A polyphosphate-rich yeast and its use in the synthesis of adenosine triphosphate

Abstract

The present application provides a kind of polyphosphate-rich yeast and its application in synthesis of adenosine triphosphate, the content of polyphosphate is 15-35wt% by dry weight of the polyphosphate-rich yeast;The present application is cultured by phosphorus starvation to the yeast, and then the method of phosphorus feeding culture is used, so that the yeast is enriched with polyphosphate, and the polyphosphate-rich yeast is obtained;Then the polyphosphate-rich yeast is used as a biological catalyst to synthesize ATP, i.e.using the related enzyme system in the polyphosphate-rich yeast itself: mainly including adenosine kinase, endo-polyphosphate kinase and exo-polyphosphate kinase, instead of exogenous polyphosphate kinase and adenosine kinase, and the method of using the polyphosphate-rich yeast as a biological catalyst to synthesize ATP does not need to add exogenous adenosine kinase and exogenous polyphosphate kinase, the cost is low, the efficiency of adenosine conversion to ATP is high, the yeast batch is stable, and the enzyme activity of the above-mentioned related enzyme system is high.

Description

Technical Field

[0001] This invention belongs to the field of biochemical technology, specifically relating to a yeast rich in polyphosphates and its application in the synthesis of adenosine triphosphate. Background Technology

[0002] Polyphosphate (PolyP) is a linear polymer of orthophosphate (Pi), linked by energy-rich phosphate anhydride bonds. It commonly exists as sodium, potassium, and calcium salts. The phosphate anhydride bonds in its molecular structure are identical to those in the high-energy compound adenosine triphosphate (ATP). PolyP is widely distributed in living organisms, participating in various biochemical reactions due to its high-energy phosphate bonds, and possesses a wide range of biological functions. In vivo, PolyP can act as a phosphate donor, catalyzing the conversion of NDP (ADP / CDP / GDP / UDP) / NMP (AMP / CMP / GMP / UMP) to NMP (AMP / CMP / GMP / UMP) / NTP (ATP / CTP / GTP / UTP). This reaction is reversible. Polyphosphate kinase (PPK) can catalyze the conversion of nucleoside triphosphate NTP (ATP / CTP / GTP / UTP) to PolyP to achieve energy cycling.

[0003] Currently, methods for synthesizing medium- and long-chain PolyPs are very limited. Existing technologies typically use enzymatic / microbial methods. Enzymatic synthesis is very expensive, requiring large amounts of ATP as substrates, and the purification and preservation of enzymes are difficult. Microbial synthesis is relatively cheaper than enzymatic methods, but the yield is severely insufficient, generally requiring kilogram-level feed to synthesize milligram-level products, and the product purity is low, typically between 85% and 90%. Existing chemical synthesis methods still have significant drawbacks. These methods rely on expensive equipment, have high fixed costs, are complex to operate, and require large amounts of acids, alkalis, and halides in the production process, resulting in significant pollution. Furthermore, ATP synthesis methods typically use enzymes from brewer's yeast sludge, an industrial byproduct, to phosphorylate adenosine to ATP step by step, which is very inefficient. Brewer's yeast sludge also suffers from large fluctuations in enzyme activity and a short shelf life. Summary of the Invention

[0004] The problem with the existing technology is that in the ATP synthesis process, adenosine is often phosphorylated into ATP through enzymes related to brewer's yeast sludge, an industrial byproduct. This results in large fluctuations in conversion efficiency and low activity of the enzymes related to brewer's yeast sludge.

[0005] To address the problems existing in the prior art, this invention proposes a polyphosphate-rich yeast and its application in the synthesis of adenosine triphosphate (ATP). This method is low-cost, highly efficient, and simple to operate. The specific technical solution is as follows: Technical Solution 1: A yeast rich in polyphosphate, characterized in that, based on the dry matter weight of the yeast rich in polyphosphate, the content of polyphosphate is 15-35 wt%.

[0006] Technical Solution 2: The polyphosphate-rich yeast according to Technical Solution 1, characterized in that, based on the dry matter weight of the polyphosphate-rich yeast, the polyphosphate content is 30-35 wt%. Preferably, the polyphosphate-rich yeast is prepared by a method comprising the following steps: (1) The yeast was inoculated into a seed culture medium and cultured to obtain the first yeast sludge; (2) The first yeast sludge obtained in step (1) is inoculated into phosphorus starvation medium and cultured to obtain the second yeast sludge; (3) The second yeast sludge obtained in step (2) is inoculated into a phosphorus-fed culture medium and cultured to obtain yeast rich in polyphosphate.

[0007] Technical Solution 3: The polyphosphate-rich yeast according to Technical Solution 1 or 2, characterized in that, in step (1), the yeast is Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae ) FX-2 strain.

[0008] Technical Solution 4: The polyphosphate-rich yeast according to any one of Technical Solutions 1-3, characterized in that, in step (2), the phosphorus-starved culture medium does not contain a phosphorus source; Preferably, per L of phosphorus-starved medium, the phosphorus-starved medium comprises 10-20 g glucose, 1-2 g (NH4)2SO4, 0.1-0.2 g KCl, 0.2-0.5 g CaCl2, 1-1.5 g sodium succinate, 0.01-0.1 g Na2EDTA, 0.02-0.1 g H3BO3, 0.01-0.05 g ZnSO4, 0.01-0.05 g FeSO4, 0.01-0.05 g CuSO4, 0.01-0.1 g inositol, 0.02-0.1 g nicotinic acid, 0.005-0.02 g D-calcium pantothenate, 0.0001-0.001 g D-biotin, and 0.001-0.005 g p-aminobenzoic acid, with the balance being water; More preferably, the phosphorus-starved culture medium is sterilized by membrane filtration, and even more preferably, the membrane has a pore size of 0.2-0.3 μm.

[0009] Technical Solution 5: A method for producing polyphosphate-rich yeast according to any one of technical solutions 1-4, characterized in that the method includes the following steps: (1) The yeast was inoculated into a seed culture medium and cultured to obtain the first yeast sludge; (2) The first yeast sludge obtained in step (1) is inoculated into phosphorus starvation medium and cultured to obtain the second yeast sludge; (3) The second yeast sludge obtained in step (2) is inoculated into a phosphorus-fed culture medium and cultured to obtain yeast rich in polyphosphate.

