Fermentation method for producing pyruvic acid by applying fermentation precursor substance to fermentation method and fermentation method of pyruvic acid
By adding dichloroacetic acid or metformin as the fermentation precursor substance to the fermentation method, the problem of low pyruvate yield in the fermentation broth is solved, and the effect of efficient production of pyruvate is achieved.
Patent Information
- Application Number
- CN202411712945.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-27
- Publication Date
- 2025-02-07
AI Technical Summary
In the process of pyruvate production by fermentation, how to increase the yield of pyruvate in the fermentation broth and reduce the impurity content has become the core pursuit.
The pyruvate yield is increased by adding dichloroacetic acid or/and metformin as fermentation precursor substances to the fermentation medium.
The yield of pyruvate in the fermentation broth was significantly improved. Compared with the condition that no precursor substance was added, the content of pyruvate in the fermentation broth was significantly increased.
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Abstract
Description
Technical Field
[0001] The present invention relates to pyruvic acid production, and in particular to a fermentation method for producing pyruvic acid by fermentation using a fermentation precursor substance. Background Art
[0002] Pyruvate, also known as α-oxopropionic acid or 2-oxopropionic acid, is an organic compound with the chemical formula C 3 H 4 O 3 , the structural formula is CH 3 COCOOH. Pyruvic acid, also known as α-oxopropionic acid or 2-oxopropionic acid, is an organic compound with the chemical formula C 3 H 4 O 3 , the structural formula is CH3COCOOH.
[0003] Pyruvate is an intermediate product produced by glucose metabolism in the liver and muscles. During glycolysis, glucose undergoes a series of enzymatic reactions and is ultimately converted into pyruvate.
[0004] The production process of pyruvate includes chemical synthesis, enzyme conversion and biological fermentation. The chemical synthesis process is simple and easy, and is the main method for producing pyruvate in China. However, this method has serious environmental pollution, large equipment loss, and the pyruvate product is a racemate, with low substrate conversion rate and high production cost; the enzyme conversion method has the advantages of low raw material cost, high product purity and mild reaction conditions, but it has great difficulties in industrialization; the biological fermentation method produces less waste and pollution in the process of producing pyruvate. Compared with the chemical synthesis method, the fermentation method does not require high temperature, high pressure and toxic solvents, reducing the risk of environmental pollution. At the same time, the emission of greenhouse gases such as carbon dioxide produced during the fermentation process is also relatively low, which helps to achieve green production.
[0005] The production of pyruvate by fermentation is an important development direction for the future production of pyruvate because of its advantages of environmental protection, low cost and high quality. In the process of producing pyruvate by fermentation, finding out how to increase the pyruvate content in the fermentation broth and reduce the impurity content in the fermentation broth has become the core pursuit of the production of pyruvate by fermentation.
[0006] The above background technology is for facilitating the understanding of the present invention and is not a known technology disclosed to the general public before the application of the present invention. Summary of the invention
[0007] In view of the above-mentioned defects, the present invention provides a fermentation precursor material for use in producing pyruvic acid by fermentation, thereby increasing the yield of pyruvic acid in the fermentation broth during the fermentation process.
[0008] The technical solution is: a fermentation precursor substance is used to produce pyruvic acid by fermentation, and the fermentation precursor substance is dichloroacetic acid and / or metformin.
[0009] Furthermore, the dichloroacetic acid is used in an amount of 0.1-1 g / L.
[0010] Furthermore, the dosage of metformin is 0.1~1 g / L.
[0011] Furthermore, the sum of the amounts of dichloroacetic acid and metformin is 0.1-2 g / L, and the mass ratio of dichloroacetic acid: metformin is 0.1-1:0.1-1.
[0012] The invention also provides a pyruvic acid fermentation method.
[0013] The technical solution is: a pyruvic acid fermentation method, comprising the following steps: S1, taking a fermentation precursor substance and a fermentation medium respectively, adding the fermentation precursor substance to the fermentation medium, and mixing them uniformly to form a mixture; S2, put the mixture of S1 into the fermentation tank, sterilize it, and inoculate the seed culture solution with an inoculation amount of 1~10v / v; S3, fermentation to obtain fermentation liquid, the fermentation conditions are: temperature 28-30°C, ventilation volume 0.5-2vvm, pH value 4.0-6.0, rotation speed 100-500r / min, fermentation time 120-160 hours, during the fermentation process, dissolved oxygen is controlled to be not less than 30% by feeding glycerol; The fermentation precursor substances are dichloroacetic acid and / or metformin.
[0014] Further, the metformin dosage is 0.1~1 g / L or the metformin dosage is 0.1~1 g / L.
[0015] Furthermore, the sum of the amounts of dichloroacetic acid and metformin is 0.1-2 g / L, and the mass ratio of dichloroacetic acid: metformin is 0.1-1:0.1-1.
[0016] Furthermore, the fermentation medium comprises: Ammonium sulfate 3~5 g / L Potassium dihydrogen phosphate 3~5 g / L Magnesium sulfate 2~3 g / L Calcium carbonate 5~8 g / L Sodium chloride 1~2 g / L Potassium hydrogen phosphate 0.1~0.5 g / L and Anhydrous sodium acetate 5~10 g / L.
