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CN-121991827-A - Culture method of methanol utilization bacteria

CN121991827ACN 121991827 ACN121991827 ACN 121991827ACN-121991827-A

Abstract

The invention provides a method for culturing methanol-utilizing bacteria, which comprises the following steps of taking grape sugar food methyl bacteria, inoculating the grape sugar food methyl bacteria into an activation culture medium, culturing to obtain an activation bacterial liquid, inoculating the activation bacterial liquid into a first-stage seed culture medium, culturing to obtain a first-stage seed liquid, inoculating the first-stage seed liquid into a second-stage seed culture medium, culturing to obtain a second-stage seed liquid, inoculating the second-stage seed liquid into a fermentation culture medium, regulating the rotating speed to enable the initial dissolved oxygen value to be 15-25%, enabling the ventilation amount to be 1.2-1.8vvm, fermenting until the dissolved oxygen value is increased by more than 15%, supplementing a feed culture medium containing methanol, and enabling the concentration of the methanol in the fermentation culture medium to be 1.5-2.0% (v/v), thus obtaining the methanol-utilizing bacteria. The culture method of the methanol utilizing bacteria can efficiently synthesize methanol protein.

Inventors

  • WANG YUJIE
  • CHAO QIANWEN
  • KONG YAJING
  • CHANG YUFENG
  • Lei Lanyue
  • SUN JIAHUI
  • CHEN YUTONG

Assignees

  • 北京德量源生物科技有限公司

Dates

Publication Date
20260508
Application Date
20260214

Claims (10)