[0010] Technical Solution 6: According to the method described in Technical Solution 5, the seed culture medium comprises 0.5-1.5% YNB medium and 1.5-2.5% glucose, with the remainder being water, in step (1) based on the weight of the substances contained in each liter (L) of seed culture medium in grams (g). Preferably, each 6.71g of the YNB culture medium specifically comprises the following components: ammonium sulfate 4.5-5.5g, biotin 1.0-3.0μg, calcium pantothenate 390.0-410.0μg, folic acid 1.0-3.0μg, inositol 1900.0-2000.0μg, niacin 390.0-410.0μg, para-aminobenzoic acid 190.0-210.0μg, pyridoxine hydrochloride 390.0-410.0μg, riboflavin 190.0-210.0μg, and thiamine hydrochloride 3... 90.0-410.0 μg, boric acid 490.0-510.0 μg, copper sulfate 35.0-45.0 μg, potassium iodide 90.0-110.0 μg, ferric chloride 190.0-210.0 μg, manganese sulfate 390.0-410.0 μg, sodium molybdate 190.0-210.0 μg, zinc sulfate 390.0-410.0 μg, potassium dihydrogen phosphate 0.1-1.0 g, magnesium sulfate 0.1-0.5 g, sodium chloride 0.05-0.1 g, and calcium chloride 0.05-0.1 g.

[0011] More preferably, the culture medium is sterilized by membrane filtration, and even more preferably, the pore size of the membrane is 0.2-0.3 μm.

[0012] Technical Solution 7: The method according to Technical Solution 5 or 6 is characterized in that, in step (1), the culture temperature is 25-35℃, and / or the culture rotation speed is 100-200r / min, and / or the culture time is 15-25h; Preferably, in step (1), the first yeast sludge is obtained by filtration after cultivation.

[0013] Technical Solution 8: The method according to any one of Technical Solutions 5-7 is characterized in that, in step (2), the mass of the first yeast sludge added to each L of phosphorus starvation culture medium is 2-5g, based on the dry matter mass of the first yeast sludge.

[0014] Technical Solution 9: The method according to any one of Technical Solutions 5-8 is characterized in that, in step (2), the culture temperature is 25-35℃, and / or the culture rotation speed is 100-200r / min, and / or the culture time is 4-6h; Preferably, the culture pH is controlled to be 5-7 by adding ammonia water. More preferably, the second yeast slurry is obtained by centrifugation after phosphorus starvation culture.

[0015] Technical Solution 10: The method according to any one of Technical Solutions 5-9 is characterized in that, in step (3), the mass of the second yeast sludge added per L of phosphorus supplementation medium is 30-50g, based on the dry matter mass of the second yeast sludge.

[0016] Technical Solution 11: The method according to any one of technical solutions 5-10 is characterized in that, in step (3), the phosphorus source in the phosphorus supplementation culture medium is KH2PO4; Preferably, the phosphorus-supplemented culture medium includes culture medium A and culture medium B. More preferably, culture medium A and culture medium B are sterilized by membrane filtration respectively; even more preferably, the pore size of the membrane is 0.2-0.3 μm; More preferably, per L of culture medium A, the culture medium A comprises 10-50 g of glucose and 10-20 g of KH2PO4, with the remainder being water; and / or per L of culture medium B, the culture medium B comprises 10-20 g of MgCl2, with the remainder being water; More preferably, before use, medium A and medium B are mixed at a volume ratio of 0.8-1.2:0.8-1.2. More preferably, before using the phosphorus-fed medium, medium A and medium B are mixed in a 1:1 volume ratio; Even more preferably, the pH value is adjusted to 4-6 after mixing.

[0017] Technical Solution 12: The method according to any one of technical solutions 5-11 is characterized in that, in step (3), the culture temperature is 25-35℃, and / or the culture rotation speed is 100-200r / min, and / or the culture time is 7-8h; Preferably, the yeast is obtained by centrifugation after culturing to obtain polyphosphate-rich yeast.

[0018] Technical Solution 13: A polyphosphate-rich yeast prepared by any one of the polyphosphate-rich yeast production methods described in any one of Technical Solutions 5-12.

[0019] Technical Solution 14: The application of any one of Technical Solutions 1-4 or the application of the polyphosphate-rich yeast described in Technical Solution 13 in the synthesis of adenosine triphosphate (ATP), in the preparation of a ATP-rich fermentation broth, or in the preparation of purified ATP products.

[0020] Technical Solution 15: A method for synthesizing adenosine triphosphate (ATP), characterized in that the method includes inoculating a yeast rich in polyphosphate, as described in any one of Technical Solutions 1-4 or Technical Solution 13, into an ATP-catalyzed culture medium for ATP catalytic culture, followed by solid-liquid separation and retention of the supernatant to obtain a ATP-rich fermentation broth, which is used to complete the synthesis of ATP.

[0021] Technical Solution 16: The method according to Technical Solution 15 is characterized in that, based on each L of the adenosine triphosphate catalytic culture medium, the adenosine triphosphate catalytic culture medium includes 8-40 g of adenosine, 0.1-1 g of magnesium chloride, 4-15 g of glucose, 1-5 mL of 5% cetyltrimethylammonium bromide solution, and the remainder is water; Preferably, the culture medium is sterilized by membrane filtration; more preferably, the pore size of the membrane is 0.2-0.3 μm.

[0022] Technical Solution 17: The method according to Technical Solution 15 or 16 is characterized in that, based on the dry matter mass of yeast, the mass of polyphosphate-rich yeast added to each L of the adenosine triphosphate catalytic culture medium is 200-500g.

[0023] Technical Solution 18: The method according to Technical Solution 17 is characterized in that the adenosine triphosphate (ATP) catalytic culture temperature is 30-40℃, and / or the ATP catalytic culture time is 240-360 min, and / or the ATP catalytic culture pH is 6-8. Preferably, hydrochloric acid or phosphoric acid with a pH of 2-4 is added after ATP catalytic culture to terminate the reaction.

[0024] Technical Solution 19: A fermentation broth rich in adenosine triphosphate (ATP), characterized in that it is prepared by any one of the methods described in Technical Solutions 15-18, wherein the ATP content in the fermentation broth is 13000.0 mg to 72000.0 mg per L of the fermentation broth, preferably, the ATP content in the fermentation broth is 13663.4 mg to 71250.4 mg per L of the fermentation broth.

[0025] Technical Solution 20: A purified adenosine triphosphate product, characterized in that it is prepared by the fermentation broth rich in adenosine triphosphate as described in Technical Solution 19.

[0026] The beneficial effects of this invention are: This invention employs a method of first culturing yeast with phosphorus starvation followed by culturing with phosphorus-fed yeast to enrich the yeast with polyphosphates, resulting in polyphosphate-rich yeast. This polyphosphate-rich yeast is then used as a biocatalyst to synthesize ATP. Specifically, it utilizes the yeast's own enzyme system, primarily adenosine kinase, endopeptidase, and exopeptidase, to replace exogenous polyphosphokinases and adenosine kinases. Furthermore, this method of using polyphosphate-rich yeast as a biocatalyst for ATP synthesis eliminates the need for additional exogenous adenosine kinases and polyphosphokinases, resulting in low cost, high efficiency in converting adenosine to ATP, batch-to-batch stability of the yeast, and high enzyme activity of the aforementioned enzyme system.

