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WO2012174978A1 - Procédés d'amélioration des souches et d'optimisation de la fermentation mixte en deux étapes de la vitamine c - Google Patents

Procédés d'amélioration des souches et d'optimisation de la fermentation mixte en deux étapes de la vitamine c Download PDF

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Publication number
WO2012174978A1
WO2012174978A1 PCT/CN2012/076310 CN2012076310W WO2012174978A1 WO 2012174978 A1 WO2012174978 A1 WO 2012174978A1 CN 2012076310 W CN2012076310 W CN 2012076310W WO 2012174978 A1 WO2012174978 A1 WO 2012174978A1
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Prior art keywords
gluconobacter oxydans
medium
solution
bacillus megaterium
seed
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PCT/CN2012/076310
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English (en)
Chinese (zh)
Inventor
元英进
邹旸
马倩
高赟
杜瑾
杨洁
李霞
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Tianjin University
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Tianjin University
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Priority claimed from CN2011101656813A external-priority patent/CN102331474B/zh
Priority claimed from CN2011103156412A external-priority patent/CN103045710A/zh
Priority claimed from CN 201110314740 external-priority patent/CN102352403B/zh
Priority claimed from CN201110346058.8A external-priority patent/CN102520184B/zh
Priority claimed from CN2011104070781A external-priority patent/CN102492762A/zh
Priority claimed from CN201210044060.4A external-priority patent/CN102586386B/zh
Priority claimed from CN2012100494407A external-priority patent/CN102590324A/zh
Priority claimed from CN2012101096286A external-priority patent/CN102634562A/zh
Priority claimed from CN201210148033.1A external-priority patent/CN102680562B/zh
Priority to DE112012002557.1T priority Critical patent/DE112012002557B4/de
Application filed by Tianjin University filed Critical Tianjin University
Publication of WO2012174978A1 publication Critical patent/WO2012174978A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/58—Aldonic, ketoaldonic or saccharic acids
    • C12P7/60—2-Ketogulonic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/11—Bacillus megaterium

Definitions

  • the invention belongs to the field of industrial microorganisms.
  • the present invention relates to a method for detecting changes in proteins, phospholipid groups, nutrient environments, and small molecule metabolites during the passage of vitamin C industrial production strains.
  • the invention also relates to a method for increasing the industrial yield of the precursor 2-keto-L-gulonic acid of vitamin C, comprising the method of evolutionary subculture of mixed bacteria, enhancing the interaction of two bacteria to increase the yield of 2-keto-L-gulonic acid, and Molecular biological genetic modification methods and methods of adding glutathione.
  • the present invention also provides quality control indicators for raw corn syrup for fermentation. Background technique
  • vitamin C is a highly effective antioxidant, which is involved in the important biosynthesis process of human metabolism and is essential for maintaining nutrients and growth. It also has a wide range of applications in the areas of pharmaceuticals, food and cosmetics.
  • the method for producing vitamin C in China is “two-step fermentation method”.
  • the first step of fermentation uses black bacillus to convert behenyl alcohol into L-behenylose
  • the second step is fermentation of Bacillus megaterium and Gluconobacter oxydans. Fermentation, conversion of hawthorn sugar to the precursor of vitamin C 2-keto-L-gulonic acid.
  • Gluconobacter oxydans is an acid-producing bacterium
  • Bacillus megaterium is a companion. If Gluconobacter oxydans is used alone, growth is not carried out.
  • the communication between them is first transmitted to the intracellular space through the cell membrane that senses the external signal, and a series of signal events, including changes in intracellular proteins and changes in the phospholipid group of the cell membrane, are generated.
  • the nutrient environment of the culture fluid is constantly changing, and then transmitted to the cells, resulting in a series of different growth fermentation behaviors. The study and determination of these changes will provide useful information for revealing the mechanisms by which subcultures enhance the interaction of two bacteria to promote acid production, and provide support for further optimization of the production process.
  • the fermented raw corn syrup also has a great influence on the fermented product.
  • Corn syrup is a by-product obtained during the impregnation of corn during the production of corn starch. It contains a variety of nutrients such as amino acids, organic acids, soluble sugars, vitamins and metal salts. It is an important raw material in the production of antibiotics and other media, providing a natural source of organic nitrogen for microbial growth. Due to its low price and rich nutrition, it is widely used in industrial microbial fermentation. Application. The existing technical quality testing methods for corn syrup are prescribed by each manufacturer and there is no national uniform standard.
  • a first aspect of the invention provides a method for increasing the yield of 2-keto-L-gulonic acid by subculture of mixed bacteria, comprising the steps of:
  • the Bacillus megaterium and Gluconobacter oxydans cultured in step (1) were separately transferred to seed medium at 28-35.
  • C 200-280 r / min shaker shaking culture, 24h-48h, obtaining Bacillus megaterium seed solution and Gluconobacter oxydans seed solution;
  • the subcultured mixed strain obtained in the step (2) is streaked and purified, and then inoculated on a solid medium, and cultured at 28-35 e C for 24-48 hours; and then transferred to a seed medium at 28-35.
  • C 200-280 r / min shaker shaking culture for 24h-48h, the evolution of the Bacillus megaterus seed solution and the evolved Gluconobacter oxydans seed solution;
  • the solid medium is prepared by weighing: 10-50 g of L-xanthine, 2-10 g of corn syrup, 2-10 g of beef bone, 2-10 g of yeast dip powder, 0.5-5 g of urea, 2-12 g of peptone, agar 10-50g, KH 2 PO 4 0.5-5g, MgSO 4 0.1-0.7g, CaCO 3 0.5-5g, add water to 1L, adjusted to pH 6.5-7.0, 121. C was sterilized for 20 min to prepare a solid medium.