  1. 1. A method for culturing a methanol-utilizing bacterium, comprising the steps of: (1) Taking grape sugar food methyl bacteria, inoculating the grape sugar food methyl bacteria into an activation culture medium, and culturing for 22-26 hours to obtain an activation bacterial liquid; the activation culture medium takes glucose and methanol as carbon sources and ammonium salt as inorganic nitrogen sources; (2) Inoculating the activated bacterial liquid obtained in the step (1) into a primary seed culture medium, and culturing for 22-26 hours to obtain a primary seed liquid; the primary seed culture medium takes glucose and methanol as carbon sources, and ammonium salt and nitrate as inorganic nitrogen sources; (3) Inoculating the primary seed liquid obtained in the step (2) into a secondary seed culture medium, and culturing for 22-26 hours to obtain a secondary seed liquid; the secondary seed culture medium takes glucose and methanol as carbon sources, and ammonium salt and nitrate as inorganic nitrogen sources; (4) Inoculating the secondary seed liquid obtained in the step (3) into a fermentation culture medium, fermenting until the dissolved oxygen value rises by more than 15%, supplementing a feed culture medium containing methanol, and fermenting for 60-72h to obtain the final product, wherein the concentration of the methanol in the fermentation culture medium is 1.5-2.0% (v/v).
  2. 2. The method for culturing a methanol-utilizing bacterium according to claim 1, wherein the activation medium comprises glucose 0.8 to 1.0g/L, dipotassium hydrogen phosphate 0.8 to 1.2g/L, potassium dihydrogen phosphate 0.1 to 0.2g/L, ammonium sulfate 0.8 to 1.2g/L, sodium chloride 0.4 to 0.6g/L, magnesium sulfate heptahydrate 0.15 to 0.30g/L, methanol 0.2 to 0.4% (v/v), and pH 7.0 to 7.5.
  3. 3. The method for culturing a methanol-utilizing bacterium according to claim 2, wherein the primary seed medium comprises 1.0 to 1.2g/L glucose, 1.2 to 1.4g/L dipotassium hydrogen phosphate, 0.3 to 0.4g/L potassium dihydrogen phosphate, 0.8 to 1.2g/L ammonium sulfate, 0.2 to 0.3g/L sodium nitrate, 0.4 to 0.6g/L sodium chloride, 0.15 to 0.30g/L magnesium sulfate heptahydrate, 0.5 to 0.7% (v/v) methanol, and a pH value of 7.0 to 7.5.
  4. 4. The method for culturing a methanol-utilizing bacterium according to claim 3, wherein the secondary seed medium comprises 1.2 to 1.5g/L glucose, 1.4 to 1.6g/L dipotassium hydrogen phosphate, 0.4 to 0.5g/L potassium dihydrogen phosphate, 0.8 to 1.2g/L ammonium sulfate, 0.4 to 0.5g/L sodium nitrate, 0.6 to 0.8g/L sodium chloride, 0.15 to 0.30g/L magnesium sulfate heptahydrate, 0.8 to 1.0% (v/v) methanol, and a pH of 7.0 to 7.5.
  5. 5. The method for culturing a methanol-utilizing bacterium according to claim 4, wherein the fermentation medium comprises 1.2 to 1.5g/L glucose, 1.4 to 1.6g/L dipotassium hydrogen phosphate, 0.4 to 0.5g/L potassium dihydrogen phosphate, 0.8 to 1.2g/L ammonium sulfate, 0.6 to 0.8g/L sodium nitrate, 0.6 to 0.8g/L sodium chloride, 0.15 to 0.30g/L magnesium sulfate heptahydrate, 1.0 to 2.0% (v/v) methanol, and a pH of 7.0 to 7.5.
  6. 6. The method according to claim 5, wherein the activation medium, the primary seed medium, the secondary seed medium, and the fermentation medium further include trace elements; Optionally, the microelements comprise 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 the ferrous sulfate heptahydrate, the zinc sulfate heptahydrate, the sodium molybdate dihydrate, the cobalt chloride hexahydrate, the anhydrous copper sulfate, the manganese chloride tetrahydrate and the 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 2.5-3.5mg/L.
  7. 7. The method according to claim 1, wherein the activation medium, the primary seed medium, the secondary seed medium, and the fermentation medium include one or more of glycine, calcium taurate chelate, inositol, betaine, and glutathione.
  8. 8. The method for culturing a methanol-utilizing bacterium according to claim 7, wherein the activation medium contains 1 to 5mg/L of betaine and 2 to 5mg/L of glutathione; Optionally, the primary seed culture medium comprises 3-8mg/L glycine, 7-12mg/L taurine chelated calcium, 0.2-0.4mg/L inositol, 5-10mg/L betaine and 5-10mg/L glutathione; Optionally, the secondary seed culture medium comprises 5-10mg/L glycine, 10-15mg/L taurine chelated calcium, 0.5-1.0mg/L inositol, 10-15mg/L betaine and 10-20mg/L glutathione; Optionally, 10-15mg/L of taurine chelated calcium, 15-20mg/L of betaine and 25-35mg/L of glutathione are included in the fermentation medium.
  9. 9. The method for culturing a methanol-utilizing bacterium according to claim 1, wherein the feed medium comprises 8 to 12% (v/v) methanol, 1.4 to 1.6g/L dipotassium hydrogen phosphate, 0.4 to 0.5g/L potassium dihydrogen phosphate, 1.0 to 1.5g/L ammonium sulfate, 0.20 to 0.35g/L magnesium sulfate heptahydrate, and has a pH value of 7.0 to 7.5.
  10. 10. The method according to claim 1, wherein in the step (1), the grape sugar-consuming food methyl bacteria are inoculated into an activation medium so that the number of living grape sugar-consuming food methyl bacteria is 1 x 10 6 CFU/mL-8×10 6 CFU/mL, and the culture is carried out by shaking at 27-37 ℃ at a shaking table rotation speed of 180-220rpm; Optionally, in the step (2), the activated bacteria liquid is inoculated into a primary seed culture medium in an inoculation amount of 4-6%, and is subjected to shaking culture at a temperature of 27-37 ℃ and a shaking table rotating speed of 180-220rpm; optionally, in the step (3), the primary seed liquid is inoculated into a secondary seed culture medium in an inoculation amount of 9-11%, and is subjected to shaking culture at a temperature of 27-37 ℃ and a shaking table rotating speed of 180-220rpm; Optionally, in the step (4), the secondary seed liquid is inoculated into a fermentation medium in an inoculum size of 4-6%, and is cultured at a temperature of 27-37 ℃ at a rotating speed of 200-300rpm and a ventilation amount of 1.2-1.8 vvm.

