutagenesis and high-concentration cycloheximide mutagenesis. The specific mutagenesis process comprises: taking the Aspergillus niger strain CGMCC NO.10142 as the starting strain, subjecting its spore suspension to ultraviolet mutagenesis, and preliminarily screening to obtain positive mutants; subjecting the screened positive mutants to spore suspensions for mutagenesis culture in a culture medium containing different concentrations of cycloheximide for 24 hours, respectively, diluting and coating them on a primary screening culture medium plate, and screening positive mutants, and finally obtaining the Aspergillus niger strain of the present invention.

[0008] In a second aspect, the present invention provides the use of the above-mentioned high-yield L-malic acid strain in the fermentation production of L-malic acid.

[0009] In a third aspect, the present invention provides a method for producing L-malic acid by fermentation of the high-yield L-malic acid strain, comprising:

[0010] Fresh spores were obtained by slant culture of Aspergillus niger strains, washed with sterile ddH2O, collected and dispersed by filtration to obtain a spore suspension, which was inoculated into the seed culture medium to make the initial spore number reach 1×10 6 -1×10 7 / mL, and then culture at 35-37°C and 180-220rpm for 22-24h; add fermentation medium and continue to culture for 44-144h, and stop the culture when the L-malic acid production rate reaches the highest or begins to gradually decrease.

[0011] Preferably, the volume of the supplementary fermentation medium is 2-3 times that of the seed medium.

[0012] Preferably, the spore suspension is inoculated so that the initial spore count in the seed culture medium reaches 1×10 7 Pieces / mL.

[0013] Preferably, the seed culture medium has C:N=45-55, most preferably C:N=45.

[0014] More preferably, the seed culture medium consists of: glucose 80 g / L, (NH4)2SO4 2.5 g / L, trypsin 2 g / L, KH2PO4 0.65 g / L, MgSO4·7H2O 0.4 g / L, FeSO4·7H2O 0.6 g / L, NaCl 0.05 g / L, CaCl2 0.13 g / L, and the pH is adjusted to 4.0.

[0015] More preferably, the fermentation medium consists of: 160 g / L glucose, 2.5 g / L (NH4)2SO4, 2 g / L trypsin, 0.65 g / L KH2PO4, 0.4 g / L MgSO4·7H2O, 0.6 g / L FeSO4·7H2O, 0.05 g / L NaCl, 0.13 g / L CaCl2, and 8% CaCO3.

[0016] This application has the following beneficial effects.

[0017] The present application screened a strain of Aspergillus niger with high yield of L-malic acid by ultraviolet mutagenesis and high concentration of actinomycin iterative mutagenesis. After optimizing the fermentation conditions, after about 144h of experimental fermentation, the fermentation supernatant and precipitate were centrifuged and separated, and HPLC analysis was performed by diluting 20 times with 10mM H2SO4. The main acid generated by the fermentation supernatant was L-malic acid, and the yield could reach 72.9g / L or even higher, and the purity could reach 93.4% or higher. It can be seen that the strain of the present invention and the method for producing L-malic acid by fermentation are very suitable for industrial application, providing a better fermentation production route for L-malic acid and promoting the development of the industry.

[0018] Collection Information

[0019] Preservation time: March 31, 2023;

[0020] Name of depository: General Microbiology Center of China Microbiological Culture Collection Administration;

[0021] Deposit number: CGMCC NO.40550;

[0022] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0023] Classification and nomenclature: Aspergillus niger. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1This is a comparison of the acid-producing transparent circles on the plates of the Aspergillus niger strain of the present invention and its starting strain in Example 1;

[0025] Figure 2 This is a comparison chart of the HPLC analysis of the acid production of the Aspergillus niger strain of the present invention and its starting strain in Example 1;

[0026] Figure 3 This is a microscopic observation of the bacterial ball morphology during the fermentation process at different speeds in the speed optimization experiment in Example 2;

[0027] Figure 4 This is a comparison chart of acid production after 72 hours of fermentation at different seed inoculation amounts and different carbon-nitrogen ratios in Example 2;

[0028] Figure 5 HPLC analysis of the fermentation broth at 96 h and 120 h and DL-malic acid standard in Example 3;

[0029] Figure 6 HPLC analysis of acid production of the fermentation supernatant and precipitate at different fermentation times in Example 3. DETAILED DESCRIPTION

[0030] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies implemented based on the above contents of the present invention belong to the scope of the present invention. Unless otherwise specified, the following examples are all completed using conventional existing technologies. The experimental methods used in the following preparation examples and examples are conventional methods unless otherwise specified; the materials, reagents, etc. used in the following preparation examples and examples, unless otherwise specified, can all be obtained from commercial channels.

