Culture method of methanol-utilizing bacteria

Abstract

The invention provides a culture method of methanol-utilizing bacteria, which comprises the following steps: taking glucose-eating methylotroph, inoculating the glucose-eating methylotroph into an activation culture medium, and culturing to obtain an activated bacteria solution; inoculating the activated bacterial liquid into a first-stage seed culture medium, and culturing to obtain a first-stage seed liquid; inoculating the first-stage seed solution into a second-stage seed culture medium, and culturing to obtain a second-stage seed solution; inoculating the secondary seed solution into a fermentation culture medium, adjusting the rotating speed to enable the initial dissolved oxygen value to be 15-25% and the ventilatory capacity to be 1.2-1.8 vvm, fermenting until the dissolved oxygen value is increased by more than 15%, supplementing a supplementary culture medium containing methanol, enabling the methanol concentration in the fermentation culture medium to be 1.5-2.0% (v / v), and fermenting to obtain a finished product. According to the culture method of the methanol utilizing bacteria, methanol protein can be efficiently synthesized.

Description

Technical Field

[0001] This invention relates to the field of microbial culture technology, and in particular to a method for culturing methanol-utilizing bacteria. Background Technology

[0002] Methanol is an important basic raw material in organic chemicals and can be produced from various raw materials (such as natural gas, coal, oil, and acetylene tail gas). Currently, the relevant synthesis technologies for coal-to-methanol and natural gas-to-methanol are relatively mature, and the cost of methanol is decreasing day by day. Developing high-value-added downstream products is of great significance.

[0003] Methanol-utilizing bacteria are methyltrophic bacteria that can obtain energy from methanol, convert it into bacterial protein, and synthesize various secondary metabolites. Among these products, feed-grade methanol protein produced from methanol has broad application prospects in alleviating feed protein shortages due to its low raw material cost, high protein content, and independence from grain resources.

[0004] Currently, common microorganisms capable of synthesizing methanol proteins using methanol as a carbon source include *Pichia pastoris*, *Corynebacterium glutamicum*, and *Methylglucosidase*. In the methanol metabolism pathway of *Methylglucosidase*, methanol is first oxidized to formaldehyde. A portion of the formaldehyde enters the dissimilatory pathway, where it is further oxidized to carbon dioxide and water; another portion enters the assimilatory pathway, promoting bacterial growth and metabolism. However, the methanol metabolism pathway of *Methylglucosidase* is complex and difficult to regulate, and methanol itself is biotoxic, resulting in significant carbon loss. This prevents the efficient and targeted conversion of methanol metabolic flux into bacterial proteins, leading to low methanol protein synthesis efficiency. Summary of the Invention

[0005] The purpose of this invention is to provide a method for culturing methanol-utilizing bacteria to solve the problem of low methanol protein synthesis efficiency in existing methods for culturing methanol-utilizing bacteria.

[0006] This invention provides the following technical solution: A method for culturing methanol-utilizing bacteria includes the following steps: (1) Take glucose-eating methyl bacteria, inoculate them in an activation medium, and culture for 22-26 hours to obtain an activated bacterial solution; The activation culture medium uses glucose and methanol as carbon sources and ammonium salts as inorganic nitrogen sources. (2) The activated bacterial solution obtained in step (1) is inoculated into the primary seed culture medium and cultured for 22-26 hours to obtain the primary seed solution; The primary seed culture medium uses glucose and methanol as carbon sources and ammonium salts and nitrates as inorganic nitrogen sources. (3) The primary seed culture obtained in step (2) is inoculated into the secondary seed culture medium and cultured for 22-26 hours to obtain the secondary seed culture. The secondary seed culture medium uses glucose and methanol as carbon sources and ammonium salts and nitrates as inorganic nitrogen sources. (4) Inoculate the secondary seed liquid obtained in step (3) into the fermentation medium and ferment until the dissolved oxygen value increases by more than 15%. Then add a feed medium containing methanol and make the methanol concentration in the fermentation medium 1.5-2.0% (v / v). Ferment for 60-72 hours to obtain the final product.

[0007] Preferably, in step (1), the activation culture medium comprises: glucose 0.8-1.0 g / L, dipotassium hydrogen phosphate 0.8-1.2 g / L, potassium dihydrogen phosphate 0.1-0.2 g / L, ammonium sulfate 0.8-1.2 g / L, sodium chloride 0.4-0.6 g / L, magnesium sulfate heptahydrate 0.15-0.30 g / L, methanol 0.2-0.4% (v / v); and a pH value of 7.0-7.5.

[0008] Preferably, the primary seed culture medium comprises: glucose 1.0-1.2 g / L, dipotassium hydrogen phosphate 1.2-1.4 g / L, potassium dihydrogen phosphate 0.3-0.4 g / L, ammonium sulfate 0.8-1.2 g / L, sodium nitrate 0.2-0.3 g / L, sodium chloride 0.4-0.6 g / L, magnesium sulfate heptahydrate 0.15-0.30 g / L, and methanol 0.5-0.7% (v / v); with a pH of 7.0-7.5.

[0009] Preferably, the secondary seed culture medium comprises: glucose 1.2-1.5 g / L, dipotassium hydrogen phosphate 1.4-1.6 g / L, potassium dihydrogen phosphate 0.4-0.5 g / L, ammonium sulfate 0.8-1.2 g / L, sodium nitrate 0.4-0.5 g / L, sodium chloride 0.6-0.8 g / L, magnesium sulfate heptahydrate 0.15-0.30 g / L, and methanol 0.8-1.0% (v / v); with a pH of 7.0-7.5.

[0010] Preferably, the fermentation medium comprises: glucose 1.2-1.5 g / L, dipotassium hydrogen phosphate 1.4-1.6 g / L, potassium dihydrogen phosphate 0.4-0.5 g / L, ammonium sulfate 0.8-1.2 g / L, sodium nitrate 0.6-0.8 g / L, sodium chloride 0.6-0.8 g / L, magnesium sulfate heptahydrate 0.15-0.30 g / L, and methanol 1.0-2.0% (v / v); with a pH of 7.0-7.5.

[0011] Preferably, the activation culture medium, the primary seed culture medium, the secondary seed culture medium, and the fermentation culture medium further include trace elements; Optionally, the trace elements include one or more of ferrous sulfate heptahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, cobalt chloride hexahydrate, anhydrous copper sulfate, manganese chloride tetrahydrate, and boric acid. Optionally, the weight ratio of ferrous sulfate heptahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, cobalt chloride hexahydrate, anhydrous copper sulfate, manganese chloride tetrahydrate, and boric acid is 10:(2-5):(0.08-0.12):(0.03-0.09):(0.5-0.8):(1-3):(0.2-0.8). Optionally, the final concentration of the ferrous sulfate heptahydrate in the culture medium is 2.5-3.5 mg / L.

[0012] Preferably, the activation culture medium, the primary seed culture medium, the secondary seed culture medium, and the fermentation culture medium further include one or more of glycine, taurine chelated calcium, inositol, betaine, and glutathione.

[0013] Preferably, the activation culture medium includes 1-5 mg / L of betaine and 2-5 mg / L of glutathione; Optionally, the primary seed culture medium comprises 3-8 mg / L glycine, 7-12 mg / L taurine chelate calcium, 0.2-0.4 mg / L inositol, 5-10 mg / L betaine, and 5-10 mg / L glutathione. Optionally, the secondary seed culture medium includes 5-10 mg / L glycine, 10-15 mg / L taurine chelate calcium, 0.5-1.0 mg / L inositol, 10-15 mg / L betaine, and 10-20 mg / L glutathione. Optionally, the fermentation medium includes 10-15 mg / L of taurine chelate calcium, 15-20 mg / L of betaine, and 25-35 mg / L of glutathione.