[0017] Compared with the prior art, the present invention has the following beneficial effects: In the process of producing pyruvate by fermentation, the present invention increases the yield of pyruvate in the fermentation broth by adding dichloroacetic acid or / and metformin as fermentation precursors in the fermentation medium. Metformin prevents pyruvate from entering the tricarboxylic acid cycle, and dichloroacetic acid inhibits the activity of pyruvate dehydrogenase kinase (dichloroacetic acid inhibits enzyme activity and thus inhibits pyruvate oxidation) to inhibit pyruvate oxidation. DETAILED DESCRIPTION
[0018] As used herein: "Prepared from" is synonymous with "comprising." As used herein, the terms "comprising," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.
[0019] The conjunction "consisting of excludes any unspecified element, step, or component. If used in a claim, this phrase renders the claim closed-ended so that it does not include materials other than those described, except for conventional impurities associated therewith. When the phrase "consisting of" appears in a clause of the body of a claim rather than immediately following the subject matter, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.
[0020] When an amount, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper preferred values and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted as including ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described in this article, unless otherwise stated, the range is intended to include its end values and all integers and fractions within the range.
[0021] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0022] Those skilled in the art will appreciate that the following examples are only used to illustrate the present application and should not be considered to limit the scope of the present application. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents, instruments or microorganisms used is not specified, they are all conventional products that can be purchased commercially.
[0023] In these examples, parts and percentages are by mass unless otherwise indicated.
[0024] "Parts by mass" refers to the basic unit of measurement for expressing the mass ratio of multiple components. 1 part can represent any unit mass, such as 1g or 2.689g. If we say that the mass of component A is a parts and the mass of component B is b parts, it means the ratio of the mass of component A to the mass of component B is a:b. Alternatively, it means that the mass of component A is aK and the mass of component B is bK (K is an arbitrary number, indicating a multiple factor). It should not be misunderstood that, unlike the mass parts, the sum of the mass of all components is not limited to 100 parts.
[0025] "And / or" is used to indicate that one or both of the stated situations may occur, for example, A and / or B includes (A and B) and (A or B).
[0026] Example 1 Preparation of seed solution The preserved pyruvate biofermentation strain is inoculated into a YPD medium, and cultured at 28-30° C. and 180-220 rpm / min for 18-20 hours to obtain a seed culture solution.
[0027] In the following Examples 2 to 10 and Comparative Example 1, the volume of the fermentation tank is 50 L. In the fermentation tank, the loading factor is 70%, which means the loading volume is 35 L. When the fermentation tank is loaded, after the fermentation medium or the fermentation medium + the fermentation precursor is loaded, water is added to replenish the liquid level to 35 L (basic common sense in this field).
[0028] Example 2 A pyruvic acid fermentation method comprises the following steps: S1, taking dichloroacetic acid and fermentation medium respectively, adding dichloroacetic acid to the fermentation medium, and mixing evenly to form a mixture.
[0029] S2, the mixture of S1 is loaded into a fermenter (the loading factor is 70%), and after sterilization, the seed culture solution of Example 1 is inoculated at an inoculation amount of 5v / v%.
[0030] S3, fermentation to obtain fermentation liquid, the fermentation conditions are: temperature about 30 ° C, ventilation volume 1vvm, pH value 4.0-6.0, rotation speed 300r / min, fermentation time 144 hours, and tank storage. During the fermentation process, dissolved oxygen is controlled to be not less than 30% by feeding glycerol.
[0031] In this embodiment, the fermentation precursor substances and fermentation medium are shown in Table 1: Table 1 The pyruvic acid in the fermentation broth of the lower tank was detected. The results are shown in Table 2.
[0032] Comparative Example 1 The only difference between this comparative example and Example 2 is that no dichloroacetic acid is added to S1.
[0033] The pyruvic acid in the fermentation broth of the lower tank was detected. The results are shown in Table 2.
[0034] Example 3 The difference between this embodiment and embodiment 2 is that the amount of dichloroacetic acid is 0.6 g / L.
[0035] The pyruvic acid in the fermentation broth of the lower tank was detected. The results are shown in Table 2.
[0036] Example 4 The only difference between this embodiment and embodiment 2 is that the amount of dichloroacetic acid is 1 g / L.
[0037] The pyruvic acid in the fermentation broth of the lower tank was detected. The results are shown in Table 2.
[0038] Example 5 The only difference between this embodiment and embodiment 2 is that in S1, dichloroacetic acid is replaced by metformin, and the amount of metformin is 0.3 g / L.
[0039] The pyruvic acid in the fermentation broth of the lower tank was detected. The results are shown in Table 2.
[0040] Example 6 The only difference between this embodiment and embodiment 2 is that in S1, dichloroacetic acid is replaced by metformin, and the amount of metformin is 0.6 g / L.
[0041] The pyruvic acid in the fermentation broth of the lower tank was detected. The results are shown in Table 2.
[0042] Example 7 The only difference between this embodiment and embodiment 2 is that in S1, dichloroacetic acid is replaced by metformin, and the amount of metformin is 1 g / L.