[0027] This invention prepares polyphosphate-rich yeast through aerobic fermentation, specifically by converting potassium dihydrogen phosphate into polyphosphate using yeast. This polyphosphate-rich yeast is then used as a biocatalyst to synthesize ATP, solving the problem of high cost in ATP preparation using polyphosphate in existing technologies. Furthermore, it addresses the issues of low efficiency, large fluctuations in conversion efficiency, low enzyme activity in brewer's yeast sludge, and cumbersome operation steps associated with the use of enzymes from industrial byproducts like brewer's yeast sludge to phosphorylate adenosine into ATP in existing technologies. Attached Figure Description

[0028] Figure 1 The changes in polyphosphate content in yeast during 8 hours of shaking culture were observed in Examples 1-3 and Comparative Example 1.

[0029] Figure 2 A schematic diagram illustrating the principle of polyphosphokinase (PPK)-mediated ATP regeneration cycle.

[0030] Information on microbial strains The brewing yeast used in this invention ( Saccharomyces cerevisiae FX-2, deposited on August 1, 2016 at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: M2016418, address: Wuhan University, Wuhan, China, postcode: 430072; telephone: 027-68754052. This strain is already in the patent application process with publication number CN109207384A. Detailed Implementation

[0031] The technical solutions of the present invention will be further described in detail below with reference to specific embodiments and comparative examples, but the present invention is not limited to the following technical solutions.

[0032] In the polyphosphokinase (PPK)-mediated ATP regeneration cycle, the substrate is converted into the product under the action of a biocatalyst (wherein, the substrate refers to a starting compound that requires ATP / GTP consumption, such as molecules that are phosphorylated or activated in reactions of kinases, synthases, etc., such as glucose, amino acids, nucleosides, etc.; the biocatalyst refers to an ATP / GTP-dependent enzyme, such as ligase, polymerase, etc.; the product mainly refers to the phosphorylated product), while simultaneously consuming ATP / GTP and generating ADP / GDP. ADP / GDP is then converted into the product under the catalysis of polyphosphokinase (PPK, specifically PPK2), utilizing (poly P... n As a phosphate group donor, ATP / GTP is resynthesized, while (poly P) is used. n Transformed into shorter chains (polyP) n-1 This completes the energy cycle. The entire process embodies the regeneration mechanism of ATP / GTP, where n represents the degree of polymerization (polyP). n-1 This indicates that after the PPK catalytic reaction, the original (polyP) n Losing a phosphate group shortens the chain length to n-1. Specifically, as shown below... Figure 2 As shown, Figure 2 A schematic diagram illustrating the principle of polyphosphokinase (PPK)-mediated ATP regeneration cycle.

[0033] This invention uses brewing yeast ( Saccharomyces cerevisia After seed culture, strain FX-2 was inoculated into phosphorus-free medium for phosphorus starvation culture, followed by high-phosphorus stress culture using phosphorus-fed medium to enrich the *Saccharomyces cerevisiae* FX-2 with large amounts of polyphosphates. Among the phosphorus sources, potassium dihydrogen phosphate was preferred over disodium hydrogen phosphate because yeast absorbs monovalent anions of H₂PO₄. 1- HPO4 does not absorb divalent anions. 2- Inorganic phosphate ions absorbed by yeast are stored in metachromatic granules in the form of polyphosphates (polyP). Then, *Saccharomyces cerevisiae* FX-2 is collected and directly converted into ATP. Since polyphosphates are a high-energy phosphorus source, under the action of the enzyme system of *Saccharomyces cerevisiae* FX-2, adenosine can be efficiently phosphorylated into ATP.

[0034] In some specific embodiments, the present invention provides a polyphosphate-rich yeast, wherein the polyphosphate content is 15-35 wt% based on the dry weight of the polyphosphate-rich yeast.

[0035] Preferably, based on the dry weight of the polyphosphate-rich yeast, the polyphosphate content may be 15.0 wt%, 15.1 wt%, 15.2 wt%, 15.3 wt%, 15.4 wt%, 15.5 wt%, 15.6 wt%, 15.7 wt%, 15.8 wt%, 15.9 wt%, 16.0 wt%, 16.1 wt%, 16.2 wt%, 16.3 wt%, 16.4 wt%, 16.5 wt%, 16.6 wt%, 16.7 wt%, 16.8 wt%, 16.9 wt%, 17.0 wt%, 17.1 wt%, 17.2 wt%, 17.3 wt%, 17.4 wt%, 17.5 wt%, 17.6 wt%. 17.7wt%, 17.8wt%, 17.9wt%, 18.0wt%, 18.1wt%, 18.2wt%, 18.3wt%, 18.4wt %, 18.5wt%, 18.6wt%, 18.7wt%, 18.8wt%, 18.9wt%, 19.0wt%, 19.1wt%, 19.2 wt%, 19.3wt%, 19.4wt%, 19.5wt%, 19.6wt%, 19.7wt%, 19.8wt%, 19.9wt%, 20 .0wt%, 20.1wt%, 20.2wt%, 20.3wt%, 20.4wt%, 20.5wt%, 20.6wt%, 20.7wt%, 2 0.8wt%, 20.9wt%, 21.0wt%, 21.1wt%, 21.2wt%, 21.3wt%, 21.4wt%, 21.5wt% , 21.6wt%, 21.7wt%, 21.8wt%, 21.9wt%, 22.0wt%, 22.1wt%, 22.2wt%, 22.3w t%, 22.4wt%, 22.5wt%, 22.6wt%, 22.7wt%, 22.8wt%, 22.9wt%, 23.0wt%, 23. 1wt%, 23.2wt%, 23.3wt%, 23.4wt%, 23.5wt%, 23.6wt%, 23.7wt%, 23.8wt%, 23 .9wt%, 24.0wt%, 24.1wt%, 24.2wt%, 24.3wt%, 24.4wt%, 24.5wt%, 24.6wt%, 24.7wt%, 24.8wt%, 24.9wt%, 25.0wt%, 25.1wt%, 25.2wt%, 25.3wt%, 25.4wt %, 25.5wt%, 25.6wt%, 25.7wt%, 25.8wt%, 25.9wt%, 26.0wt%, 26.1wt%, 26.2 wt%, 26.3wt%, 26.4wt%, 26.5wt%, 26.6wt%, 26.7wt%, 26.8wt%, 26.9wt%, 27.0wt%, 27.1wt%, 27.2wt%, 27.3wt%, 27.4wt%, 27.5wt%, 27.6wt%, 27.7wt%, 27.8wt%, 27.9wt%, 28.0wt% , 28.1wt%, 28.2wt%, 28.3wt%, 28.4wt%, 28.5wt%, 28.6wt%, 28.7wt%, 28.8wt%, 28.9wt%, 29.0wt%, 29.1 wt%, 29.2wt%, 29.3wt%, 29.4wt%, 29.5wt%, 29.6wt%, 29.7wt%, 29.8wt%, 29.9wt%, 30.0wt%, 30.1wt%, 3 0.2wt%, 30.3wt%, 30.4wt%, 30.5wt%, 30.6wt%, 30.7wt%, 30.8wt%, 30.9wt%, 31.0wt%, 31.1wt%, 31.2wt %, 31.3wt%, 31.4wt%, 31.5wt%, 31.6wt%, 31.7wt%, 31.8wt%, 31.9wt%, 32.0wt%, 32.1wt%, 32.2wt%, 32 .3wt%, 32.4wt%, 32.5wt%, 32.6wt%, 32.7wt%, 32.8wt%, 32.9wt%, 33.0wt%, 33.1wt%, 33.2wt%, 33.3wt% The content of polyphosphate within a numerical range consisting of 33.4 wt%, 33.5 wt%, 33.6 wt%, 33.7 wt%, 33.8 wt%, 33.9 wt%, 34.0 wt%, 34.1 wt%, 34.2 wt%, 34.3 wt%, 34.4 wt%, 34.5 wt%, 34.6 wt%, 34.7 wt%, 34.8 wt%, 34.9 wt%, or 35.0 wt%, or any two of the above specific values ​​as endpoints.