[0031] The "seed inoculum amount" described in the following examples refers to the initial spore number after inoculation into the culture medium.

[0032] The seed culture medium formula involved below is: glucose 80g / L, (NH4)2SO4 2.5g / L, trypsin 2g / L, KH2PO4 0.65g / L, MgSO4·7H2O 0.4g / L, FeSO4·7H2O 0.6g / L, NaCl 0.05g / L, CaCl2 0.13g / L, pH adjusted to 4.0.

[0033] The fermentation medium formula involved below is: glucose 160g / L, (NH4)2SO4 2.5g / L, trypsin 2g / L, KH2PO4 0.65g / L, MgSO4·7H2O 0.4g / L, FeSO4·7H2O 0.6g / L, NaCl 0.05g / L, CaCl2 0.13g / L, 8% CaCO3.

[0034] Example 1

[0035] Screening of high-yield L-malic acid industrial Aspergillus niger strains by ultraviolet mutagenesis and high-concentration cycloheximide iterative mutagenesis:

[0036] (1) UV mutagenesis and initial screening of positive mutants

[0037] Using Aspergillus niger strain CGMCC NO.10142 as the starting strain, fresh spores grown on a slant for 6 days were washed with sterile ddH2O and collected by filtration through Miracloth, and then dispersed with small glass beads to obtain a spore suspension. The spores were incubated at 30°C and 180 r / min for 0.5, 1, 2, 3, 4, and 5 h, respectively, for water absorption and swelling, and counted using a hemocytometer. The spores were diluted to a concentration of 10 7 Take 100 μL of the incubated spore suspension and place it in a mutagenesis dish for UV irradiation. Irradiate for 1-15 min at 1-min intervals. Dilute and spread on the primary screening culture medium plate to analyze the UV mutagenesis lethality and screen positive mutants.

[0038] Primary screening culture medium: 50×ASP+N solution 20mL / L, glucose 10g / L, 1M MgSO4 2mL / L, culture medium trace elements 1mL, casein hydrolysate 1g / L, yeast extract 5g / L, agar powder 20g / L.

[0039] 50×ASP+N solution: Dissolve 74.8g KH2PO4, 297.5g NaNO3, and 26.1g KCl in appropriate amount of distilled water, adjust the pH to 5.5 with KOH, and make up to 1000mL.

[0040] Trace elements in the culture medium: H3BO3 1.1g, MnCl2·4H2O 0.5g, CoCl2·4H2O 0.17g, FeSO4·H2O0.5g, CuSO4·5H2O 0.16g, ZnSO4·H2O 2.1g, Na2MoO4·2H2O 0.15g, EDTA 5.1g, make up to 1L.

[0041] (2) High-concentration cycloheximide mutagenesis and initial screening of positive mutants

[0042] Fresh spores of the positive mutants screened by UV mutagenesis were grown on a slant for 6 days, washed with sterile ddH2O and collected by filtration through Miracloth, then dispersed with small glass beads to obtain a spore suspension, which was diluted to a concentration of 10 8 1 mL of spore suspension was taken and cultured in a medium containing different concentrations of 2-10 μg / ml cycloheximide for 24 h, and then diluted and coated on a primary screening medium plate to screen positive mutants. The screened positive mutants (i.e., the Aspergillus niger strain of the present invention) were passaged, and the spore suspension was spotted on a primary screening plate containing 6 μg / ml cycloheximide, and acid production transparent zone analysis (such as Figure 1 The starting strain was used as the control strain. The control strain did not form a transparent circle, while the positive mutant strain could form an obvious transparent circle. When the strain grew on the plate for 4 and 8 days, 1 cm of the acid-producing transparent circle formed on the plate was taken. 2 , dissolved in 10mM H2SO4, and used for HPLC acid production analysis (such as Figure 2 The results showed that the positive mutant strain produced a large amount of L-malic acid in the transparent zone on the 8th day.