[0043] The pyruvic acid in the fermentation broth of the lower tank was detected. The results are shown in Table 2.
[0044] Example 8 The only difference between this embodiment and embodiment 2 is that in S1, in addition to dichloroacetic acid, metformin is also added, wherein the amount of dichloroacetic acid is 0.3 g / L and the amount of metformin is 0.3 g / L.
[0045] The pyruvic acid in the fermentation broth of the lower tank was detected. The results are shown in Table 2.
[0046] Example 9 The only difference between this embodiment and embodiment 8 is that in S1, the amount of dichloroacetic acid is 0.4 g / L, and the amount of metformin is 0.2 g / L.
[0047] The pyruvic acid in the fermentation broth of the lower tank was detected. The results are shown in Table 2.
[0048] Example 10 The only difference between this embodiment and embodiment 8 is that in S1, the fermentation medium is different.
[0049] In this embodiment, the fermentation medium is as shown in Table 3.
[0050] The pyruvic acid in the fermentation broth of the lower tank was detected. The results are shown in Table 2.
[0051] Comparative Example 2 The only difference between this comparative example and Example 10 is that in S1, no dichloroacetic acid or metformin was added.
[0052] Table 2 Pyruvic acid in fermentation broth Table 3 Fermentation medium of Example 10 Comparing Example 2 to Example 4 with Comparative Example 1, after adding dichloroacetic acid as a precursor, the content of pyruvic acid in the fermentation broth was increased. Comparing Example 5 to Example 7 with Comparative Example 1, after adding metformin as a precursor, the content of pyruvic acid in the fermentation broth was also increased. Example 3, Example 6 are the same as Example 8 and Example 9. After adding the same amount of precursor, adding dichloroacetic acid and metformin at the same time is better than adding dichloroacetic acid or metformin alone. Comparing Example 10 with Comparative Example 2, the addition of dichloroacetic acid and metformin also increases the content of pyruvic acid in the fermentation broth.
[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A fermentation precursor material is used for producing pyruvic acid by fermentation, characterized in that: The fermentation precursor substance is dichloroacetic acid and / or metformin.
2. The fermentation precursor material according to claim 1 is used to produce pyruvic acid by fermentation, characterized in that: The dichloroacetic acid dosage is 0.1-1 g / L.
3. The fermentation precursor material according to claim 1 is used to produce pyruvic acid by fermentation, characterized in that: The dosage of metformin is 0.1~1 g / L.
4. The fermentation precursor according to claim 1 is used for producing pyruvic acid by fermentation, characterized in that: The sum of the amounts of dichloroacetic acid and metformin used is 0.1-2 g / L, and the mass ratio of dichloroacetic acid to metformin is 0.1-1:0.1-1.
5. The fermentation precursor according to any one of claims 1 to 4 is used to produce pyruvic acid by fermentation, characterized in that: The dichloroacetic acid prevents pyruvate from entering the tricarboxylic acid cycle during the fermentation process. The dichloroacetic acid inhibits the activity of pyruvate dehydrogenase kinase during the fermentation process and inhibits pyruvate oxidation.
6. A pyruvic acid fermentation method, characterized in that: The following steps are involved: S1, taking a fermentation precursor substance and a fermentation medium respectively, adding the fermentation precursor substance to the fermentation medium, and mixing them uniformly to form a mixture; S2, put the mixture of S1 into the fermentation tank, sterilize it, and inoculate the seed culture solution with an inoculation amount of 1~10v / v; S3, fermentation to obtain fermentation liquid, the fermentation conditions are: temperature 28-30°C, ventilation volume 0.5-2vvm, pH value 4.0-6.0, rotation speed 100-500r / min, fermentation time 120-160 hours, during the fermentation process, dissolved oxygen is controlled to be not less than 30% by feeding glycerol; The fermentation precursor substances are dichloroacetic acid and / or metformin.
7. The pyruvic acid fermentation method according to claim 6, characterized in that: The metformin dosage is 0.1-1 g / L or the metformin dosage is 0.1-1 g / L.
8. The pyruvic acid fermentation method according to claim 6, characterized in that: The sum of the amounts of dichloroacetic acid and metformin used is 0.1-2 g / L, and the mass ratio of dichloroacetic acid to metformin is 0.1-1:0.1-1.
9. The pyruvic acid fermentation method according to claim 6, characterized in that: The dichloroacetic acid prevents pyruvate from entering the tricarboxylic acid cycle during the fermentation process. The dichloroacetic acid inhibits the activity of pyruvate dehydrogenase kinase during the fermentation process and inhibits pyruvate oxidation.
10. The pyruvic acid fermentation method according to any one of claims 6 to 9, characterized in that: The fermentation medium comprises: Ammonium sulfate 3~5 g / L Potassium dihydrogen phosphate 3~5 g / L Magnesium sulfate 2~3 g / L Calcium carbonate 5~8 g / L Sodium chloride 1~2 g / L Potassium hydrogen phosphate 0.1~0.5 g / L and Anhydrous sodium acetate 5~10 g / L.