  • the solid medium was prepared by weighing: 20 g of L-xanthine, 3 g of corn syrup, 3 g of beef bone, 3 g of yeast dipping powder, urea lg, peptone 10 g , agar 20 g , KH 2 P0 4 lg, MgSO 4 0.2 g, CaC0 3 lg, water was added to 1 L, pH was adjusted to 6.8, and sterilized at 121 ° C for 20 min to prepare a solid medium.
  • the seed culture medium is prepared as follows: Weigh 10-50 g of L-xanthine, 2-10 g of corn syrup, 2-10 g of beef bone, 2-10 g of yeast dip powder, 0.5-5 g of urea, 2-12 g of peptone, KH 2 PO 4 0.5-5g, MgSO 4 0.1-0.7g, CaCO 3 0.5-5g, add water to 1L, adjusted to pH 6.5-7.0, 121. C was sterilized for 20 min to prepare a seed medium.
  • Preferred prepared seed medium is: Weigh scale mountain Qian L- sugar 20g, corn syrup 3 g, beef bone 3 g, yeast extract 3 g, urea lg, peptone 10 g, KH 2 P0 4 l g, MgSO 4 0.2 g, CaCO 3 lg, water was added to 1 L, and the pH was adjusted to 6.8, 121. C was sterilized for 20 min to prepare a seed medium.
  • the fermentation medium is prepared as follows: L-xanthose 40-120g, corn syrup is weighed proportionally 10-50 g, urea 10-25 g, KH 2 PO 4 0.5-3 g, MgSO 4 0.2-1.2 g, CaCO 3 0.5-5 g Water was added to 1 L, and the pH was adjusted to 6.5-7.5, 121. C was sterilized for 20 min to prepare a fermentation medium.
  • the preparation of the fermentation medium is preferably: weigh 80 g of L-wheat sugar, 20 g of corn syrup, 12 g of urea, 12 g of urea, KH 2 P0 4 lg, MgSO 4 0.5 g, CaC0 3 lg, add water to 1 L, adjust the pH to 7.0. , 121. C was sterilized for 20 min to prepare a fermentation medium.
  • a second aspect of the invention provides a method of enhancing the interaction of two bacteria to increase the yield of 2-keto-L-gulonic acid, comprising the steps of:
  • the Bacillus megaterium and Gluconobacter oxydans cultured in step (1) were separately transferred to seed medium at 28-35.
  • C 200-280 r / min shaker shaking culture, 24h-48h, obtaining Bacillus megaterium seed solution and Gluconobacter oxydans seed solution;
  • the subcultured mixed strain obtained in the step (2) is streaked and purified, and then inoculated on a solid medium, and cultured at 28-35 e C for 24-48 hours; and then transferred to a seed medium at 28-35.
  • C 200-280 r / min shaker shaking culture for 24h-48h, the evolution of the Bacillus megaterus seed solution and the evolved Gluconobacter oxydans seed solution;
  • the evolved Bacillus megaterium and the evolved Gluconobacter oxydans were inoculated into a fermentation medium, and the density of the evolved Bacillus megaterium was 2 ⁇ 10 7 -2 ⁇ 10 10 cfu/ml, and the evolved oxidized glucose was obtained.
  • the density of the bacillus is xloS xloUcfu/ml, at 28-35.
  • C 200-280 r/min shaker shaking culture for 72h-96h, obtaining 2-keto-L-gulonic acid or inoculating the original Bacillus megaterium and evolved Gluconobacter oxydans into the fermentation medium, so that The density of B.
  • megaterium was 2xl0 7 -2> ⁇ 10 1() cfu/ml
  • density of the evolved Gluconobacter oxydans was 2x10 7 -2xl 9 cfu/ml at 28-35.
  • C 200-280 r / min shaker shaking culture for 72h-96h, to obtain 2-keto-L-gulonic acid.
  • the solid medium is prepared by weighing: 10-50 g of L-xanthine, 2-10 g of corn syrup, 2-10 g of beef bone, 2-10 g of yeast dip powder, 0.5-5 g of urea, 2-12 g of peptone, agar 10-50g, KH 2 PO 4 0.5-5g, MgSO 4 0.1-0.7g, CaCO 3 0.5-5g, add water to 1L, adjusted to pH 6.5-7.0, 121. C was sterilized for 20 min to prepare a solid medium.
  • the solid medium was prepared by weighing: 20 g of L-xanthine, 3 g of corn syrup, 3 g of beef bone, 3 g of yeast dipping powder, urea lg, peptone 10 g , agar 20 g , KH 2 P0 4 lg, MgSO 4 0.2 g, CaC0 3 lg, water was added to 1 L, pH was adjusted to 6.8, and sterilized at 121 ° C for 20 min to prepare a solid medium.
  • the seed medium is prepared as follows: 10-50 g of L-xanthine, 2-10 g of corn syrup, 2-10 g of beef bone, 2-10 g of yeast dip powder, 0.5-5 g of urea, and 2-12 g of peptone. KH 2 PO 4 0.5-5g, MgSO 4 0.1-0.7g, CaCO 3 0.5-5g, add water to 1L, adjusted to pH 6.5-7.0, 121. C was sterilized for 20 min to prepare a seed medium.
  • Preferred prepared seed medium is: Weigh scale mountain Qian L- sugar 20g, corn syrup 3 g, beef bone 3 g, yeast extract 3 g, urea lg, peptone 10 g, KH 2 P0 4 l g, MgSO 4 0.2 g, CaCO 3 lg, water was added to 1 L, and the pH was adjusted to 6.8, 121. C was sterilized for 20 min to prepare a seed medium.