[0014] Preferably, the supplemental culture medium comprises 8-12% (v / v) methanol, 1.4-1.6 g / L dipotassium hydrogen phosphate, 0.4-0.5 g / L potassium dihydrogen phosphate, 1.0-1.5 g / L ammonium sulfate, and 0.20-0.35 g / L magnesium sulfate heptahydrate; the pH value is 7.0-7.5.

[0015] Preferably, in step (1), the glucose-eating methyl-eating bacteria are inoculated into an activation culture medium to achieve a viable count of 1 × 10⁻⁶. 6 CFU / mL - 8 × 10 6 CFU / mL, cultured with shaking at 27-37℃, with a shaking speed of 180-220 rpm; Optionally, in step (2), the activated bacterial solution is inoculated into the primary seed culture medium at an inoculation rate of 4-6%, and cultured by shaking at 27-37°C with a shaking speed of 180-220 rpm. Optionally, in step (3), the primary seed culture is inoculated into the secondary seed culture medium at an inoculation rate of 9-11%, and cultured by shaking at 27-37°C with a shaking speed of 180-220 rpm. Optionally, in step (4), the secondary seed liquid is inoculated into the fermentation medium at an inoculation rate of 4-6% and cultured at 27-37℃, a rotation speed of 200-300rpm, and an aeration rate of 1.2-1.8vvm.

[0016] It should be noted that the percentage of inoculum amount mentioned above refers to % (v / v); dissolved oxygen value refers to (measured DO value / saturated DO value) × 100%, and the fermentation until the dissolved oxygen value increases by more than 15% is based on the initial dissolved oxygen value after the secondary seed liquid is inoculated into the fermentation medium.

[0017] The above-described solution of the present invention has at least the following beneficial effects: (1) The method for culturing methanol-utilizing bacteria of the present invention includes the following steps: taking glucose-eating methyl-eating bacteria, inoculating them into an activation culture medium, and culturing them to obtain an activated bacterial solution; inoculating the activated bacterial solution into a primary seed culture medium and culturing it to obtain a primary seed solution; inoculating the primary seed solution into a secondary seed culture medium and culturing it to obtain a secondary seed solution; inoculating the secondary seed solution into a fermentation culture medium, adjusting the rotation speed to make the initial dissolved oxygen value 15-25%, the aeration rate 1.2-1.8 vvm, fermenting until the dissolved oxygen value rises by more than 15%, adding a feed culture medium containing methanol, and making the methanol concentration in the fermentation culture medium 1.5-2.0% (v / v), and fermenting to obtain methanol-utilizing bacteria.

[0018] The method for cultivating methanol-utilizing bacteria described in this invention uses glucose and methanol as carbon sources and ammonium salts as inorganic nitrogen sources during the activation culture stage. Rapid glucose metabolism provides the bacteria with basic energy and a carbon skeleton, allowing them to adapt to the methanol environment. Ammonium salts provide rapid nitrogen supply to quickly restore activity. In both primary and secondary seed cultures, glucose and methanol are used as carbon sources, and ammonium salts and nitrates as inorganic nitrogen sources. Methanol induces the synthesis of key enzymes in the methanol assimilation pathway, while glucose provides energy. The combination of the rapid nitrogen source ammonium salt and the nitrogen source nitrate (which needs to be reduced) regulates the balance between the synthesis of key enzymes in the assimilation pathway and bacterial proliferation, ensuring sufficient synthesis of these enzymes rather than initiating high-intensity metabolism. This prevents excessive consumption of carbon sources in the dissimilatory pathway due to excessive ammonium salts, which would hinder the induction of key enzyme synthesis, and also avoids interruption of key enzyme synthesis due to insufficient ammonium salts. In the fermentation culture, methanol is used as the carbon source to achieve efficient synthesis of methanol proteins.

[0019] (2) The method for culturing methanol-utilizing bacteria of the present invention, wherein the activated culture medium, the primary seed culture medium, the secondary seed culture medium and the fermentation culture medium are adjusted by adjusting the ratio of carbon source glucose to methanol, the ratio of nitrogen source ammonium salt to nitrate, and the concentration of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride and magnesium sulfate heptahydrate, to achieve multiple gradient changes, adapt to different stages of cultivation, improve the methanol metabolism efficiency of the strain, and achieve efficient synthesis of methanol protein.

[0020] (3) The method for culturing methanol-utilizing bacteria of the present invention further includes one or more of glycine, taurine chelated calcium, inositol, betaine, and glutathione in the activation culture medium, the primary seed culture medium, the secondary seed culture medium, and the fermentation culture medium.

[0021] Methanol is a small polar molecule that can easily pass through the cell membrane and enter the cell. The betaine can maintain the osmotic pressure difference between the inside and outside of the cell and enhance the cell's tolerance to methanol.

[0022] Since the methanol metabolism of glucose-eating methyl-eating bacteria requires the conversion of methanol to formaldehyde, this process continuously generates free radicals that damage the active site of the enzyme. The addition of glutathione can protect the activity of the bacteria and maintain the structural stability of key enzymes in the assimilation pathway.

[0023] Compared to inorganic nitrogen sources that freely diffuse into the bacterial cell and are indiscriminately captured by various intracellular metabolic pathways, glycine, as an organic nitrogen source, can improve protein synthesis efficiency and achieve enzyme enrichment. In synergy with ammonium salts and nitrates, which also serve as nitrogen sources, the total nitrogen source concentration can be controlled, ensuring stable nitrogen metabolism in the bacterial cell and matching the synthesis rates of various metabolic pathways and enzyme systems.

[0024] The taurine chelated calcium can synergistically work with inositol to achieve a balance in bacterial proliferation, inhibit the diversion of methanol via the dissimilatory pathway, and promote the absorption and metabolism of methanol by the bacteria at a faster rate.

[0025] During the activation culture stage, betaine protects against inhibited cell proliferation, while glutathione ensures enzyme synthesis and activity. In the primary and secondary seed cultures, the concentrations of various substances in the culture medium are increased, including methanol. Based on betaine and glutathione, glycine enhances protein synthesis efficiency and enriches the enzyme system. Taurine-chelated calcium and inositol inhibit excessive cell proliferation, improving the quality of the seed culture and preventing the cells from initiating high-intensity catabolism. During the fermentation culture stage, taurine-chelated calcium regulates cell proliferation balance, betaine protects against inhibited cell proliferation, and glutathione ensures enzyme synthesis and activity. Detailed Implementation

[0026] Unless otherwise specified in the embodiments of this invention, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available products; different manufacturers and models of raw materials do not affect the implementation of the technical solution or the achievement of the technical effect of this invention.

[0027] The raw materials used in the following examples are all commercially available products or can be prepared using existing technologies. The Latin name of the glucose-eating methyl-eating bacterium is Methylovorus glucosotrophus, for example, glucose-eating methyl-eating bacterium with the number ATCC49758 or NCIMB 13222 can be used.

[0028] The taurine-chelated calcium can be prepared by methods in the prior art, such as chelating taurine and calcium oxide in a molar ratio of 2:1.