[0036] In some specific embodiments, the present invention provides a method for synthesizing adenosine triphosphate (ATP), the method comprising inoculating the polyphosphate-rich yeast into an ATP-catalyzed culture medium for ATP-catalyzed culture, followed by solid-liquid separation and retention of the supernatant to obtain a ATP-rich fermentation broth for completing the synthesis of ATP.

[0037] Preferably, the adenosine triphosphate (ATP) content in the fermentation broth is 13000.0 mg to 72000.0 mg per liter of the fermentation broth.

[0038] More preferably, the adenosine triphosphate (ATP) content in the fermentation broth, calculated per L, may be 13000.0 mg, 14000.0 mg, 15000.0 mg, 16000.0 mg, 17000.0 mg, 18000.0 mg, 19000.0 mg, 20000.0 mg, 21000.0 mg, 22000.0 mg, 23000.0 mg, 24000.0 mg, 25000.0 mg, or 26000.0 mg. mg, 27000.0mg, 28000.0mg, 29000.0mg, 30000.0mg, 31000.0mg, 32000.0mg, 33000.0mg, 34000.0mg, 35000.0mg, 36000.0mg, 37000.0mg, 38000.0mg, 39000.0mg, 40000.0mg, 41000.0mg, 42000.0mg, 4300 0.0mg, 44000.0mg, 45000.0mg, 46000.0mg, 47000.0mg, 48000.0mg, 49000.0mg, 50000.0mg, 51000.0 mg, 52000.0mg, 53000.0mg, 54000.0mg, 55000.0mg, 56000.0mg, 57000.0mg, 58000.0mg, 59000.0mg, 6 The adenosine triphosphate (ATP) content in the fermentation broth is defined as 0000.0 mg, 61000.0 mg, 62000.0 mg, 63000.0 mg, 64000.0 mg, 65000.0 mg, 66000.0 mg, 67000.0 mg, 68000.0 mg, 69000.0 mg, 70000.0 mg, 71000.0 mg, or 72000.0 mg, or any two of the above specific values ​​as endpoints.

[0039] Unless otherwise stated, all reagents and instruments used in the embodiments and comparative examples of this invention are conventional commercially available products. Information on the sources of the instruments and reagents used in the embodiments and comparative examples of this invention is shown in Table 1 below.

[0040]

[0041] The YNB culture medium used in this embodiment of the invention specifically comprises the following components in every 6.71g of the YNB culture medium: 5.0g ammonium sulfate, 2.0μg biotin, 400.0μg calcium pantothenate, 2.0μg folic acid, 2000.0μg inositol, 400.0μg nicotinic acid, 200.0μg para-aminobenzoic acid, 400.0μg pyridoxine hydrochloride, 200.0μg riboflavin, 400.0μg thiamine hydrochloride, 500.0μg boric acid, 40.0μg copper sulfate, 100.0μg potassium iodide, 200.0μg ferric chloride, 400.0μg manganese sulfate, 200.0μg sodium molybdate, 400.0μg zinc sulfate, 1.0g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 0.1g sodium chloride, and 0.1g calcium chloride.

[0042] In the embodiments and comparative examples of this invention, the content of adenosine, AMP, ADP, and ATP was detected using liquid chromatography. 1. Sample testing The samples were detected by liquid chromatography using a C18 column. The mobile phase consisted of a gradient elution of 0.025 mol / L triethylamine solution (adjusted to pH 6.0 with phosphoric acid) and acetonitrile at a flow rate of 1 mL / min for 40 min. During the first 20 min, the ratio of triethylamine buffer to acetonitrile in the mobile phase was reduced from 99:1 to 95:5 and maintained for 5 min. Then, over the next 15 min, the ratio was changed back from 95:5 to 99:1. The UV detection wavelength was 254 nm, the column temperature was 30 °C, and the sample injection volume was 20 μL. The peak areas of adenosine, AMP, ADP, and ATP in the sample were obtained.

[0043] 2. Establishment of Standard Curve (1) Weigh appropriate amounts of adenosine, AMP, ADP and ATP standards respectively, and prepare chromatographic grade standard solutions with concentrations of 20 mg / L, 40 mg / L, 60 mg / L, 80 mg / L and 100 mg / L respectively.

[0044] (2) Perform instrumental analysis on the above standard solutions of different concentrations and record the peak areas of adenosine, AMP, ADP and ATP at each concentration.

[0045] (3) Plot the standard curves with the concentrations of adenosine, AMP, ADP, and ATP as the x-axis and the corresponding peak areas as the y-axis, respectively. Normally, the standard curve will show a linear relationship. The equation of the standard curve can be obtained through linear regression analysis. The general form of the standard curve is: y = kx + b, where y is the peak area, x is the ATP concentration, k is the slope, and b is the intercept.

[0046] (3) Calculation of adenosine, AMP, ADP and ATP content Substitute the peak areas of adenosine, AMP, ADP and ATP in the sample obtained in step (1) into the standard curve equation (y = kx + b) of adenosine, AMP, ADP and ATP obtained in step (2) respectively, calculate the corresponding x value, and obtain the concentration of adenosine, AMP, ADP and ATP in the sample.

[0047] Example 1 (1) Seed culture One loop of *Saccharomyces cerevisiae* strain FX-2, preserved in a test tube, was inoculated into 0.2 L of seed culture medium and incubated at 30 °C with shaking at 150 rpm for 20 h to obtain the seed culture solution. The seed culture medium, calculated in grams per liter (L), consisted of 1% YNB medium and 2% glucose, with water as the solvent. The pH of the medium was adjusted to 5.4, and the solution was sterilized by filtration through a 0.22 μm filter membrane. The seed culture solution was then filtered to obtain the first yeast sludge. A sterilized filter paper was placed on a Buchner funnel, with the bottom connected to an SHB-III circulating water multi-purpose vacuum pump. The seed culture solution was poured onto the filter paper. Under negative pressure, water and culture medium rapidly passed through the filter paper, while yeast cells were retained, resulting in the first yeast sludge.