  • the fermentation medium is prepared as follows: 40-120 g of L-xanthose, 10-50 g of corn syrup, 10-25 g of urea, KH 2 PO 4 0.5-3 g, MgSO 4 0.2-1.2 g, CaCO 3 0.5-5 g Add water to 1 L and adjust the pH to 6.5 - 7.5, 121. C was sterilized for 20 min to prepare a fermentation medium.
  • the preparation of the fermentation medium is preferably: weigh 80 g of L-wheat sugar, 20 g of corn syrup, 12 g of urea, 12 g of urea, KH 2 P0 4 lg, MgSO 4 0.5 g, CaC0 3 lg, add water to 1 L, adjust the pH to 7.0. , 121. C was sterilized for 20 min to prepare a fermentation medium.
  • a third aspect of the invention provides a method for detecting changes in different algebraic proteins of a vitamin C industrial mixed strain, comprising the steps of:
  • the Bacillus megaterium and Gluconobacter oxydans cultured in the step (1) 1 were separately transferred to a seed medium at 28-35.
  • C 200-280 r / min shaker shaking culture for 24h-48h, obtaining Bacillus megaterium seed solution and Gluconobacter oxydans seed solution;
  • the 3-4 samples of the mixed cells obtained in the step (1) 2 were streaked and separately inoculated on a solid medium, 28-35.
  • the evolved Bacillus megaterium and the evolved Gluconobacter oxydans obtained in the step (1) 3 were inoculated separately into the seed culture medium, and the density of the evolved Bacillus megaterium was 2 ⁇ 10 7 -2 ⁇ 10 W CFU/mL, and evolved.
  • the density of Gluconobacter oxydans is 2x10 8 -2 x 10 n CFU / mL, at 28-35. C, 200-280 r / min shaker shaking culture for 10-15h;
  • each 80-100mg is placed in a centrifuge tube, add 0.5 ⁇ 1mL cell lysate to each tube, mix well, intermittent ultrasonic sonication on ice for 20 ⁇ 50s; add 5 ⁇ 15 ⁇ mass ratio is 2 ⁇ 4 :1
  • C to stand the reaction for 10 ⁇ 30min; add 5 ⁇ 15 ⁇ 80 ⁇ 120mM benzyl sulfonyl isopropyl alcohol solution, 4 .
  • C is allowed to stand for l ⁇ 3h; centrifuge at 15000rpm for 25 ⁇ 40min; take the supernatant to obtain protein solution;
  • the cell lysate is: 8 mol-L 1 urea, 4% by mass of 3-[(3-cholamidopropyl)-diethylamine]-propanesulfonic acid, 40 mM Tris, the balance being water ;
  • the bovine serum albumin was added to the Coomassie Brilliant Blue G-250 solution from a low concentration to a high concentration using a Bradford kit, and the absorbance at 595 nm of each protein solution obtained in the step (2) 2 was determined to establish a standard curve; The concentration of the solution protein;
  • Step (2) were taken protein containing 50-100 ⁇ ⁇ 2 respective protein solution obtained, was added to each 4 to 6 volumes of -20.
  • C ⁇ -40. C acetone, at -20.
  • C ⁇ -40. Place under the conditions of C for 12 ⁇ 20h; centrifuge, discard the supernatant, and use -20 for precipitation.
  • C ⁇ -40. Washing with a volumetric concentration of C of 70%-85% in acetone; drying to obtain a dry protein powder;
  • the two groups of the mixture obtained in (2)7 were subjected to Q-Tof mass identification to obtain a protein spectrum, and differentially expressed proteins of each mixed group sample were obtained by quantitative determination;
  • the candidate differential protein content obtained in step (3) is plotted according to time series, and the regularity of these protein changes is observed and analyzed, and then the mixed culture evolution culture is found to improve the production of 2-keto-L-gulonic acid by Gluconobacter oxydans. The role.
  • a fourth aspect of the invention provides a method of analyzing a change in a phospholipid group during the passage of a vitamin C producing strain, comprising the steps of:
  • the Bacillus megaterium and the Gluconobacter oxydans cultured in the step (1) are respectively transferred into a seed culture medium, and shake cultured at 28-35 ° C, shaking at 200-280 rpm for 24 h to 48 h to obtain a Bacillus megaterus seed solution and Gluconobacter oxydans seed solution;
  • the Bacillus megaterium and Gluconobacter oxydans was inoculated into a new seed medium, the density of the Bacillus megaterium is 2xl0 7 -2xl0 10 cfu / mL, so that the density of Gluconobacter oxydans 2xl0 8 _2xlO u cfu / mL, Shake the culture at 28-35 ° C, shaking at 200-280 rpm, using 24h-48h as the passage cycle, and inserting the new seed medium with the volume ratio of 1%-10% for passage, passaging for 100-150 days, 0-100 or 0-150 days to choose 3-5 ⁇ time to take 3-5 samples;
  • the 3-5 subculture strains obtained in the step (2) are streaked and purified, and then inoculated on a solid medium, cultured at 28-35 ° C for 24 h to 48 h; and then transferred to the seed medium, respectively.