[0029] The CAS number of the inositol is 87-89-8; The CAS number of the betaine is 107-43-7; The CAS number of the glutathione is 70-18-8.

[0030] Example 1 The method for culturing methanol-utilizing bacteria in this embodiment includes the following steps: (1) Take glucose-eating methyl-eating bacteria and inoculate them into an activation culture medium to make the viable count of the glucose-eating methyl-eating bacteria 1×10⁻⁶. 6 CFU / mL, shaken at 27℃ with a shaking speed of 180 rpm, cultured for 23 h to obtain activated bacterial solution; The activation medium uses glucose and methanol as carbon sources and ammonium salts as inorganic nitrogen sources. Specifically, in this embodiment, the activation medium includes: 0.8 g / L glucose, 0.9 g / L dipotassium hydrogen phosphate, 0.1 g / L potassium dihydrogen phosphate, 0.8 g / L ammonium sulfate, 0.5 g / L sodium chloride, 0.15 g / L magnesium sulfate heptahydrate, 0.3% (v / v) methanol, trace elements, 2 mg / L betaine, and 4 mg / L glutathione; the pH value is 7.0.

[0031] The trace elements are a mixture of ferrous sulfate heptahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, cobalt chloride hexahydrate, anhydrous copper sulfate, manganese chloride tetrahydrate, and boric acid in a weight ratio of 10:5:0.12:0.04:0.5:2:0.8; the final concentration of ferrous sulfate heptahydrate in the culture medium is 2.8 mg / L.

[0032] (2) The activated bacterial solution obtained in step (1) was inoculated into the primary seed culture medium at an inoculation rate of 4%, and cultured at 27°C with shaking at a speed of 190 rpm for 23 h to obtain the primary seed solution. The primary seed culture medium uses glucose and methanol as carbon sources and ammonium salts and nitrates as inorganic nitrogen sources. Specifically, in this embodiment, the primary seed culture medium includes: glucose 1.2 g / L, dipotassium hydrogen phosphate 1.2 g / L, potassium dihydrogen phosphate 0.3 g / L, ammonium sulfate 0.8 g / L, sodium nitrate 0.3 g / L, sodium chloride 0.4 g / L, magnesium sulfate heptahydrate 0.15 g / L, methanol 0.5% (v / v), trace elements, glycine 8 mg / L, taurine chelated calcium 12 mg / L, inositol 0.2 mg / L, betaine 10 mg / L, and glutathione 10 mg / L; the pH value is 7.0.

[0033] The trace elements are a mixture of ferrous sulfate heptahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, cobalt chloride hexahydrate, anhydrous copper sulfate, manganese chloride tetrahydrate, and boric acid in a weight ratio of 10:2:0.12:0.03:0.5:1:0.2; the final concentration of ferrous sulfate heptahydrate in the culture medium is 2.8 mg / L.

[0034] (3) The primary seed culture obtained in step (2) is inoculated into the secondary seed culture medium at an inoculation rate of 10%, and cultured at 27°C with shaking at a speed of 220 rpm for 25 h to obtain the secondary seed culture. The secondary seed culture medium uses glucose and methanol as carbon sources and ammonium salts and nitrates as inorganic nitrogen sources. Specifically, in this embodiment, the secondary seed culture medium includes: glucose 1.5 g / L, dipotassium hydrogen phosphate 1.4 g / L, potassium dihydrogen phosphate 0.4 g / L, ammonium sulfate 0.8 g / L, sodium nitrate 0.5 g / L, sodium chloride 0.6 g / L, magnesium sulfate heptahydrate 0.30 g / L, methanol 0.8% (v / v), trace elements, glycine 10 mg / L, taurine chelated calcium 10 mg / L, inositol 1.0 mg / L, betaine 15 mg / L, and glutathione 10 mg / L; the pH value is 7.0.

[0035] The trace elements are a mixture of ferrous sulfate heptahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, cobalt chloride hexahydrate, anhydrous copper sulfate, manganese chloride tetrahydrate, and boric acid in a weight ratio of 10:4:0.08:0.03:0.7:1:0.6; the final concentration of ferrous sulfate heptahydrate in the culture medium is 2.5 mg / L.

[0036] (4) The secondary seed liquid obtained in step (3) is inoculated into the fermentation medium at an inoculation rate of 5%, and cultured at 27°C. The rotation speed is adjusted to 200 rpm and the aeration rate is 1.6 vvm. Fermentation continues until the dissolved oxygen value increases by more than 15%. Then, a feed medium containing methanol is added, with methanol as the carbon source, and the methanol concentration in the fermentation medium is 1.5% (v / v). Fermentation continues for 65 hours to obtain the final product.

[0037] The fermentation medium comprises: glucose 1.2 g / L, dipotassium hydrogen phosphate 1.5 g / L, potassium dihydrogen phosphate 0.4 g / L, ammonium sulfate 1.1 g / L, sodium nitrate 0.6 g / L, sodium chloride 0.7 g / L, magnesium sulfate heptahydrate 0.15 g / L, methanol 1.8% (v / v), trace elements, taurine chelated calcium 10 mg / L, betaine 20 mg / L, glutathione 25 mg / L; and a pH of 7.1.

[0038] The trace elements are a mixture of ferrous sulfate heptahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, cobalt chloride hexahydrate, anhydrous copper sulfate, manganese chloride tetrahydrate, and boric acid in a weight ratio of 10:4:0.08:0.09:0.5:2:0.8; the final concentration of ferrous sulfate heptahydrate in the culture medium is 2.8 mg / L.

[0039] The feed medium consists of 8% methanol (v / v), 1.5 g / L dipotassium hydrogen phosphate, 0.4 g / L potassium dihydrogen phosphate, 1.5 g / L ammonium sulfate, and 0.20 g / L magnesium sulfate heptahydrate; the pH value is 7.3.

[0040] Example 2 The method for culturing methanol-utilizing bacteria in this embodiment includes the following steps: (1) Take glucose-eating methyl-eating bacteria and inoculate them into an activation culture medium to make the viable count of the glucose-eating methyl-eating bacteria 8 × 10⁻⁶. 6 CFU / mL, shaken at 30℃ with a shaking speed of 220 rpm, cultured for 22 h to obtain activated bacterial solution; The activation medium uses glucose and methanol as carbon sources and ammonium salts as inorganic nitrogen sources. Specifically, in this embodiment, the activation medium includes: 1.0 g / L glucose, 0.8 g / L dipotassium hydrogen phosphate, 0.2 g / L potassium dihydrogen phosphate, 1.2 g / L ammonium sulfate, 0.6 g / L sodium chloride, 0.30 g / L magnesium sulfate heptahydrate, 0.4% (v / v) methanol, trace elements, 1 mg / L betaine, and 5 mg / L glutathione; the pH value is 7.5.

[0041] The trace elements are a mixture of ferrous sulfate heptahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, cobalt chloride hexahydrate, anhydrous copper sulfate, manganese chloride tetrahydrate, and boric acid in a weight ratio of 10:2:0.08:0.09:0.8:1:0.2; the final concentration of ferrous sulfate heptahydrate in the culture medium is 2.5 mg / L.