[0048] (2) Phosphorus starvation culture The phosphorus-starved medium, calculated in grams (g) per liter (L), comprises 20 g / L glucose, 1 g / L (NH₄)₂SO₄, 0.1 g / L KCl, 0.4 g / L CaCl₂, 1.5 g / L sodium succinate, 0.1 g / L Na₂EDTA, 0.1 g / L H₃BO₃, 0.01 g / L ZnSO₄, 0.03 g / L FeSO₄, 0.03 g / L CuSO₄, 0.1 g / L inositol, 0.02 g / L nicotinic acid, 0.01 g / L D-calcium pantothenate, 0.005 g / L p-aminobenzoic acid, and 0.001 g / L D-biotin, in water, totaling 0.2 L. After aseptic filtration through a 0.22 µm filter membrane, it is stored at 4 °C.

[0049] After drying the first yeast sludge obtained in step (1), add 1g of the first yeast sludge to the phosphorus-starved medium, based on the calculation that the mass (g) of the first yeast sludge added per liter (L) of phosphorus-starved medium is 5g / L. Control the pH to 6 by adding ammonia water, and culture at 30℃ and 150r / min with shaking for 5 hours. Centrifuge at 4000r / min for 10min to collect the second yeast sludge, and store it at 4℃ for later use.

[0050] (3) Phosphorus-fed culture The phosphorus-fed medium consists of two media, A and B, with the composition expressed in grams (g) per liter (L). Medium A comprises 50 g / L glucose and 20 g / L KH₂PO₄ in water, totaling 0.25 L. The pH is adjusted to 5, and the medium is aseptically filtered through a 0.22 µm membrane and stored at 4 °C. Medium B comprises 20 g / L MgCl₂ in water, also totaling 0.25 L, and is aseptically filtered through a 0.22 µm membrane and stored at 4 °C. To use, mediums A and B are mixed to obtain 0.5 L of phosphorus-fed medium, with the pH adjusted to 5.

[0051] After washing the second yeast sludge obtained in step (2) with sterile double-distilled water, and based on the dry matter mass of the second yeast sludge, and the mass (g) of the second yeast sludge added per liter (L) of phosphorus-fed culture medium, the amount of second yeast sludge added to the phosphorus-fed culture medium was calculated to be 20g. The mixture was then cultured at 30℃ and 150r / min with shaking for 8 hours. Then, after centrifugation at 4000r / min for 10min, yeast rich in polyphosphate was obtained. Specifically, the precipitate obtained after centrifugation to remove the supernatant is the yeast rich in polyphosphate.

[0052] During the 8-hour shaking culture period, the polyphosphate content in the yeast was measured every hour to track hourly changes in polyphosphate levels. The polyphosphate content was determined as follows: the yeast was washed twice with sterile double-distilled water, centrifuged, and air-dried on filter paper for 5 minutes before polyphosphate detection. The polyphosphate determination followed the method specified in GB / T 9695.9-2009 "Determination of Polyphosphate in Meat and Meat Products". The changes in polyphosphate content in the yeast during the 8-hour shaking culture period are shown below. Figure 1 As shown.

[0053] (4) ATP catalysis The components contained in each liter (L) of ATP catalytic medium are expressed in grams (g) or milliliters (mL). The ATP catalytic medium includes 8 g / L adenosine, 0.1 g / L magnesium chloride, 4 g / L glucose, 1 ml / L 5% CTAB solution, and water as the solvent for the ATP catalytic medium. The total volume is 1 L. After sterile filtration through a 0.22 µm filter membrane, it is stored at 4 °C.

[0054] The polyphosphate-rich yeast obtained in step (3) was added to ATP-catalyzing medium for ATP catalysis. The amount of polyphosphate-rich yeast added per liter (L) of ATP-catalyzing medium was 200 g / L, based on the dry matter mass of the yeast. After adding the polyphosphate-rich yeast, the pH was adjusted to 7.0, and catalysis was carried out at 36℃ with shaking at 250 r / min for 6 h. Then, concentrated hydrochloric acid at pH 3.0 was added to stop the reaction, and the fermentation supernatant was collected. The actual contents of adenosine, AMP, ADP, and ATP in the fermentation supernatant were detected, and the ATP yield was calculated. The results are shown in Table 2.

[0055] Example 2 (1) Seed culture One loop of *Saccharomyces cerevisiae* strain FX-2, preserved in a test tube, was inoculated into 0.2 L of seed culture medium and incubated at 25 °C with shaking at 100 rpm for 15 h to obtain the seed culture solution. The seed culture medium, calculated in grams per liter (L), consisted of 0.5% YNB medium and 1.5% glucose, with water as the solvent. The pH of the medium was adjusted to 5, and the solution was sterilized by filtration through a 0.2 μm filter membrane. The seed culture solution was then filtered to obtain the first yeast sludge. A sterilized filter paper was placed on a Buchner funnel, with the bottom connected to an SHB-III circulating water multi-purpose vacuum pump. The seed culture solution was poured onto the filter paper. Under negative pressure, water and culture medium rapidly passed through the filter paper, while yeast cells were retained, resulting in the first yeast sludge.

[0056] (2) Phosphorus starvation culture The phosphorus-starved medium formula, based on the weight in grams (g) of each substance contained in each liter (L), is as follows: 10 g / L glucose, 1 g / L (NH4)2SO4, 0.1 g / L KCl, 0.2 g / L CaCl2, 1.5 g / L sodium succinate, 0.1 g / L Na2EDTA, 0.1 g / L H3BO3, 0.05 g / L ZnSO4, 0.05 g / L FeSO4, 0.05 g / L CuSO4, 0.01 g / L inositol, 0.1 g / L nicotinic acid, 0.005 g / L D-calcium pantothenate, 0.005 g / L p-aminobenzoic acid, and 0.001 g / L D-biotin, with water as the solvent, totaling 0.2 L. After aseptic filtration through a 0.22 µm filter membrane, it is stored at 4 °C.

[0057] After drying the first yeast sludge obtained in step (1), add 0.4 g of the first yeast sludge to the phosphorus-starved medium, based on a mass (g) of 2 g / L of the first yeast sludge added per liter (L) of phosphorus-starved medium. Control the pH to 5 by adding ammonia water, and culture at 25℃ and 100 r / min for 4 hours with shaking. Centrifuge at 4000 r / min for 10 min to collect the second yeast sludge, and store it at 4℃ for later use.

[0058] (3) Phosphorus-fed culture The phosphorus-supplemented culture medium is divided into medium A and medium B, with the weight of substances contained in each liter (L) of medium expressed in grams (g). Medium A consists of 10 g / L glucose and 10 g / L KH2PO4 in water, totaling 0.25 L. The pH is adjusted to 5, and after aseptic filtration through a 0.22 µm filter membrane, it is stored at 4 °C. Medium B consists of 10 g / L MgCl2 in water, totaling 0.25 L. After aseptic filtration through a 0.22 µm filter membrane, it is stored at 4 °C. When using, medium A and medium B are mixed to obtain 0.5 L of phosphorus-supplemented culture medium, and the pH is adjusted to 4.