  • the cultured Bacillus megaterium seed solution and the evolved Gluconobacter oxydans seed solution were obtained by shaking culture at 28-35 ° C, shaking at 200-280 rpm for 24 h_48 h;
  • the evolved Bacillus megaterium, the evolved Gluconobacter oxydans, and the mixed evolved Bacillus megaterium and the evolved Gluconobacter oxydans were respectively inoculated into a fermentation medium to make the evolved Bacillus megaterium Density is 2 ⁇ 10 7 -2 ⁇ 10 1 ⁇ Cfu/mL, the density of the evolved Gluconobacter oxydans is 2X108-2X10 11 cfu/mL, and shaken at 28-35 ° C, shaking at 200-280 rpm for 10 h-15 h;
  • step 2 The cells obtained in step 1 are made into a dry powder, and 200-300 mg of cell dry powder is weighed, placed in a centrifuge tube A, and added with 0.5-0.8 mL of ultrapure water, and thoroughly mixed;
  • step 3 Repeat step 3 2-4 times until the cells are completely settled
  • the total phospholipid extraction mixture is dissolved in 0.2-2 mL of the stock solution to prepare a sample, and stored at -40 ° C or below;
  • the phospholipid standard is added to the sample, so that the final concentration of the phospholipid standard is 0.5-1.5 g / mL;
  • the extract is a chloroform/methanol solution containing dibutylhydroxytoluene, the volume ratio of the chloroform/methanol is 1-2:1, and the mass fraction of the dibutylhydroxytoluene is 0.005-0.01%;
  • the storage solution is a chloroform/methanol solution containing dibutylhydroxytoluene, the volume ratio of the chloroform/methanol is 1-4:1, and the mass fraction of the dibutylhydroxytoluene is 0.005-0.01%;
  • the sample containing the phospholipid standard obtained in the step (5) was tested by LC-MS to obtain the molecular structure and content data of the phospholipid group in the passage process of the vitamin C producing strain;
  • the molecular weight data of the phospholipid group of the vitamin C producing strain obtained in the step (6) were subjected to multivariate statistical analysis to obtain differential phospholipid molecular markers for distinguishing the vitamin C producing strains of different passage times;
  • the content of the differential phospholipid molecular markers obtained in step (7) is plotted according to different passage times, and the changes of these brick lipid molecules are observed and analyzed, and the phospholipid molecules which play a key role in the subculture process of mixed bacteria and related are found.
  • the metabolic pathway provides direction for strain modification and optimization of culture conditions for the purpose of improving 2-keto-L-gulonic acid.
  • the method of making the cells into a dry powder is preferably grinding the cells in a mortar using liquid nitrogen.
  • the distillation in the step (5) 7 is preferably a vacuum distillation at 30 to 40 °C.
  • the phospholipid standard is at least two of phosphatidylglycerol, phosphatidylethanolamine, hemolytic phosphatidylethanolamine, and phosphatidic acid.
  • the phosphatidylglycerol, phosphatidylethanolamine, hemolytic phosphatidylethanolamine or phospholipid has a fatty acid hydrophobic tail length of from 10 to 20 carbon atoms per strip.
  • the LC-MS detection conditions are preferably:
  • Injection volume 10 ⁇ ;
  • Mobile phase A (%): chloroform (89.5), methanol (10), ammonium hydroxide (0.5); mobile phase B (%): chloroform (55), methanol (39), ammonium hydroxide (0.5), water ( 5.5);
  • Ionization mode ESI (negative ion mode);
  • Ion source temperature 100 ° C;
  • Desolvent gas flow 400 L/Hr;
  • Inlet/outlet energy 50;
  • HM1/LM1/HM2/LM2 15.0;
  • the multivariate statistical analysis method performs principal component analysis after Pareto pretreatment.
  • the invention provides a method for analyzing the changes of phospholipid groups in the process of vitamin C production strains, which involves the extraction and analysis of total phospholipids of cells, and the multi-statistical method for analyzing the obtained phospholipidomics data through the vitamins of different passage numbers.
  • the qualitative and quantitative analysis of the phospholipid molecules of the C-producing strains under separate and mixed culture conditions, respectively, and the analysis of phospholipid molecules and metabolic pathways associated with enhancing the interaction between the two strains of vitamin C production strains provide a method for improving 2-ketone It is of great significance to modify the strains and optimize the culture conditions for the purpose of basal-L-gulonic acid.
  • a fifth aspect of the invention provides a method for detecting a change in a nutrient environment during the passage of a vitamin C producing strain, comprising the steps of:
  • the Bacillus megaterium and Gluconobacter oxydans cultured in the step (1) 1 were separately transferred to a seed medium at 28-35.
  • the 3-4 samples of the mixed cells obtained in the step (1) 2 were streaked and purified, and then inoculated separately on a solid medium, 28-35.
  • the Bacillus megaterium and Gluconobacter oxydans obtained in the step (1) 3 and the mixed bacteria in which the two bacteria are mixed together are inoculated into a new seed medium, so that the density of Bacillus megaterium is 2 ⁇ 10 7 -2 ⁇ 10 10
  • the density of CFU/mL, Gluconobacter oxydans is 2xl0 8 -2xl n n CFU/mL, at 28-35.
  • step (2) obtained filtrate 10-50 in a centrifuge tube, add 50-200 ⁇ , 0.04-0.14 mg / ml ⁇ mark succinic acid methanol solution as an internal standard, freeze-dry; add 40-100 A pyridine solution of methoxyamine hydrochloride at a concentration of 20 mg/mL was at 30. C-40.
  • the hydration reaction in a C water bath is 60-120 min; and 50-100 LN-methyl-N-trimethylsilane trifluoroacetamide is added to 35.
  • C -40 C water bath for silanization reaction 30-60 min;
  • the sample obtained in step (2) 2 is introduced into the gas phase color, and the color column is For DB-5MS, the color column has a specification of 30 mx 0.25 mm id and an inlet temperature of 250. C-280.
  • the carrier gas is high-purity helium
  • the flow rate is 0.6-0.8ml/min
  • the split ratio is 3: 1-20: 1.
  • the temperature rise program of the column oven is: initial 50. C-80. C, hold 2 min-5 min, 4 ° C / min -8. The speed of C/min rose to 260. C-300.