[0042] (2) The activated bacterial solution obtained in step (1) was inoculated into the primary seed culture medium at an inoculation rate of 6%, and cultured at 30°C with shaking at a speed of 220 rpm for 26 h to obtain the primary seed solution. The primary seed culture medium uses glucose and methanol as carbon sources and ammonium salts and nitrates as inorganic nitrogen sources. Specifically, in this embodiment, the primary seed culture medium includes: glucose 1.0 g / L, dipotassium hydrogen phosphate 1.4 g / L, potassium dihydrogen phosphate 0.4 g / L, ammonium sulfate 1.2 g / L, sodium nitrate 0.2 g / L, sodium chloride 0.6 g / L, magnesium sulfate heptahydrate 0.30 g / L, methanol 0.7% (v / v), trace elements, glycine 3 mg / L, taurine chelated calcium 7 mg / L, inositol 0.4 mg / L, betaine 5 mg / L, and glutathione 5 mg / L; the pH value is 7.5.

[0043] The trace elements are a mixture of ferrous sulfate heptahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, cobalt chloride hexahydrate, anhydrous copper sulfate, manganese chloride tetrahydrate, and boric acid in a weight ratio of 10:5:0.08:0.09:0.8:3:0.8; the final concentration of ferrous sulfate heptahydrate in the culture medium is 3.5 mg / L.

[0044] (3) The primary seed culture obtained in step (2) is inoculated into the secondary seed culture medium at an inoculation rate of 9%, and cultured at 30°C with shaking at a speed of 180 rpm for 22 h to obtain the secondary seed culture. The secondary seed culture medium uses glucose and methanol as carbon sources and ammonium salts and nitrates as inorganic nitrogen sources. Specifically, in this embodiment, the secondary seed culture medium includes: glucose 1.2 g / L, dipotassium hydrogen phosphate 1.6 g / L, potassium dihydrogen phosphate 0.5 g / L, ammonium sulfate 1.2 g / L, sodium nitrate 0.4 g / L, sodium chloride 0.8 g / L, magnesium sulfate heptahydrate 0.15 g / L, methanol 1.0% (v / v), trace elements, glycine 5 mg / L, taurine chelated calcium 15 mg / L, inositol 0.7 mg / L, betaine 10 mg / L, and glutathione 20 mg / L; the pH value is 7.5.

[0045] The trace elements are a mixture of ferrous sulfate heptahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, cobalt chloride hexahydrate, anhydrous copper sulfate, manganese chloride tetrahydrate, and boric acid in a weight ratio of 10:5:0.12:0.09:0.8:3:0.8; the final concentration of ferrous sulfate heptahydrate in the culture medium is 3.5 mg / L.

[0046] (4) The secondary seed liquid obtained in step (3) is inoculated into the fermentation medium at an inoculation rate of 6%, and cultured at 30°C. The rotation speed is adjusted to 300 rpm and the aeration rate is 1.2 vvm. Fermentation continues until the dissolved oxygen value increases by more than 15%. Then, a feed medium containing methanol is added, with methanol as the carbon source, and the methanol concentration in the fermentation medium is 2.0% (v / v). Fermentation continues for 60 h to obtain the final product.

[0047] The fermentation medium comprises: glucose 1.5 g / L, dipotassium hydrogen phosphate 1.6 g / L, potassium dihydrogen phosphate 0.5 g / L, ammonium sulfate 1.2 g / L, sodium nitrate 0.8 g / L, sodium chloride 0.8 g / L, magnesium sulfate heptahydrate 0.30 g / L, methanol 2.0% (v / v), trace elements, taurine chelated calcium 15 mg / L, betaine 15 mg / L, glutathione 35 mg / L; and a pH of 7.5.

[0048] The trace elements are a mixture of ferrous sulfate heptahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, cobalt chloride hexahydrate, anhydrous copper sulfate, manganese chloride tetrahydrate, and boric acid in a weight ratio of 10:5:0.12:0.03:0.8:3:0.2; the final concentration of ferrous sulfate heptahydrate in the culture medium is 2.5 mg / L.

[0049] The feed medium consists of 12% methanol (v / v), 1.6 g / L dipotassium hydrogen phosphate, 0.5 g / L potassium dihydrogen phosphate, 1.0 g / L ammonium sulfate, and 0.35 g / L magnesium sulfate heptahydrate; the pH value is 7.5.

[0050] Example 3 The method for culturing methanol-utilizing bacteria in this embodiment includes the following steps: (1) Take glucose-eating methyl-eating bacteria and inoculate them into an activation culture medium to make the viable count of the glucose-eating methyl-eating bacteria 3 × 10⁻⁶. 6 CFU / mL, shaken at 33℃ with a shaking speed of 190 rpm, cultured for 26 h to obtain activated bacterial solution; The activation medium uses glucose and methanol as carbon sources and ammonium salts as inorganic nitrogen sources. Specifically, in this embodiment, the activation medium includes: glucose 0.9 g / L, dipotassium hydrogen phosphate 1.2 g / L, potassium dihydrogen phosphate 0.13 g / L, ammonium sulfate 1.1 g / L, sodium chloride 0.4 g / L, magnesium sulfate heptahydrate 0.20 g / L, methanol 0.2% (v / v), trace elements, betaine 5 mg / L, glutathione 2 mg / L; and a pH of 7.3.

[0051] The trace elements are a mixture of ferrous sulfate heptahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, cobalt chloride hexahydrate, anhydrous copper sulfate, manganese chloride tetrahydrate, and boric acid in a weight ratio of 10:4:0.09:0.03:0.6:3:0.6; the final concentration of ferrous sulfate heptahydrate in the culture medium is 3.5 mg / L.

[0052] (2) The activated bacterial solution obtained in step (1) was inoculated into the primary seed culture medium at an inoculation rate of 4%, and cultured at 33°C with shaking at a speed of 180 rpm for 22 h to obtain the primary seed solution. The primary seed culture medium uses glucose and methanol as carbon sources and ammonium salts and nitrates as inorganic nitrogen sources. Specifically, in this embodiment, the primary seed culture medium includes: glucose 1.1 g / L, dipotassium hydrogen phosphate 1.2 g / L, potassium dihydrogen phosphate 0.3 g / L, ammonium sulfate 1.1 g / L, sodium nitrate 0.25 g / L, sodium chloride 0.5 g / L, magnesium sulfate heptahydrate 0.20 g / L, methanol 0.5% (v / v), trace elements, glycine 5 mg / L, taurine chelated calcium 10 mg / L, inositol 0.3 mg / L, betaine 8 mg / L, and glutathione 6 mg / L; the pH value is 7.3.

[0053] The trace elements are a mixture of ferrous sulfate heptahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, cobalt chloride hexahydrate, anhydrous copper sulfate, manganese chloride tetrahydrate, and boric acid in a weight ratio of 10:4:0.11:0.05:0.4:1.5:0.4; the final concentration of ferrous sulfate heptahydrate in the culture medium is 2.5 mg / L.

[0054] (3) The primary seed culture obtained in step (2) is inoculated into the secondary seed culture medium at an inoculation rate of 11%, and cultured at 33°C with shaking at a speed of 200 rpm for 26 h to obtain the secondary seed culture. The secondary seed culture medium uses glucose and methanol as carbon sources and ammonium salts and nitrates as inorganic nitrogen sources. Specifically, in this embodiment, the secondary seed culture medium includes: glucose 1.4 g / L, dipotassium hydrogen phosphate 1.5 g / L, potassium dihydrogen phosphate 0.5 g / L, ammonium sulfate 1.1 g / L, sodium nitrate 0.4 g / L, sodium chloride 0.7 g / L, magnesium sulfate heptahydrate 0.20 g / L, methanol 0.9% (v / v), trace elements, glycine 7 mg / L, taurine chelated calcium 12 mg / L, inositol 0.5 mg / L, betaine 13 mg / L, and glutathione 18 mg / L; the pH value is 7.3.