[0059] After washing the second yeast sludge obtained in step (2) with sterile double-distilled water, and based on the dry matter weight of the second yeast sludge, and calculating that the mass (g) of the second yeast sludge added per liter (L) of phosphorus-fed culture medium is 30 g / L, the amount of the second yeast sludge added to the phosphorus-fed culture medium is 15 g. The medium is then cultured at 25°C and 100 r / min with shaking for 8 hours. Then, the yeast rich in polyphosphate is obtained by centrifugation at 4000 r / min for 10 min; that is, the precipitate obtained after centrifugation to remove the supernatant is the yeast rich in polyphosphate.

[0060] During the 8-hour shaking culture period, the polyphosphate content in the yeast was measured every hour to track hourly changes in polyphosphate levels. The polyphosphate content was determined as follows: the yeast was washed twice with sterile double-distilled water, centrifuged, and air-dried on filter paper for 5 minutes before polyphosphate detection. The polyphosphate determination followed the method specified in GB / T 9695.9-2009 "Determination of Polyphosphate in Meat and Meat Products". The changes in polyphosphate content in the yeast during the 8-hour shaking culture period are shown below. Figure 1 As shown.

[0061] (4) ATP catalysis The components contained in each liter (L) of ATP catalytic medium are expressed in grams (g) or milliliters (ml). The ATP catalytic medium includes 40 g / L adenosine, 0.5 g / L magnesium chloride, 10 g / L glucose, and 5 ml / L 5% CTAB solution. The solvent of the ATP catalytic medium is water. The total volume is 1 L. After sterile filtration through a 0.22 µm filter membrane, it is stored at 4 °C.

[0062] The polyphosphate-rich yeast obtained in step (3) was added to ATP-catalyzing medium for ATP catalysis. The amount of polyphosphate-rich yeast added per liter (L) of ATP-catalyzing medium was 300 g / L. The pH was adjusted to 6.0, and catalysis was carried out at 30℃ with shaking at 300 rpm for 4 h. Then, concentrated hydrochloric acid at pH 2.0 was added to stop the reaction, and the fermentation supernatant was collected. The actual contents of adenosine, AMP, ADP, and ATP in the fermentation supernatant were detected, and the ATP yield was calculated. The results are shown in Table 2.

[0063] Example 3 (1) Seed culture One loop of *Saccharomyces cerevisiae* strain FX-2, preserved in a test tube, was inoculated into 0.2 L of seed culture medium and incubated at 35°C with shaking at 200 rpm for 25 h to obtain the seed culture solution. The seed culture medium consisted of 1.5% YNB medium and 2.5% glucose, with the remainder being water, calculated by weight per liter (L). The pH of the medium was adjusted to 6, and the solution was sterilized by filtration through a 0.3 μm filter membrane. The seed culture solution was then filtered to obtain the first yeast sludge. A sterilized filter paper was placed on a Buchner funnel, with the lower part connected to an SHB-III circulating water multi-purpose vacuum pump. The seed culture solution was poured onto the filter paper. Under negative pressure, water and culture medium rapidly passed through the filter paper, while yeast cells were retained, resulting in the first yeast sludge.

[0064] (2) Phosphorus starvation culture The phosphorus-starved medium formula, based on the weight in grams (g) of each substance contained in each liter (L), is as follows: 20 g / L glucose, 2 g / L (NH4)2SO4, 0.2 g / L KCl, 0.5 g / L CaCl2, 1.5 g / L sodium succinate, 0.1 g / L Na2EDTA, 0.1 g / L H3BO3, 0.05 g / L ZnSO4, 0.05 g / L FeSO4, 0.05 g / L CuSO4, 0.1 g / L inositol, 0.1 g / L nicotinic acid, 0.02 g / L D-calcium pantothenate, 0.005 g / L p-aminobenzoic acid, and 0.001 g / L D-biotin, with water as the solvent, totaling 0.2 L. After aseptic filtration through a 0.22 µm filter membrane, it is stored at 4 °C.

[0065] After drying the first yeast sludge obtained in step (1), add 1g of the first yeast sludge to the phosphorus-starved medium, based on the calculation that the mass (g) of the first yeast sludge added per liter (L) of phosphorus-starved medium is 5g / L. Control the pH to 7 by adding ammonia water, and culture at 30℃ and 200r / min with shaking for 6 hours. Centrifuge at 4000r / min for 10min to collect the second yeast sludge, and store it at 4℃ for later use.

[0066] (3) Phosphorus-fed culture The phosphorus-supplemented culture medium is divided into medium A and medium B, with the weight of substances contained in each liter (L) of medium expressed in grams (g). Medium A consists of 50 g / L glucose and 20 g / L KH2PO4 in water, totaling 0.25 L. The pH is adjusted to 6, and after aseptic filtration through a 0.22 µm filter membrane, it is stored at 4 °C. Medium B consists of 20 g / L MgCl2 in water, totaling 0.25 L. After aseptic filtration through a 0.22 µm filter membrane, it is stored at 4 °C. When using, medium A and medium B are mixed to obtain 0.5 L of phosphorus-supplemented culture medium, and the pH is adjusted to 6.

[0067] After washing the second yeast sludge obtained in step (2) with sterile double-distilled water, and calculating based on the dry matter mass of the second yeast sludge, and the mass (g) of the second yeast sludge added per liter (L) of phosphorus-fed culture medium, the amount of second yeast sludge added to the phosphorus-fed culture medium was 25g. The mixture was then cultured at 35℃ and 200 r / min for 8 hours with shaking. Then, the yeast rich in polyphosphate was obtained by centrifugation at 4000 r / min for 10 min; that is, the precipitate obtained after centrifugation to remove the supernatant is the yeast rich in polyphosphate.

[0068] During the 8-hour shaking culture period, the polyphosphate content in the yeast was measured every hour to track hourly changes in polyphosphate levels. The polyphosphate content was determined as follows: the yeast was washed twice with sterile double-distilled water, centrifuged, and air-dried on filter paper for 5 minutes before polyphosphate detection. The polyphosphate determination followed the method specified in GB / T 9695.9-2009 "Determination of Polyphosphate in Meat and Meat Products". The changes in polyphosphate content in the yeast during the 8-hour shaking culture period are shown below. Figure 1 As shown.

[0069] (4) ATP catalysis The components contained in each liter (L) of ATP catalytic medium are expressed in grams (g) or milliliters (ml). The ATP catalytic medium includes 20 g / L adenosine, 1 g / L magnesium chloride, 15 g / L glucose, 5 ml / L 5% CTAB solution, and water as the solvent for the ATP catalytic medium. The total volume is 1 L. After sterile filtration through a 0.22 µm filter membrane, it is stored at 4 °C.