[0043] Example 2

[0044] Optimization of fermentation culture conditions for high-yield L-malic acid Aspergillus niger strains:

[0045] Fresh spores of the positive mutant strain screened in Example 1 grown on a slant for 6 days were washed with sterile ddH2O, collected by filtration through Miracloth, and dispersed with small glass beads to obtain a spore suspension, which was inoculated into the seed culture medium.

[0046] Other condition settings:

[0047] (1) Speed ​​optimization: Five groups of experiments were set up. The speed of the seed culture process in each group was set at 300 rpm, 280 rpm, 250 rpm, 220 rpm, 200 rpm, and 180 rpm, respectively. The seed inoculation amount was 1 × 10 7 / mL, culture temperature 37℃, seed culture medium 30mL, culture to 24h. Figure 3 (Observed under a 40x microscope) As shown, when the rotation speed is higher than 250rpm, the fungus ball cannot be formed normally and the hyphae are easy to break. When the rotation speed is between 180-220rpm, a normal fungus ball can be formed. When the rotation speed is 220rpm, the fungus ball shape and size reach the optimal state.

[0048] (2) Optimization of inoculation amount: Three groups of experiments were set up, and the seed inoculation amount of each group of seed culture medium was set to 1×10 8 , 1×10 7 , 1×10 6The three groups were divided into three groups with different concentrations, the rotation speed was 200r / min, the culture temperature was 37℃, the seed culture medium was 30mL, and the culture was continued for 24h. Then 60mL of fermentation culture medium was added and the culture was continued for 72h. The acid production was analyzed. Figure 4 As shown in A, the seed inoculation amount is 1×10 6 -1×10 7 The optimum seed inoculation amount is 1×10 7 / mL, and the highest acid production reached 18.3g / L.

[0049] (3) C:N optimization: Four groups of experiments were set up, and the carbon source: nitrogen source (i.e., C:N) ratio in the seed culture medium of each group was 60, 55, 50, and 45, respectively. The specific operation was as follows: the main nitrogen source in the seed culture medium was (NH4)2SO4, with a content of 2.5 g / L, and the corresponding glucose concentrations were adjusted to 79.5 g / L, 72.9 g / L, 66.3 g / L, and 59.6 g / L, respectively. Other conditions: the spore inoculation amount was 1×10 7 / mL, 30mL seed culture medium, 200r / min rotation speed, 37℃ culture temperature, culture for 24h, add 60mL fermentation medium, continue culture for 72h, and analyze the acid production. The acid production results for 72h are as follows Figure 4 As shown in B, the better C:N=45-55, the most suitable C:N=45, and the highest acid production reaches 22.6g / L.

[0050] The optimal seed culture conditions were finally determined: seed inoculation volume 1×10 7 / mL, C:N=45, rotation speed 220rpm.

[0051] The change of Aspergillus niger from being unable to form a ball to forming a normal ball during the fermentation process. Before optimization, the ball was formed irregularly and the edge hyphae were too long. After optimization, a regular ball was formed and the edge hyphae were relatively short and had many branches. Under the condition of a high speed of 300rpm, the edge hyphae were easily destroyed. After reducing the speed, the hyphae could be better protected, but the production of malic acid would be affected after the speed was reduced.

[0052] Example 3

[0053] High-yield L-malic acid Aspergillus niger strain fermentation to produce L-malic acid:

[0054] Based on the optimized seed culture conditions, fresh spores of the Aspergillus niger strain grown on a slant for 6 days were washed with sterile ddH2O and collected by filtration through Miracloth, and then dispersed with small glass beads to obtain a spore suspension, which was inoculated into 5 groups of 30 ml seed culture medium to make the spore count reach 10 7 / ml, 37℃, rotation speed 220rpm for 24h, add 60ml fermentation medium, continue to culture, stop culture at 44h, 72h, 96h, 120h and 144h respectively, one of the groups was analyzed for acid production, the fermentation broth that stopped culture was re-converted to 90ml, centrifuged to separate the fermentation supernatant and precipitate, and diluted 20 times with 10mM H2SO4 for HPLC analysis. Figure 6 shown.