  • EI ionization source source temperature 230 °C-260 °C, detector voltage 2300 V-2700V, ionization voltage 60 eV-80 eV, current 30 ⁇ -50 ⁇ ; quality transfer detection Range 50-800 m/z; Identification of nutrient environmental substances using the NIST 2005 database, quality data processing and relative determination of nutrient environmental substances using Masslynx 4.1 software; and by integrating the peak area of the chromatogram, and with the internal standard The peak area is controlled to obtain the relative content of nutrient environmental substances;
  • the relative content of the differential nutrient environmental markers was plotted according to different passage times. The changes of these substances were observed and analyzed, and the changes of nutrient environment during the passage of vitamin C production strains were detected.
  • a sixth aspect of the invention provides an assay for detecting a vitamin C producing strain
  • a method for small molecule metabolite changes during subculture comprising the following steps:
  • Gluconobacter oxydans stored in a glycerin aqueous solution having a volume concentration of 15-30% and Bacillus megaterium deposited in a lactic acid aqueous solution having a volume concentration of 15-30% were inoculated into solid culture respectively.
  • the Bacillus megaterium and Gluconobacter oxydans cultured in the step (1) 1 were respectively transferred into a seed culture medium, and shake cultured at 28-35, 200-280 rpm for 24 to 48 hours, respectively, to obtain a Bacillus megaterus seed solution and Gluconobacter oxydans seed solution;
  • the 3-5 samples of the mixed cells obtained in the step (1) 2 are streaked and purified, and then inoculated on a solid medium, and cultured in 28-35 for 24-48 hours; and then transferred to a new seed medium, respectively.
  • the evolved Bacillus megaterium seed solution and Gluconobacter oxydans seed solution were separately obtained; and stored in a glycerin aqueous solution having a volume concentration of 15-30%;
  • the evolved Bacillus megaterium and Gluconobacter oxydans obtained in the step (1) 3 and the mixed bacteria in which the two bacteria are mixed together are inoculated into a new seed medium.
  • the density of the evolved Bacillus megaterium is xlO ⁇ xli ⁇ CFU/mL
  • the density of the evolved Gluconobacter oxydans is 2x10 8 -2x10 n CFU/mL, shaking at 28-35, 200-280 rpm shaker. Cultivate for 10-15 h;
  • the cells obtained in the step (2) 1 are made into a dry powder, and 30-60 mg of the cell dry powder are weighed, respectively, placed in three centrifuge tubes, and then 0.5-1.5 mL of the extract is added, and the concentration of 30-70 ⁇ is 0.020-0.060.
  • step (2) 2 to obtain three centrifuge tubes, respectively, add 40-100 concentration of 10-30 mg / mL of methamine hydrochloride pyridine solution in a water bath "chemical reaction 60-120 mill; then join 50-100 ⁇ ⁇ -methyl- ⁇ -trimethylsilane trifluoroacetamide in a 35-40 water bath for silanization reaction 30-60 min;
  • the extract is an aqueous methanol solution having a volume fraction of 50-70%;
  • the sample obtained in step (2)3 is introduced into the gas phase color, the color transfer column is DB-5MS, the color column is 3 m ⁇ ⁇ 30 m 0.25 mm id, the inlet temperature is ISO -ISOX , carrier gas For high purity helium, constant pressure 80-100KPa, split ratio 3: 1-20: 1, column oven heating procedure is: initial ⁇ - ⁇ , hold 3 min-6 min, to 4* ⁇ /min-S / Min rises to ⁇ ⁇ for 3 min-8 min, using EI ionization source, source temperature 230* ⁇ -260 ⁇ ⁇ , detector voltage 2300 V-2700V, ionization voltage 60 eV-80 eV, current 30 ⁇ - 50 ⁇ ; mass transfer detection range 50-800 m/z; small molecule Metabolite identification was performed using the NIST 2005 database, mass spectrometry data processing and metabolite relative content determination using Masslynx 4.1 software; and by integrating the color peak area and comparing with the peak area of the
  • the content of small molecule metabolite markers was graphed according to different passage times, and the changes of small molecule metabolite markers were observed and analyzed, and the changes of intracellular small molecule metabolites during the passage of vitamin C producing strains were detected.
  • the method in which the cells are made into a dry powder is liquid nitrogen-milled cells.
  • the seventh aspect of the present invention provides a method for transforming Gluconobacter oxydans by metabolic engineering, increasing the copy number of the kaempferol dehydrogenase gene, and improving the fermentation of Gluconobacter oxydans and Bacillus megaterium to transform L-mountain
  • the method in which the sugar is a property of 2-keto-L-gulonic acid It includes the following steps:
  • the bacterial genome extraction kit was used to extract the genome of Gluconobacter oxydans; the genomic solution 1 ⁇ 1, 5xFastPfu buffer 4 ⁇ 1, 20mM dNTP 2 ⁇ 1, 20 ⁇ primer 1 0 ⁇ 4 ⁇ 1, 20 ⁇ primer 2 0 ⁇ 4 ⁇ 1, sterile water 11.8 ⁇ 1 , FastPfu enzyme 0.4 ⁇ 1, mixed evenly in the PCR tube; PCR tube; ⁇ PCR instrument for amplification cycle, the amplification procedure is: 95. C 2min; 95 ° C 20s, 55 ° C 35s, 72 ° C 2min, a total of 25 cycles; 72. C 5min; 4. C 30min.
  • the PCR product was subjected to agarose gel electrophoresis, and the PCR product strip was excised, and the PCR was purified by agarose gel recovery kit. Amplification product.