[0055] The trace elements are a mixture of ferrous sulfate heptahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, cobalt chloride hexahydrate, anhydrous copper sulfate, manganese chloride tetrahydrate, and boric acid in a weight ratio of 10:2:0.09:0.05:0.5:2:0.2; the final concentration of ferrous sulfate heptahydrate in the culture medium is 3.3 mg / L.

[0056] (4) The secondary seed liquid obtained in step (3) is inoculated into the fermentation medium at an inoculation rate of 4%, and cultured at 35°C. The rotation speed is adjusted to 260 rpm and the aeration rate is 1.8 vvm. Fermentation continues until the dissolved oxygen value increases by more than 15%. Then, a feed medium containing methanol is added, with methanol as the carbon source, and the methanol concentration in the fermentation medium is 1.8% (v / v). Fermentation continues for 72 hours to obtain the final product.

[0057] The fermentation medium comprises: glucose 1.4 g / L, dipotassium hydrogen phosphate 1.4 g / L, potassium dihydrogen phosphate 0.5 g / L, ammonium sulfate 0.8 g / L, sodium nitrate 0.7 g / L, sodium chloride 0.6 g / L, magnesium sulfate heptahydrate 0.20 g / L, methanol 1.0% (v / v), trace elements, taurine chelated calcium 12 mg / L, betaine 17 mg / L, glutathione 28 mg / L; and a pH of 7.0.

[0058] The trace elements are a mixture of ferrous sulfate heptahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, cobalt chloride hexahydrate, anhydrous copper sulfate, manganese chloride tetrahydrate, and boric acid in a weight ratio of 10:2:0.11:0.05:0.7:1:0.6; the final concentration of ferrous sulfate heptahydrate in the culture medium is 3.5 mg / L.

[0059] The feed medium consists of 11% methanol (v / v), 1.4 g / L dipotassium hydrogen phosphate, 0.44 g / L potassium dihydrogen phosphate, 1.2 g / L ammonium sulfate, and 0.25 g / L magnesium sulfate heptahydrate; the pH value is 7.0.

[0060] Example 4 The method for culturing methanol-utilizing bacteria in this embodiment includes the following steps: (1) Take glucose-eating methyl-eating bacteria and inoculate them into an activation culture medium to make the viable count of the glucose-eating methyl-eating bacteria 5 × 10⁻⁶. 6 CFU / mL, shaken at 30℃ with a shaking speed of 200 rpm, cultured for 24 h to obtain activated bacterial solution; The activation medium uses glucose and methanol as carbon sources and ammonium salts as inorganic nitrogen sources. Specifically, in this embodiment, the activation medium includes: glucose 0.9 g / L, dipotassium hydrogen phosphate 1.0 g / L, potassium dihydrogen phosphate 0.15 g / L, ammonium sulfate 1.0 g / L, sodium chloride 0.5 g / L, magnesium sulfate heptahydrate 0.25 g / L, methanol 0.3% (v / v), trace elements, betaine 3 mg / L, glutathione 3 mg / L; and a pH of 7.2.

[0061] The trace elements are a mixture of ferrous sulfate heptahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, cobalt chloride hexahydrate, anhydrous copper sulfate, manganese chloride tetrahydrate, and boric acid in a weight ratio of 10:3:0.10:0.06:0.7:2:0.5; the final concentration of ferrous sulfate heptahydrate in the culture medium is 3.0 mg / L.

[0062] (2) The activated bacterial solution obtained in step (1) is inoculated into the primary seed culture medium at an inoculation rate of 5%, and cultured at 30°C with shaking at a speed of 200 rpm for 24 h to obtain the primary seed solution. The primary seed culture medium uses glucose and methanol as carbon sources and ammonium salts and nitrates as inorganic nitrogen sources. Specifically, in this embodiment, the primary seed culture medium includes: glucose 1.1 g / L, dipotassium hydrogen phosphate 1.3 g / L, potassium dihydrogen phosphate 0.35 g / L, ammonium sulfate 1.0 g / L, sodium nitrate 0.25 g / L, sodium chloride 0.5 g / L, magnesium sulfate heptahydrate 0.25 g / L, methanol 0.6% (v / v), trace elements, glycine 6 mg / L, taurine chelated calcium 9 mg / L, inositol 0.3 mg / L, betaine 7 mg / L, and glutathione 7 mg / L; the pH value is 7.2.

[0063] The trace elements are a mixture of ferrous sulfate heptahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, cobalt chloride hexahydrate, anhydrous copper sulfate, manganese chloride tetrahydrate, and boric acid in a weight ratio of 10:3:0.10:0.07:0.6:2:0.5; the final concentration of ferrous sulfate heptahydrate in the culture medium is 3.0 mg / L.

[0064] (3) The primary seed culture obtained in step (2) is inoculated into the secondary seed culture medium at an inoculation rate of 10%, and cultured at 30°C with shaking at a speed of 200 rpm for 24 h to obtain the secondary seed culture. The secondary seed culture medium uses glucose and methanol as carbon sources and ammonium salts and nitrates as inorganic nitrogen sources. Specifically, in this embodiment, the secondary seed culture medium includes: glucose 1.3 g / L, dipotassium hydrogen phosphate 1.5 g / L, potassium dihydrogen phosphate 0.4 g / L, ammonium sulfate 1.0 g / L, sodium nitrate 0.5 g / L, sodium chloride 0.7 g / L, magnesium sulfate heptahydrate 0.25 g / L, methanol 0.9% (v / v), trace elements, glycine 8 mg / L, taurine chelated calcium 13 mg / L, inositol 0.8 mg / L, betaine 12 mg / L, and glutathione 17 mg / L; the pH value is 7.2.

[0065] The trace elements are a mixture of ferrous sulfate heptahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, cobalt chloride hexahydrate, anhydrous copper sulfate, manganese chloride tetrahydrate, and boric acid in a weight ratio of 10:3:0.1:0.06:0.6:2:0.5; the final concentration of ferrous sulfate heptahydrate in the culture medium is 3.0 mg / L.

[0066] (4) The secondary seed liquid obtained in step (3) is inoculated into the fermentation medium at an inoculation rate of 5%, and cultured at 30°C. The rotation speed is adjusted to 260 rpm and the aeration rate is 1.5 vvm. Fermentation continues until the dissolved oxygen value increases by more than 15%. Then, a feed medium containing methanol is added, with methanol as the carbon source, and the methanol concentration in the fermentation medium is 1.7% (v / v). Fermentation continues for 68 hours to obtain the final product.

[0067] The fermentation medium comprises: glucose 1.3 g / L, dipotassium hydrogen phosphate 1.5 g / L, potassium dihydrogen phosphate 0.45 g / L, ammonium sulfate 1.0 g / L, sodium nitrate 0.7 g / L, sodium chloride 0.7 g / L, magnesium sulfate heptahydrate 0.25 g / L, methanol 1.5% (v / v), trace elements, taurine chelated calcium 13 mg / L, betaine 18 mg / L, glutathione 30 mg / L; and a pH of 7.2.

[0068] The trace elements are a mixture of ferrous sulfate heptahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, cobalt chloride hexahydrate, anhydrous copper sulfate, manganese chloride tetrahydrate, and boric acid in a weight ratio of 10:3:0.10:0.06:0.7:2:0.5; the final concentration of ferrous sulfate heptahydrate in the culture medium is 3.0 mg / L.