[0070] The polyphosphate-rich yeast obtained in step (3) was added to ATP-catalyzing medium for ATP catalysis. The amount of polyphosphate-rich yeast added per liter (L) of ATP-catalyzing medium was 500 g / L. After adding the polyphosphate-rich yeast, the pH was adjusted to 8.0, and catalysis was carried out at 40℃ with shaking at 250 r / min for 6 h. Then, concentrated hydrochloric acid at pH 4.0 was added to stop the reaction. The fermentation supernatant was collected after the reaction. The actual contents of adenosine, AMP, ADP, and ATP in the fermentation supernatant were detected, and the ATP yield was calculated. The results are shown in Table 2.

[0071] Comparative Example 1 (1) Seed culture is the same as in Example 1.

[0072] (2) Yeast culture Compared to Example 1, the yeast culture medium in Comparative Example 1 replaced (NH4)2SO4 in the phosphorus-starved medium of Example 1 with NH4H2PO4. The remaining components and contents of the yeast culture medium in Comparative Example 1 were the same as those in the phosphorus-starved medium of Example 1. After aseptic filtration through a 0.22µm filter membrane, it was stored at 4°C.

[0073] After drying the first yeast sludge obtained in step (1), add 1g of the first yeast sludge to the phosphorus-starved medium, based on the calculation that the mass (g) of the first yeast sludge added per liter (L) of phosphorus-starved medium is 5g / L. Control the pH to 6 by adding ammonia water, and culture at 30℃ and 150r / min with shaking for 5 hours. Centrifuge at 4000r / min for 10min to collect the second yeast sludge, and store it at 4℃ for later use.

[0074] (3) Yeast fed culture After washing the second yeast sludge obtained in step (2) with sterile double-distilled water, the inoculation amount of the second yeast sludge in the yeast fed culture medium was 40 g / L, based on the dry matter mass of the second yeast sludge. The composition of the yeast fed culture medium was the same as that of the phosphorus-fed culture medium. The mixture was cultured at 30°C and 150 r / min with shaking for 8 hours. Then, the yeast rich in polyphosphate was obtained by centrifugation at 4000 r / min for 10 min. That is, the precipitate obtained after centrifugation to remove the supernatant is the yeast rich in polyphosphate.

[0075] During the 8-hour shaking culture period, the polyphosphate content in the yeast was measured every hour to track hourly changes in polyphosphate levels. The polyphosphate content was determined as follows: the yeast was washed twice with sterile double-distilled water, centrifuged, and air-dried on filter paper for 5 minutes before polyphosphate detection. The polyphosphate determination followed the method specified in GB / T 9695.9-2009 "Determination of Polyphosphate in Meat and Meat Products". The changes in polyphosphate content in the yeast during the 8-hour shaking culture period are shown below. Figure 1 As shown.

[0076] (4) The ATP catalysis method is the same as in Example 1. The actual contents of adenosine, AMP, ADP and ATP in the fermentation supernatant were detected and the ATP yield was calculated. The ATP catalysis results are shown in Table 2.

[0077] Technical effect evaluation 1. For example Figure 1 As shown, Figure 1 Examples 1-3 and Comparative Example 1 show the changes in polyphosphate content in yeast during 8 hours of shaking culture. Figure 1 The comparative example in the example is represented as Comparative Example 1. From... Figure 1 It can be seen that after 8 hours of cultivation, the polyphosphate content in the polyphosphate-rich yeasts obtained in Examples 1-3 was 15-35 wt% (on a dry matter basis). In particular, in Example 3, during the phosphorus starvation phase, the polyP level decreased from 2% in "normal" cells to 0.2% in completely starved cells, a decrease of about 10 times. During the phosphorus feeding phase, the polyP content in Example 3 increased more than 150 times in just 5 hours, reaching more than 30%, while the polyP content in the comparative examples remained almost unchanged at the normal level.

[0078] In Example 3 of this invention, during the phosphorus replenishment stage, the polyP content increased by more than 150 times in just 5 hours, reaching more than 30%. The main reasons for this phenomenon are analyzed as follows. It should be noted that the following analysis is only one reasoning of this invention, and its exact mechanism is not limited by the following analysis: (1) When yeast is in a phosphorus-starved state, the polyphosphate (polyP) in the cell is degraded and stored as an endogenous phosphate reserve for cell use. When phosphorus is replenished, the yeast cell will initiate an overcompensation mechanism to synthesize more polyP than before the starvation to cope with the possible recurrence of phosphorus deficiency in the future. (2) Polyphosphokinase (PPK, specifically PPK1) plays a key role in the synthesis of polyP. It can catalyze the transfer of the γ-phosphate group of ATP to polyP, thereby promoting the synthesis of polyP. When phosphorus is replenished after phosphorus starvation, the activity of PPK in the yeast cell will increase significantly. (3) During the compensatory accumulation phase following phosphorus starvation, the activity of phosphatases in yeast cells changes. The activity of low molecular weight exophosphatase (40 kD) in the cytoplasm decreases, while the activity of high molecular weight exophosphatase (830 kD) increases. This change in activity is conducive to the accumulation of polyP. (4) After phosphorus supplementation, the metabolic activity of yeast cells is enhanced, and the energy supply is sufficient. The presence of carbon sources such as glucose can provide energy for the synthesis of polyP, promoting the rapid accumulation of polyP.

[0079] 2. Actual adenosine content, actual AMP content, actual ADP content, actual ATP content, and ATP yield, where ATP yield = actual ATP content / theoretical ATP content, and theoretical ATP content = initial adenosine content / (267.25 × 507.2 × 1000). The results are shown in Table 2 below.

[0080]

[0081] The results showed that the actual ATP content in the fermentation supernatant obtained by the method of the present invention was 13663.4 mg / L-71250.4 mg / L, and the ATP yield was 90.0%-95.6%, significantly higher than that of Comparative Example 1. Furthermore, under the condition that the initial adenosine concentration was 8 g / L in both Example 1 and Comparative Example 1, the actual ATP content in the fermentation supernatant obtained by the method of Example 1 was 23.2% higher than that of Comparative Example 1. This demonstrates that the method provided by the present invention achieves both high yield and high ATP synthesis, significantly improving the efficiency of adenosine conversion to ATP.

[0082] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention. Any simple modifications, alterations, or equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A yeast rich in polyphosphates, characterized in that, Based on the dry weight of the aforementioned polyphosphate-rich yeast, the polyphosphate content is 28.1-35 wt%. The polyphosphate-rich yeast is prepared by a method comprising the following steps: (1) The yeast was inoculated into a seed culture medium and cultured to obtain the first yeast sludge; (2) The first yeast sludge obtained in step (1) is inoculated into phosphorus starvation medium and cultured to obtain the second yeast sludge; (3) The second yeast sludge obtained in step (2) was inoculated into a phosphorus-fed culture medium and cultured to obtain yeast rich in polyphosphate. In step (1), the yeast is *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Saccharomyces cerevisiae FX-2 strain, preservation number CCTCC NO: M2016418; In step (2), the phosphorus-starved medium does not contain a phosphorus source. Specifically, per L of phosphorus-starved medium, the medium comprises 2g of (NH4)2SO4, 10-20g of glucose, 0.1-0.2g of KCl, 0.2-0.5g of CaCl2, 1-1.5g of sodium succinate, 0.01-0.1g of Na2EDTA, and 0.02-0.1g of H3BO3. 0.01-0.05g ZnSO4, 0.01-0.05g FeSO4, 0.01-0.05g CuSO4, 0.01-0.1g inositol, 0.02-0.1g nicotinic acid, 0.005-0.02g D-calcium pantothenate, 0.0001-0.001g D-biotin, and 0.001-0.005g p-aminobenzoic acid, with the balance being water.