  • the sequence of the primer 1 is: 5,-CCCAAGCTTGACTGGCAGCAGCGCAAC-3'
  • the sequence of the primer 2 is: 5,- CGCGGATCCCCGTGATAGCGGCACATGTC-3'
  • step (1) Take the PCR amplification product solution obtained in step (1) 8 ⁇ 1, and mix it evenly with ⁇ lOxdigest buffer, 0.5 ⁇ 1 Hindlll enzyme, 0.5 ⁇ 1 BamHI enzyme, at 37.
  • the digestion reaction was carried out for 2 hours under C conditions; the product after the reaction was subjected to agarose gel electrophoresis, and the cut product strip was excised, and the PCR product was purified by agarose gel recovery kit.
  • the enzyme digestion reaction was carried out for 2 hours under C conditions; the product after the reaction was subjected to agarose gel electrophoresis, and the cut product strip was excised, and the vector product was purified by agarose gel recovery kit.
  • the PCR product was subjected to digestion with 5.5 ⁇ l, and the product was digested with 3 ⁇ l, lOxligase buffer ⁇ , DNA ligase 0.5 ⁇ l, and mixed uniformly at 22.
  • the ligation reaction was carried out for 30 min under C conditions.
  • the ligation product was transformed into Escherichia coli DH5a by chemical transformation, and applied to an antibiotic-containing LB solid medium, and cultured at 37 ° C for 12 h. The single colonies obtained after the culture were in LB liquid medium 37 .
  • the plasmid was extracted with a plasmid extraction kit to obtain a gene encoding the kaempferol dehydrogenase gene.
  • Gluconobacter oxidans was coated on solid medium at 30. Incubate for 48 hours under C conditions; wash the cells with 2 ml of sterile water, and ice bath for 10 min, 4. C, centrifuge at 5 rpm for 5 min, collect the cells, wash once with pre-cooled sterile water, wash twice with 10% glycerol, resuspend the cells with ⁇ 10% glycerol; and obtain the kaempferol dehydrogenase gene vector obtained from step (2).
  • the aqueous solution is uniformly mixed in an electric rotor, ice bath for 5 minutes, and the electric cup is turned Place the 1800V electric shock in the electro-rotator; mix the electric shock product with 900 ⁇ 1 seed medium, at 30.
  • the solid medium is: L-mountain 20 g , corn syrup 3 g , beef bone 3 g , yeast dip powder 3 g, urea lg, peptone 10 g, agar 20 g, KH 2 PO 4 lg, MgSO 4 0.2 g, CaC0 3 lg, add water to 1L, adjust pH to 6.8, 121. C sterilized for 20 min.
  • the seed medium is: L-mountain 20 g , corn syrup 3 g , beef bone 3 g , yeast dipping powder 3 g, urea lg, peptone 10 g, KH 2 P0 4 lg, MgSO 4 0.2 g, CaC0 3 lg Add water to 1L and adjust pH to 6.8, 121. C sterilized for 20 min.
  • the Gluconobacter oxydans and Bacillus megaterium containing the vector encoding the kaempferol dehydrogenase gene obtained in the step (3) are inoculated into a fermentation medium to obtain Gluconobacter oxydans containing a gene encoding a kaempferol dehydrogenase gene.
  • the density is 4x10 8 cfu/ml
  • the density of Bacillus megaterium is 4x10 8 cfu/ml at 30.
  • C cultured at 250 rpm for 120 h to obtain 2-keto-L-gulonic acid.
  • the fermentation medium is: 80 g of L-wheat sugar, 20 g of corn syrup, 12 g of urea, 12 g of urea, KH 2 P0 4 lg, MgSO 4 0.5 g, CaC0 3 lg, water to 1 L, pH adjusted to 7.0, 121. C sterilized for 20 min.
  • the method can improve the sugar acid conversion rate of 2-keto-L-gulonic acid produced by the fermentation of G. oxidans and Bacillus megaterium.
  • An eighth aspect of the invention relates to a method for detecting glutathione for increasing intracellular protein changes during the production of 2-keto-L-gulonic acid by Gluconobacter oxydans, comprising the steps of: (1) determination of intracellular proteins: 1 cell collection and quenching:
  • i L fermentation medium A is: 80 gL 1 hawthorn sugar, 20 gL 1 corn syrup, 1 g * L 1 KH 2 P0 4 , 0.2 g * L 1 MgS0 4 , ⁇ 12 g * L 1 urea, balance For water;
  • i L fermentation medium B is: 80 gL 1 hawthorn sugar, 20 gL 1 corn syrup, 1 gL 1 KH 2 P0 4 , 0.2 gL 1 MgS0 4 , and 12 g*L 1 urea, 0.8 ⁇ 1.5 mg*mL 1 glutathione, the balance is water;
  • step 1 Take the crushed cells obtained in step 1. Place 100 ⁇ 200mg each in a centrifuge tube, add 0.5 ⁇ 2ml cell lysate to each tube, mix well, and sonicly break on ice for 20 ⁇ 50s; add 5 ⁇ 15 ⁇ mass ratio 2 ⁇ 4:1 DNase I / RNaseA enzyme mixed solution, mix, 4. C to stand the reaction for 10 ⁇ 30min; add 5 ⁇ 15 ⁇ 80 ⁇ 120mM phenylmethylsulfonyl isopropanol solution, 4 .
  • the bovine serum albumin was added to the Coomassie Brilliant Blue G-250 solution from a low concentration to a high concentration using a Bradford kit.
  • the absorbance at 595 nm of each protein solution obtained in Step 2 was determined to establish a standard curve; the protein of each protein solution was determined. concentration;
  • the solution obtained in the step solution 6 is mixed to obtain 3-5 parts of the mixed solution, and each mixed solution includes the labeled protein which is obtained by the fermentation of the crushed cells after the fermentation for 1 h from the medium A, and then after the steps 2-6.