[0069] The feed medium consists of 10% methanol (v / v), 1.5 g / L dipotassium hydrogen phosphate, 0.45 g / L potassium dihydrogen phosphate, 1.6 g / L ammonium sulfate, and 0.30 g / L magnesium sulfate heptahydrate; the pH value is 7.2.

[0070] Example 5 The method for cultivating methanol-utilizing bacteria in this embodiment uses the same raw materials, the same amount of each raw material, and the same steps as in Example 4. The only difference is that the fermentation culture medium does not contain taurine chelated calcium, betaine, or glutathione.

[0071] Example 6 The method for cultivating methanol-utilizing bacteria in this embodiment uses the same raw materials, the same amount of each raw material, and the same steps as in Example 4. The only difference is that the activation culture medium, the primary seed culture medium, the secondary seed culture medium, and the fermentation culture medium do not contain betaine.

[0072] Example 7 The method for cultivating methanol-utilizing bacteria in this embodiment uses the same raw materials, the same amount of each raw material, and the same steps as in Example 4. The only difference is that the activation culture medium, the primary seed culture medium, the secondary seed culture medium, and the fermentation culture medium do not contain glutathione.

[0073] Example 8 The method for cultivating methanol-utilizing bacteria in this embodiment uses the same raw materials, the same amount of each raw material, and the same steps as in Example 4. The only difference is that the primary seed culture medium and the secondary seed culture medium do not contain glycine.

[0074] Example 9 The method for cultivating methanol-utilizing bacteria in this embodiment uses the same raw materials, the same amount of each raw material, and the same steps as in Example 4. The only difference is that the primary seed culture medium, the secondary seed culture medium, and the fermentation culture medium do not contain taurine chelated calcium.

[0075] Example 10 The method for cultivating methanol-utilizing bacteria in this embodiment uses the same raw materials, the same amount of each raw material, and the same steps as in Example 4. The only difference is that the primary seed culture medium, the secondary seed culture medium, and the fermentation culture medium do not contain inositol.

[0076] Example 11 The method for cultivating methanol-utilizing bacteria in this embodiment uses the same raw materials, the same amount of each raw material, and the same steps as in Example 4. The only difference is that the activation culture medium, the primary seed culture medium, the secondary seed culture medium, and the fermentation culture medium do not contain glycine, taurine chelated calcium, inositol, or glutathione, but contain betaine.

[0077] Example 12 The method for cultivating methanol-utilizing bacteria in this embodiment uses the same raw materials, the same amount of each raw material, and the same steps as in Example 4. The only difference is that the activation culture medium, the primary seed culture medium, the secondary seed culture medium, and the fermentation culture medium do not contain glycine, taurine chelated calcium, inositol, or betaine, but contain glutathione.

[0078] Example 13 The method for cultivating methanol-utilizing bacteria in this embodiment uses the same raw materials, the same amount of each raw material, and the same steps as in Example 4. The only difference is that the activation culture medium, the primary seed culture medium, the secondary seed culture medium, and the fermentation culture medium do not contain glycine, inositol, betaine, or glutathione, but contain taurine chelated calcium.

[0079] Example 14 The method for cultivating methanol-utilizing bacteria in this embodiment uses the same raw materials, the same amount of each raw material, and the same steps as in Example 4. The only difference is that neither the primary seed culture medium nor the secondary seed culture medium contains glycine, taurine chelated calcium, betaine, or glutathione, but contains inositol.

[0080] Comparative Example 1 The cultivation method for the methanol-utilizing bacteria in this comparative example uses the same raw materials and amounts as in Example 4, and is prepared using the same method. The only difference is that the primary seed culture medium, the secondary seed culture medium, and the fermentation culture medium do not contain sodium nitrate; ammonium sulfate is used as the inorganic nitrogen source, and the sodium nitrate is replaced by an equimolar amount of ammonium sulfate added to each culture medium. Furthermore, the primary seed culture medium and the secondary seed culture medium do not contain glycine.

[0081] Comparative Example 2 The cultivation method for the methanol-utilizing bacteria in this comparative example uses the same raw materials and amounts as in Example 4, and is prepared using the same method. The only difference is that the primary seed culture medium, the secondary seed culture medium, and the fermentation culture medium do not contain sodium nitrate; ammonium sulfate is used as the inorganic nitrogen source. Furthermore, the primary seed culture medium and the secondary seed culture medium do not contain glycine.

[0082] Comparative Example 3 The cultivation method for the methanol-utilizing bacteria in this comparative example uses the same raw materials and amounts as in Example 4, and is prepared using the same method. The only difference is that the primary seed culture medium, the secondary seed culture medium, and the fermentation culture medium do not contain sodium nitrate; only ammonium sulfate is used as the inorganic nitrogen source, and the sodium nitrate is replaced with an equimolar amount of ammonium sulfate added to each culture medium. Additionally, the primary seed culture medium and the secondary seed culture medium contain glycine.

[0083] Comparative Example 4 The method for culturing the methanol-utilizing bacteria in this comparative example is the same as that used in Example 4, with the same amount of each raw material and the same preparation method. The only difference is that the activation culture medium, the primary seed culture medium, and the secondary seed culture medium do not contain methanol.

[0084] Comparative Example 5 The method for culturing the methanol-utilizing bacteria in this comparative example is the same as that used in Example 4, with the same raw materials, the same amount of each raw material, and the same preparation method. The only difference is that the concentration of methanol in the activation culture medium, the primary seed culture medium, and the secondary seed culture medium is 1.5% (v / v).

[0085] Comparative Example 6 The cultivation method of methanol-utilizing bacteria in this comparative example uses the same raw materials, the same amount of each raw material, and the same steps as in Example 4. The only difference is that the primary seed culture medium, the secondary seed culture medium, the activation culture medium, the primary seed culture medium, the secondary seed culture medium, and the fermentation culture medium do not contain glycine, taurine chelated calcium, inositol, betaine, or glutathione.

[0086] Comparative Example 7 The cultivation method of methanol-utilizing bacteria in this comparative example uses the same raw materials, the same amount of each raw material, and the same steps as in Example 4. The only difference is that in the primary seed culture medium, the secondary seed culture medium, and the fermentation culture medium, the taurine chelate calcium is replaced with taurine.

[0087] Comparative Example 8 The cultivation method of the methanol-utilizing bacteria in this comparative example uses the same raw materials, the same amount of each raw material, and the same steps as in Example 4. The only difference is that the taurine chelated calcium is replaced with calcium chloride in the primary seed culture medium, the secondary seed culture medium, and the fermentation culture medium.

[0088] Effect Experiment Example To verify the technical effectiveness of the methanol-utilizing bacteria culture method described in this invention, the following experiments were conducted: (1) Dry cell biomass and crude protein content Methanol-utilizing bacteria were cultured according to the methods of Examples 1-14 and Comparative Examples 1-8. The fermentation broth was collected and centrifuged at 4500 r / min for 10 min to collect the bacterial cells. The cells were washed and dried at 55°C until the mass was constant. The weight was then measured to obtain the methanol protein. The dry cell biomass (dry weight of cells produced per liter of fermentation broth, dry weight of cells / fermentation broth, unit g / L) was calculated. The crude protein content of the dry cells was determined according to the method of GB / T6432—2018.