[0055] HPLC analysis of organic acid components in fermentation broth:

[0056] Analytical method: The fermentation broth was centrifuged to separate the fermentation supernatant and precipitate, which were diluted 20 times with 10mM H2SO4, filtered through a membrane with a diameter of 0.22μm, and then tested. The separation was performed using an American Bio-Rad Aminex organic acid analysis column (300mm×7.8mm). The mobile phase was 5mM dilute sulfuric acid, and the flow rate was adjusted to 0.6mL / min. At the ultraviolet detection wavelength of 210nm, the separation and accurate quantification of organic acids can be achieved. Different concentrations of 2mg / L, 1mg / L, 0.8mg / L, 0.6mg / L, 0.4mg / L, 0.2mg / L, 0.1mg / L, and DL-malic acid standards were selected to draw the standard curve; the fermentation broths of 96h and 144h were selected to compare the peak graph overlap with the standard DL-malic acid HPLC analysis results (such as Figure 5 As shown in the figure, it can be determined that L-malic acid is the main product in the fermentation broth supernatant, followed by a small amount of D-malic acid. The specific data are as follows:

[0057] During the entire fermentation process, the main acid produced in the fermentation supernatant of 44h, 72h, 96h, 120h and 144h was L-malic acid (the detected contents were 16.9g / L, 30.4g / L, 39.9g / L, 66.3g / L and 72.9g / L, respectively); the main by-products in the fermentation supernatant of 44h and 72h were oxalic acid (the detected contents were 1.63g / L and 1.35g / L, respectively) and D-malic acid (the detected contents were 1.59g / L and 2.26g / L, respectively); the main by-product in the fermentation supernatant of 96h, 120h and 144h was D-malic acid (the detected contents were 3.40g / L, 6.14g / L and 5.13g / L, respectively); the purity of L-malic acid was about 84% to 93.4%.

[0058] The main acid produced in the fermentation precipitates of 44h, 72h, 96h, 120h and 144h was L-malic acid (the detected contents were 6.7g / L, 11.2g / L, 10.3g / L, 17.7g / L and 15.4g / L, respectively), followed by citric acid (the detected contents were 1.69g / L, 4.30g / L, 4.96g / L, 10.05g / L and 8.49g / L, respectively), as well as a small amount of D-malic acid and oxalic acid with a concentration of less than 1g / L. The purity of L-malic acid in the precipitate was much lower than that in the supernatant, about 56.42% to 69.5%.

Claims

1. A strain with high L-malic acid production, characterized in that: This strain is Aspergillus niger ( Aspergillus niger ), deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on March 31, 2023, with the deposit number CGMCCNO. 40550.

2. Use of the strain according to claim 1 in fermentation production of L-malic acid.

3. An L-malic acid fermentation method, characterized in that: The specific steps include: Fresh spores were obtained by slant culture of Aspergillus niger strains, washed with sterile ddH2O, collected and dispersed by filtration to obtain a spore suspension, which was inoculated into the seed culture medium to make the initial spore number reach 1×10 6 -1×10 7 / mL, then culture at 35-37°C and 180-220 rpm for 22-24 hours, add fermentation medium, continue to culture for 44-144 hours, and stop culturing when the L-malic acid production rate reaches the maximum or begins to gradually decrease, wherein the Aspergillus niger strain is the strain described in claim 1.

4. The L-malic acid fermentation method according to claim 3, characterized in that: The volume of the fermentation medium added is 2-3 times that of the seed medium.

5. The L-malic acid fermentation method according to claim 3, characterized in that: The initial spore count in the seed culture medium reached 1×10 7 Pieces / mL.

6. The L-malic acid fermentation method according to claim 3, characterized in that: Seed culture medium C:N=45-55.

7. The L-malic acid fermentation method according to claim 6, characterized in that: The seed culture medium C:N=45.

8. The L-malic acid fermentation method according to claim 3, characterized in that: The seed culture medium is composed of: glucose 80 g / L, (NH4)2SO4 2.5 g / L, tryptone 2 g / L, KH2PO4 0.65 g / L, MgSO4·7H2O 0.4 g / L, FeSO4·7H2O 0.6 g / L, NaCl 0.05 g / L, CaCl2 0.13 g / L, and the pH is adjusted to 4.

0.

9. The L-malic acid fermentation method according to claim 3, characterized in that: The fermentation medium is composed of: 160 g / L glucose, 2.5 g / L (NH4)2SO4, 2 g / L tryptone, 0.65 g / L KH2PO4, 0.4 g / L MgSO4·7H2O, 0.6 g / L FeSO4·7H2O, 0.05 g / L NaCl, 0.13 g / L CaCl2, and 8% CaCO3.

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