  • the quenched broken cells were collected from the medium A for 15 h, and the labeled proteins obtained after the steps 2-6 were collected, and the quenched broken cells were collected from the medium B for 1 h to collect the labeled cells after the steps 2-6.
  • Protein and from medium B The labeled protein obtained after the step 2-6 was collected from the quenched broken cells for 15 h; C save;
  • the mixed solution obtained in 7 was subjected to Q-Tof mass identification to obtain a protein spectrum, and differentially expressed proteins of each mixed group sample were obtained by quantitative determination;
  • step (1)8 Principal component analysis is performed to obtain data categories with different variation rules, and candidate differential proteins are obtained;
  • the candidate differential protein content obtained in step (2) was plotted according to time series, and the regularity of these protein changes was observed and analyzed, and then glutathione was found to increase the production of 2-keto-L-gulonic acid by Gluconobacter oxydans. The role.
  • a ninth aspect of the invention relates to a method for finding a quality control index for a raw material corn syrup for fermentation, which comprises the steps of:
  • 0.5-2 g of the uniform raw corn syrup for fermentation is placed in a centrifuge tube, and the supernatant is collected by centrifugation; the obtained supernatant is diluted with ultrapure water 10-40 times, and 10-4 ( ⁇ L is placed in another In a centrifuge tube, add 30-50 ⁇ of 0.040 mg/mL yttrium-labeled succinic acid methanol.
  • the solution was an internal standard; after lyophilization, 50-100 ⁇ l of a pyridine solution of methoxyamine hydrochloride at a concentration of 20 mg/mL was added at 30. C-40.
  • the hydration reaction in a C water bath was carried out for 60-120 min; 60-100 ⁇ -methyl-N-trimethylsulfonyl trifluoroacetamide was further added at 35. C-40.
  • the silencing reaction of the C7j bath is carried out for 20-50 min;
  • the sample obtained in step 1 was fed to a gas chromatograph of DB-5MS having a specification of 30 mx 0.25 mm i.d. and an inlet temperature of 250. C-280.
  • the carrier gas is high-purity helium
  • the flow rate is 0.6-0.8ml/min
  • the split ratio is 30: 1-10: 1
  • the column temperature riser is: initial 50.
  • C is kept for 2-5min, to 4.
  • the method uses high-throughput high-resolution detection technology to simultaneously measure corn syrup
  • the various chemical components are simple and easy to process. After repeated batches of raw materials, the repeatability is good.
  • the method has high resolution and sensitivity, good reproducibility and stability, and the partial least squares discriminant analysis method can quickly and effectively select high-throughput data to distinguish chemical substances of different batches of raw materials and find corn.
  • the nine chemicals in the pulp such as glucose, lactic acid, proline, phosphoric acid, galactose, alanine, fructose, 5-ketoproline, and sucrose, can be considered as quality control indicators for corn syrup, which is the current corn syrup. A supplement to the quality check.
  • Figure 1 is a graph showing the change in conversion of 2-keto-L-gulonic acid during passaging according to the first aspect of the present invention.
  • ⁇ 2-keto-L-gulonic acid conversion.
  • Fig. 2 is a graph showing the results of strain fermentation after 50 days of passage according to the first aspect of the present invention.
  • Fig. 3 is a graph showing the results of cross-matching fermentation of strains after 100 days of passaging according to the second aspect of the present invention.
  • Figure 4 is a cross-column fermentation result of strains after passage for 150 days in accordance with the second aspect of the present invention.
  • Figure 5 is a cluster analysis of Gluconobacter oxydans protein of subculture 0, 50, 100, 150 passages according to the third aspect of the present invention.
  • Figure 6 is a graph showing the relative expression level of intracellular hawthorn/behenone dehydrogenase in Gluconobacter oxydans cells subcultured in accordance with the third aspect of the present invention in 0, 50, 100, and 150 passages;
  • Figure 7 is a cluster analysis of Bacillus megaterium protein of subculture 0, 50, 100, 150 passages according to the third aspect of the present invention.
  • Figure 8 is a category 2 of a B. megaterium cluster analysis of subcultures 0, 50, 100, 150 passages according to a third aspect of the present invention.
  • Figure 9 is a graph showing changes in phospholipid content of Bacillus megaterium at different passage times in accordance with the fourth aspect of the present invention
  • Figure 10 is a principal component analysis score map (Figure 10-1) and a load map ( Figure 10-2) of Bacillus megaterium at different passage times according to the fourth aspect of the present invention
  • Figure 11 is a graph showing changes in phospholipid molecular marker content of Bacillus megaterium at different passage times according to the fourth aspect of the present invention.
  • Figure 12 is a graph showing changes in phospholipid content of Gluconobacter oxydans at different passage times according to the fourth aspect of the present invention.
  • Figure 13 is a graph showing the principal component analysis scores (Fig. 13-1) and load maps (Fig. 13-2) of Gluconobacter oxydans at different passage times according to the fourth aspect of the present invention
  • Figure 14 is a graph showing changes in the phospholipid molecular marker content of Gluconobacter oxydans at different passage times according to the fourth aspect of the present invention.
  • Figure 15 is a graph showing changes in the phosphorus content of the mixed bacteria of different passage times according to the fourth aspect of the present invention.
  • Figure 16 is a graph showing the principal component analysis scores (Fig. 16-1) and load diagrams (Fig. 16-2) of the mixed bacteria of different passage times according to the fourth aspect of the present invention.
  • Fig. 17 is a graph showing changes in the content of phospholipid molecular markers of mixed bacteria at different passage times according to the fourth aspect of the present invention.