[0089] (2) Methanol protein yield Methanol-utilizing bacteria were cultured according to the methods of Examples 1-14 and Comparative Examples 1-8. The fermentation broth was collected, and the methanol content in the fermentation broth was detected by liquid chromatography. The methanol protein yield was then calculated.

[0090] The methanol-protein yield is the mass of dry bacterial cells obtained per unit mass of methanol consumed, expressed as g dry bacterial cells / g methanol, and is calculated using the following formula: ; in, Y m W represents the methanol-protein yield. DCW This refers to the dry bacterial cell mass (g). The total amount of methanol consumed (g) during the fermentation process is calculated using the following formula: =W total -W res ; W total =K(V0×C0+V f ×C f ); W res =K·V e Ce; Among them, W total W represents the total mass of methanol used in the entire fermentation process. res V0 represents the mass of residual methanol at the end of fermentation; K is the conversion factor for methanol volume fraction to mass; V0 is the initial volume of fermentation broth (L); C0 is the initial methanol volume concentration of fermentation broth; V0 f Total volume (L) of supplemental culture medium; C f Ve is the methanol volume concentration in the fed medium; Ve is the total volume at the end of fermentation (L); and Ce is the residual methanol volume concentration at the end of fermentation.

[0091] (3) Bacterial growth Methanol-utilizing bacteria were cultured according to the methods of Examples 1-14 and Comparative Examples 1-8. The OD values ​​of the primary and secondary seed cultures after the culture were compared. 600 Conduct testing.

[0092] The results of the experiment are as follows:

[0093] Based on the above results, it can be seen that the method for cultivating methanol-utilizing bacteria described in this invention can achieve efficient synthesis of methanol protein by using methanol as a carbon source during the fermentation stage.

[0094] Based on the results of Examples 4 and 5, it can be seen that when the inoculation volume is equal and the bacterial density OD is... 600 In the case of similar secondary seed culture, adding taurine chelated calcium, betaine, and glutathione to the fermentation medium can increase the dry cell biomass, crude protein content, and methanol protein yield.

[0095] Based on the results of Examples 4, 6-14, and Comparative Example 6, it can be seen that, compared with Example 4, Example 6, which does not contain betaine, has a higher bacterial density (OD) in both the primary and secondary seed cultures. 600 All parameters decreased, including dry cell biomass, crude protein content, and methanol protein yield. In Example 11, where only betaine was added, the bacterial density OD of the primary and secondary seed cultures decreased compared to Comparative Example 6. 600 The results showed some improvement, with increased dry cell biomass, crude protein content, and methanol protein yield. This indicates that betaine plays a role in ensuring cell proliferation during the seed stage, thereby guaranteeing product synthesis during fermentation. In Example 7, which did not contain glutathione, cell proliferation decreased compared to Example 4, but the impact on cell proliferation was relatively small compared to Example 6, while the impact on crude protein content and methanol protein yield was significant. In Example 12, which only added glutathione, cell proliferation was slightly increased compared to Comparative Example 6, but crude protein content and methanol protein yield were significantly increased. This indicates that glutathione has a greater impact on protein synthesis and enzyme activity in the cells. In Example 8, which did not contain glycine, the bacterial density OD of the primary and secondary seed cultures was... 600 The changes were relatively small, with a slight decrease, but the dry cell biomass, crude protein content, and methanol protein yield all decreased significantly. This indicates that glycine, as an organic nitrogen source, affects protein synthesis efficiency. Example 9, which does not contain taurine chelated calcium, shows the bacterial density OD of the primary and secondary seed cultures. 600 All parameters were improved, but dry cell biomass, crude protein content, and methanol protein yield decreased. Compared to Comparative Example 6, Example 13, which contained only taurine chelated calcium, showed a higher bacterial density (OD) in both the primary and secondary seed cultures. 600No significant changes were observed, but the dry cell biomass, crude protein content, and methanol protein yield increased slightly. This indicates that taurine-chelated calcium can regulate the cell proliferation rate, preventing rapid proliferation using inorganic nitrogen sources, increasing cell numbers while decreasing intracellular material, and diverting carbon sources to catabolism. This stabilizes cell proliferation and metabolism, matching the synthesis rates of various metabolic pathways and enzyme systems, thereby achieving efficient methanol protein synthesis during fermentation. However, without the presence of regulators such as betaine, glutathione, and glycine to ensure cell proliferation, its inhibitory effect on excessive cell proliferation is difficult to exert effectively. Example 10, which does not contain inositol, shows the bacterial density OD of the primary and secondary seed cultures. 600 The levels decreased, with a significant decrease in crude protein content and methanol protein yield. In Example 14, which contained only inositol, compared to Comparative Example 6, the bacterial density OD of the primary and secondary seed cultures was significantly lower. 600 The levels of crude protein and methanol protein increased significantly. This indicates that inositol affects bacterial cell proliferation and thus the quality of the seed culture.

[0096] Based on the results of Example 4 and Comparative Example 6, it can be seen that the addition of inositol, glycine, taurine chelated calcium, betaine, and glutathione at different stages can play a comprehensive role in regulating cell growth, carbon and nitrogen metabolic flow and balance, thereby achieving efficient synthesis of methanol protein.

[0097] Based on the results of Example 4 and Comparative Examples 1-4, it is evident that the combination of inorganic nitrogen source and glycine significantly influences bacterial metabolism and synthesis. In Comparative Examples 1 and 2, ammonium salt was used as the inorganic nitrogen source in activation, primary seed culture, and secondary seed culture. Comparative Example 1 used a higher concentration of ammonium salt. The bacterial density OD of the primary and secondary seed cultures was [not specified]. 600 The levels of ammonium salts were increased, but the dry cell biomass, crude protein content, and methanol protein yield all decreased. Comparative Example 2 had the same ammonium salt level as Example 4, but without nitrate supplementation; the bacterial density OD of the primary and secondary seed cultures was [not specified]. 600 The levels of dry cell biomass, crude protein content, and methanol protein yield all decreased. In Comparative Example 3, during activation, primary seed culture, and secondary seed culture, a high concentration of ammonium salt was used as the inorganic nitrogen source, but glycine was added. The bacterial density OD of the primary and secondary seed cultures decreased. 600 The yield of methanol protein still increased significantly, while the dry cell biomass, crude protein content, and methanol protein yield all decreased markedly. However, the changes in Comparative Example 3 were smaller than those in Comparative Example 1, indicating that glycine played a regulatory role to some extent. Under the influence of high ammonium salt concentrations, the cells proliferated rapidly, increasing the cell number but decreasing intracellular material. Glycine improved the yield and quality of methanol protein to some extent by accelerating protein synthesis efficiency. At lower ammonium salt concentrations and without nitrate supplementation, the needs of cell proliferation and metabolic synthesis could not be fully met.

[0098] Based on the results of Example 4 and Comparative Examples 4-5, it is evident that adding a low concentration of methanol to the activation medium, the primary seed medium, and the secondary seed medium is more conducive to the synthesis of methanol protein by the bacteria. Comparative Example 4, which did not contain methanol, although it increased the bacterial density (OD) during seed culture... 600 However, the synthesis of methanol protein was poor when the bacteria were inoculated into the fermenter. In Comparative Example 5, with the addition of a high concentration of methanol, growth was inhibited during the seed culture stage, and the bacterial density (OD) decreased. 600 The levels are low, making it difficult to efficiently synthesize methanol protein during the fermentation stage.