Description

Culture method of methanol utilization bacteria Technical Field The invention relates to the technical field of microorganism culture, in particular to a method for culturing methanol utilization bacteria. Background Methanol is an important organic chemical basic raw material, and can be produced by adopting various raw materials (such as natural gas, coal, oil, acetylene tail gas and the like). At present, the related synthesis technology of preparing methanol from coal and preparing methanol from natural gas is mature, the cost of methanol is gradually reduced, and the development of downstream products with high added value is significant. The methanol utilizing bacteria belong to methyl nutrition type bacteria, can utilize methanol to obtain energy, can convert the methanol into mycoprotein and synthesize various secondary metabolites. Among these products, the feed-grade methanol protein produced by using methanol as a raw material has a wide application prospect in relieving the shortage of feed protein due to low raw material cost, high protein content and no dependence on grain resources. At present, common microorganisms capable of synthesizing methanol protein by taking methanol as a carbon source comprise pichia pastoris, corynebacterium glutamicum, grape sugar food methyl bacteria and the like. In the methanol metabolic pathway of the grape sugar-eating methyl bacteria, methanol is first oxidized to formaldehyde. Part of formaldehyde enters the catabolism path and is further oxidized into carbon dioxide and water, and the other part of formaldehyde enters the anabolism path to promote the growth and metabolism of thalli. However, the methyl alcohol metabolic pathway of grape sugar food methyl bacteria is complex and difficult to regulate, and methanol itself has biotoxicity, so that the carbon loss is large, and the efficient and directional conversion of the methanol metabolic flow into mycoprotein can not be realized, so that the synthesis efficiency of the methanol protein is lower. Disclosure of Invention The invention aims to provide a method for culturing methanol-utilizing bacteria, which aims to solve the problem of low synthesis efficiency of methanol protein in the prior art. The invention provides the following technical scheme: a method for culturing methanol-utilizing bacteria comprises the following steps: (1) Taking grape sugar food methyl bacteria, inoculating the grape sugar food methyl bacteria into an activation culture medium, and culturing for 22-26 hours to obtain an activation bacterial liquid; the activation culture medium takes glucose and methanol as carbon sources and ammonium salt as inorganic nitrogen sources; (2) Inoculating the activated bacterial liquid obtained in the step (1) into a primary seed culture medium, and culturing for 22-26 hours to obtain a primary seed liquid; the primary seed culture medium takes glucose and methanol as carbon sources, and ammonium salt and nitrate as inorganic nitrogen sources; (3) Inoculating the primary seed liquid obtained in the step (2) into a secondary seed culture medium, and culturing for 22-26 hours to obtain a secondary seed liquid; the secondary seed culture medium takes glucose and methanol as carbon sources, and ammonium salt and nitrate as inorganic nitrogen sources; (4) Inoculating the secondary seed liquid obtained in the step (3) into a fermentation culture medium, fermenting until the dissolved oxygen value rises by more than 15%, supplementing a feed culture medium containing methanol, and fermenting for 60-72h to obtain the final product, wherein the concentration of the methanol in the fermentation culture medium is 1.5-2.0% (v/v). Preferably, in the step (1), the activation culture medium comprises 0.8-1.0g/L of glucose, 0.8-1.2g/L of dipotassium hydrogen phosphate, 0.1-0.2g/L of monopotassium phosphate, 0.8-1.2g/L of ammonium sulfate, 0.4-0.6g/L of sodium chloride, 0.15-0.30g/L of magnesium sulfate heptahydrate, 0.2-0.4% (v/v) of methanol and the pH value is 7.0-7.5. Preferably, the primary seed culture medium comprises 1.0-1.2g/L of glucose, 1.2-1.4g/L of dipotassium hydrogen phosphate, 0.3-0.4g/L of monopotassium phosphate, 0.8-1.2g/L of ammonium sulfate, 0.2-0.3g/L of sodium nitrate, 0.4-0.6g/L of sodium chloride, 0.15-0.30g/L of magnesium sulfate heptahydrate, 0.5-0.7% (v/v) of methanol and 7.0-7.5 of pH value. Preferably, the secondary seed culture medium comprises 1.2-1.5g/L glucose, 1.4-1.6g/L dipotassium hydrogen phosphate, 0.4-0.5g/L monopotassium phosphate, 0.8-1.2g/L ammonium sulfate, 0.4-0.5g/L sodium nitrate, 0.6-0.8g/L sodium chloride, 0.15-0.30g/L magnesium sulfate heptahydrate, 0.8-1.0% (v/v) methanol and 7.0-7.5 pH value. Preferably, the fermentation medium comprises 1.2-1.5g/L glucose, 1.4-1.6g/L dipotassium hydrogen phosphate, 0.4-0.5g/L monopotassium phosphate, 0.8-1.2g/L ammonium sulfate, 0.6-0.8g/L sodium nitrate, 0.6-0.8g/L sodium chloride, 0.15-0.30g/L magnesium sulfate heptahydrate,