2. The polyphosphate-rich yeast according to claim 1, characterized in that, The polyphosphate content is 30-35 wt% based on the dry weight of the polyphosphate-rich yeast.

3. A method for producing polyphosphate-rich yeast as described in claim 1, characterized in that, The method includes the following steps: (1) The yeast was inoculated into a seed culture medium and cultured to obtain the first yeast sludge; (2) The first yeast sludge obtained in step (1) is inoculated into phosphorus starvation medium and cultured to obtain the second yeast sludge; (3) The second yeast sludge obtained in step (2) was inoculated into a phosphorus-fed culture medium and cultured to obtain yeast rich in polyphosphate. In step (1), the yeast is *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Saccharomyces cerevisiae FX-2 strain, preservation number CCTCC NO: M2016418; In step (2), the phosphorus-starved medium does not contain a phosphorus source. Specifically, per L of phosphorus-starved medium, the medium comprises 2g of (NH4)2SO4, 10-20g of glucose, 0.1-0.2g of KCl, 0.2-0.5g of CaCl2, 1-1.5g of sodium succinate, 0.01-0.1g of Na2EDTA, and 0.02-0.1g of H3BO3. 0.01-0.05g ZnSO4, 0.01-0.05g FeSO4, 0.01-0.05g CuSO4, 0.01-0.1g inositol, 0.02-0.1g nicotinic acid, 0.005-0.02g D-calcium pantothenate, 0.0001-0.001g D-biotin, and 0.001-0.005g p-aminobenzoic acid, with the balance being water.

4. The method according to claim 3, characterized in that, In step (1), the seed culture medium consists of 0.5-1.5% YNB medium and 1.5-2.5% glucose, with the remainder being water, based on the weight of the substances contained in each liter (L) of seed culture medium in grams (g). The pH value of the seed culture medium is 5-6.

5. The method according to claim 4, characterized in that, Each 6.71g of the YNB culture medium specifically comprises the following components: ammonium sulfate 4.5-5.5g, biotin 1.0-3.0μg, calcium pantothenate 390.0-410.0μg, folic acid 1.0-3.0μg, inositol 1900.0-2000.0μg, niacin 390.0-410.0μg, para-aminobenzoic acid 190.0-210.0μg, pyridoxine hydrochloride 390.0-410.0μg, riboflavin 190.0-210.0μg, and thiamine hydrochloride 390. 0.0-410.0 μg, boric acid 490.0-510.0 μg, copper sulfate 35.0-45.0 μg, potassium iodide 90.0-110.0 μg, ferric chloride 190.0-210.0 μg, manganese sulfate 390.0-410.0 μg, sodium molybdate 190.0-210.0 μg, zinc sulfate 390.0-410.0 μg, potassium dihydrogen phosphate 0.1-1.0 g, magnesium sulfate 0.1-0.5 g, sodium chloride 0.05-0.1 g, and calcium chloride 0.05-0.1 g.

6. The method according to claim 3, characterized in that, In step (1), the culture temperature is 25-35℃, and / or the culture speed is 100-200r / min, and / or the culture time is 15-25h.

7. The method according to any one of claims 3-6, characterized in that, In step (2), the mass of the first yeast sludge added to each L of phosphorus-starved culture medium is 2-5g, based on the dry matter mass of the first yeast sludge.

8. The method according to any one of claims 3-6, characterized in that, In step (2), the culture temperature is 25-35℃, and / or the culture speed is 100-200r / min, and / or the culture time is 4-6h.

9. The method according to any one of claims 3-6, characterized in that, In step (3), the amount of second yeast sludge added per L of phosphorus supplemented culture medium is 30-50g, based on the dry matter mass of the second yeast sludge.

10. The method according to any one of claims 3-6, characterized in that, In step (3), the phosphorus source in the phosphorus supplementation medium is KH2PO4, wherein the phosphorus supplementation medium includes medium A and medium B.

11. The method according to claim 10, characterized in that, Based on each L of culture medium A, said culture medium A comprises 10-50 g glucose and 10-20 g KH2PO4, with the remainder being water; and / or based on each L of culture medium B, said culture medium B comprises 10-20 g MgCl2, with the remainder being water.

12. The method according to claim 10, characterized in that, Before using the phosphorus-supplemented culture medium, mix medium A and medium B at a volume ratio of 0.8-1.2:0.8-1.

2.

13. The method according to any one of claims 3-6, characterized in that, In step (3), the culture temperature is 25-35℃, and / or the culture speed is 100-200r / min, and / or the culture time is 7-8h.

14. A polyphosphate-rich yeast prepared by any one of claims 3-13.

15. The use of the polyphosphate-rich yeast of claim 1 or 2 or the polyphosphate-rich yeast of claim 14 in the synthesis of adenosine triphosphate (ATP) or in the preparation of a ATP-rich fermentation broth.

16. A method for synthesizing adenosine triphosphate, characterized in that, The method includes inoculating the polyphosphate-rich yeast of claim 1 or 2 or the polyphosphate-rich yeast of claim 14 into an adenosine triphosphate (ATP) catalytic medium for ATP catalytic culture, followed by solid-liquid separation and retention of the supernatant to obtain a ATP-rich fermentation broth for completing the synthesis of ATP.

17. The method according to claim 16, characterized in that, Based on each L of the adenosine triphosphate catalytic medium, the adenosine triphosphate catalytic medium comprises 8-40 g of adenosine, 0.1-1 g of magnesium chloride, 4-15 g of glucose, 1-5 mL of 5% cetyltrimethylammonium bromide solution, and the remainder being water.

18. The method according to claim 16, characterized in that, Based on the dry matter mass of yeast, the mass of polyphosphate-rich yeast added to each L of the aforementioned adenosine triphosphate catalytic medium is 200-500g.

19. The method according to claim 16, characterized in that, The adenosine triphosphate (ATP) catalytic culture temperature is 30-40℃, and / or the ATP catalytic culture time is 240-360 min, and / or the ATP catalytic culture pH is 6-8.

20. A fermentation broth rich in adenosine triphosphate, characterized in that, The fermentation broth is prepared by any one of claims 16-19, wherein the adenosine triphosphate content in the fermentation broth is 13000.0 mg to 72000.0 mg per L of the fermentation broth.

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