[0099] Based on the results of Examples 4 and 9 and Comparative Examples 7-8, it can be seen that taurine and calcium ions can play a certain regulatory role in bacterial proliferation and inhibit the diversion of methanol dissimilatory pathway, but the effect is not ideal.

[0100] As is known from common technical knowledge, this invention can be implemented through other embodiments that do not depart from its spirit or essential characteristics. Therefore, the disclosed embodiments described above are merely illustrative in all respects and are not the only ones. All modifications within the scope of this invention or its equivalents are encompassed by this invention.

Claims

1. A method for culturing methanol-utilizing bacteria, characterized in that, Includes the following steps: (1) Take glucose-eating methyl bacteria, inoculate them in an activation medium, and culture for 22-26 hours to obtain an activated bacterial solution; The activation culture medium uses glucose and methanol as carbon sources and ammonium salts as inorganic nitrogen sources. (2) The activated bacterial solution obtained in step (1) is inoculated into the primary seed culture medium and cultured for 22-26 hours to obtain the primary seed solution; The primary seed culture medium uses glucose and methanol as carbon sources and ammonium salts and nitrates as inorganic nitrogen sources. (3) The primary seed culture obtained in step (2) is inoculated into the secondary seed culture medium and cultured for 22-26 hours to obtain the secondary seed culture. The secondary seed culture medium uses glucose and methanol as carbon sources and ammonium salts and nitrates as inorganic nitrogen sources. (4) Inoculate the secondary seed liquid obtained in step (3) into the fermentation medium and ferment until the dissolved oxygen value increases by more than 15%. Then add a feed medium containing methanol and make the methanol concentration in the fermentation medium 1.5-2.0% (v / v). Ferment for 60-72 hours to obtain the final product.

2. The method for culturing methanol-utilizing bacteria according to claim 1, characterized in that, The activation culture medium comprises: glucose 0.8-1.0 g / L, dipotassium hydrogen phosphate 0.8-1.2 g / L, potassium dihydrogen phosphate 0.1-0.2 g / L, ammonium sulfate 0.8-1.2 g / L, sodium chloride 0.4-0.6 g / L, magnesium sulfate heptahydrate 0.15-0.30 g / L, and methanol 0.2-0.4% (v / v); with a pH of 7.0-7.

5.

3. The method for culturing methanol-utilizing bacteria according to claim 2, characterized in that, The primary seed culture medium comprises: glucose 1.0-1.2 g / L, dipotassium hydrogen phosphate 1.2-1.4 g / L, potassium dihydrogen phosphate 0.3-0.4 g / L, ammonium sulfate 0.8-1.2 g / L, sodium nitrate 0.2-0.3 g / L, sodium chloride 0.4-0.6 g / L, magnesium sulfate heptahydrate 0.15-0.30 g / L, and methanol 0.5-0.7% (v / v); with a pH of 7.0-7.

5.

4. The method for culturing methanol-utilizing bacteria according to claim 3, characterized in that, The secondary seed culture medium comprises: glucose 1.2-1.5 g / L, dipotassium hydrogen phosphate 1.4-1.6 g / L, potassium dihydrogen phosphate 0.4-0.5 g / L, ammonium sulfate 0.8-1.2 g / L, sodium nitrate 0.4-0.5 g / L, sodium chloride 0.6-0.8 g / L, magnesium sulfate heptahydrate 0.15-0.30 g / L, and methanol 0.8-1.0% (v / v); with a pH of 7.0-7.

5.

5. The method for culturing methanol-utilizing bacteria according to claim 4, characterized in that, The fermentation medium comprises: glucose 1.2-1.5 g / L, dipotassium hydrogen phosphate 1.4-1.6 g / L, potassium dihydrogen phosphate 0.4-0.5 g / L, ammonium sulfate 0.8-1.2 g / L, sodium nitrate 0.6-0.8 g / L, sodium chloride 0.6-0.8 g / L, magnesium sulfate heptahydrate 0.15-0.30 g / L, and methanol 1.0-2.0% (v / v); the pH value is 7.0-7.

5.

6. The method for culturing methanol-utilizing bacteria according to claim 5, characterized in that, The activation culture medium, the primary seed culture medium, the secondary seed culture medium, and the fermentation culture medium also include trace elements; Optionally, the trace elements include one or more of ferrous sulfate heptahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, cobalt chloride hexahydrate, anhydrous copper sulfate, manganese chloride tetrahydrate, and boric acid. Optionally, the weight ratio of ferrous sulfate heptahydrate, zinc sulfate heptahydrate, sodium molybdate dihydrate, cobalt chloride hexahydrate, anhydrous copper sulfate, manganese chloride tetrahydrate, and boric acid is 10:(2-5):(0.08-0.12):(0.03-0.09):(0.5-0.8):(1-3):(0.2-0.8). Optionally, the final concentration of the ferrous sulfate heptahydrate in the culture medium is 2.5-3.5 mg / L.

7. The method for culturing methanol-utilizing bacteria according to claim 1, characterized in that, The activation culture medium, the primary seed culture medium, the secondary seed culture medium, and the fermentation culture medium include one or more of glycine, taurine chelated calcium, inositol, betaine, and glutathione.

8. The method for culturing methanol-utilizing bacteria according to claim 7, characterized in that, The activation culture medium contains 1-5 mg / L betaine and 2-5 mg / L glutathione. Optionally, the primary seed culture medium comprises 3-8 mg / L glycine, 7-12 mg / L taurine chelate calcium, 0.2-0.4 mg / L inositol, 5-10 mg / L betaine, and 5-10 mg / L glutathione. Optionally, the secondary seed culture medium includes 5-10 mg / L glycine, 10-15 mg / L taurine chelate calcium, 0.5-1.0 mg / L inositol, 10-15 mg / L betaine, and 10-20 mg / L glutathione. Optionally, the fermentation medium includes 10-15 mg / L of taurine chelate calcium, 15-20 mg / L of betaine, and 25-35 mg / L of glutathione.

9. The method for culturing methanol-utilizing bacteria according to claim 1, characterized in that, The feed medium comprises 8-12% (v / v) methanol, 1.4-1.6 g / L dipotassium hydrogen phosphate, 0.4-0.5 g / L potassium dihydrogen phosphate, 1.0-1.5 g / L ammonium sulfate, and 0.20-0.35 g / L magnesium sulfate heptahydrate; the pH value is 7.0-7.

5.

10. The method for culturing methanol-utilizing bacteria according to claim 1, characterized in that, In step (1), the glucose-eating methyl-eating bacteria are inoculated into an activation culture medium to achieve a viable count of 1 × 10⁻⁶. 6 CFU / mL - 8 × 10 6 CFU / mL, cultured with shaking at 27-37℃, with a shaking speed of 180-220 rpm; Optionally, in step (2), the activated bacterial solution is inoculated into the primary seed culture medium at an inoculation rate of 4-6%, and cultured by shaking at 27-37°C with a shaking speed of 180-220 rpm. Optionally, in step (3), the primary seed culture is inoculated into the secondary seed culture medium at an inoculation rate of 9-11%, and cultured by shaking at 27-37°C with a shaking speed of 180-220 rpm. Optionally, in step (4), the secondary seed liquid is inoculated into the fermentation medium at an inoculation rate of 4-6% and cultured at 27-37℃, a rotation speed of 200-300rpm, and an aeration rate of 1.2-1.8vvm.

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