  • Figure 18 is a Principal Component Analysis score map (Figure 18-1) and load map ( Figure 18-2) of the G. oxidans glucone environment at different passage times in accordance with the fifth aspect of the present invention
  • Figure 19 is a graph showing changes in nutrient environmental markers during the subculture of Gluconobacter oxydans at different passage times in accordance with the fifth aspect of the present invention.
  • Figure 20 is a principal component analysis score diagram (Fig. 20-1) and a load diagram (Fig. 20-2) of a B. megaterium nutrient environmental substance of different passage times according to the fifth aspect of the present invention
  • Figure 21 is a graph showing changes in nutrient environmental markers during the subculture of Bacillus megaterium at different passage times in accordance with the fifth aspect of the present invention.
  • Figure 22 is a mixture of different passage times according to the fifth aspect of the present invention.
  • Fig. 23 is a difference in nutrient environment during mixed passage culture of different passage times according to the fifth aspect of the present invention a map of changes in matter;
  • Figure 24 is a graph showing the principal component analysis scores of the small molecular metabolites of Gluconobacter oxydans in different passage times according to the sixth aspect of the present invention (Fig. 24-1) and load maps (Fig. 24)
  • Figure 25 is a graph showing changes in the content of small molecule metabolite markers during subculture of Gluconobacter oxydans in different passage times according to the sixth aspect of the present invention.
  • Figure 26 is a principal component analysis score map (Figure 26-1) and load map ( Figure 26-2) of Bacillus megaterium small molecule metabolites of different passage times in accordance with the sixth aspect of the present invention
  • Figure 27 is a graph showing changes in the content of small molecule metabolite markers during the subculture of Bacillus megaterium at different passage times in accordance with the sixth aspect of the present invention.
  • FIG 28 is a analysis diagram (FIG. 28-1) and the load ( Figure 28-2) depending on the generation time of the sixth aspect of the present invention, the main component mixed small molecule metabolites of bacteria;
  • FIG. 29 according to the present The sixth aspect of the invention changes the content of small molecule metabolite markers during mixed passage culture of different passage times;
  • Figure 30 is a diagram showing the results of fermentation of Gluconobacter oxydans and Bacillus megaterium containing the gene encoding the kaempferol dehydrogenase gene according to the seventh aspect of the present invention
  • Figure 31 is a principal component analysis of four sets of sample protein (mixed solution) data according to the eighth aspect of the present invention:
  • B material load diagram
  • FIG 32 is a diagram showing the expression of a thiamine transporter and an important enzyme thereof as a coenzyme according to the eighth aspect of the present invention: GSH-1h/KV-lh, which is derived from the medium B lh Sample/sample from medium A lh; KV-15h/KV-lh, representing sample from medium A 15h / sample from medium A lh, GSH-15h/KV-lh, indicating from medium B 15h Sample / sample from medium A lh;
  • Figure 33 is a diagram showing the expression of key enzymes in the tricarboxylic acid cycle, pentose phosphate pathway according to the eighth aspect of the present invention: GSH-1h/KV-lh, representing a sample from the medium Blh/from the medium A lh Sample; KV-15h/KV-lh, representing sample from medium A 15h / sample from medium A lh, GSH-15h/KV-lh, representing sample from medium B 15h / from medium A lh sample;
  • Figure 34 is a partial least squares discriminant analysis score chart of different batches of corn syrup according to the ninth aspect of the present invention
  • Figure 34-1 is a principal component analysis of different batches of corn syrup t[l]-t[2]
  • Figure 34-2 is a plot of principal component analysis of different batches of corn syrup t[l]-u[l];
  • Figure 35 is a partial least squares discriminant analysis load of different batches of corn syrup according to the ninth aspect of the present invention; Fig.
  • 35-1 is a pp(corr) S diagram of different batches of corn syrup w*c[l Buw*c[2] scatter plot;
  • Figure 35-2 is the first principal component of different batches of corn syrup;
  • Figure 36 is a partial least squares discriminant analysis VIP map (V..1) of different batches of corn syrup according to the ninth aspect of the present invention. detailed description
  • a method for improving the yield of 2-keto-L-gulonic acid by subculture of mixed bacteria comprises the following steps: (1) Solid culture:
  • the solid medium was prepared by weighing 20 g of L-xanthine, 3 g of corn syrup, 3 g of beef bone, 3 g of yeast dipping powder, urea lg, peptone 10 g, agar 20 g, KH 2 P0 4 lg, MgSO 4 0.2 g, CaC0 3 lg water to 1L, adjusted to pH 6.8, sterilized at 121 ° C for 20min, to make a solid medium;
  • the seed medium was prepared as follows: L-xanthine 20g, corn syrup 3g, beef bone 3g, yeast dip 3g, urea lg, peptone 10g, KH 2 P0 4 lg, MgSO 4 0.2 g, CaC0 3 lg Add water to 1 L and adjust the pH to 6.8, 121. C sterilized for 20 min to prepare a seed culture medium;
  • the Bacillus megaterium and Gluconobacter oxydans cultured in the step (1) were separately transferred to a seed medium at 30°C. C, 250 r / min shaker shaking culture, 48h, the Bacillus megaterium seed solution and Gluconobacter oxydans seed solution;
  • the Bacillus megaterium and Gluconobacter oxydans was inoculated into a new seed medium, the density of the Bacillus megaterium is 2xl0 7 cfu / ml, the density of the Gluconobacter oxydans is 2xl0 9 cfu / ml, at 30.
  • C 250 r / min shaker shaking culture, 24h as the passage cycle, the volume ratio of 4% for the passage ratio into the new seed medium, passage for 50 days;
  • the subcultured mixed strain obtained in the step (2) is streaked and purified, and then inoculated on a solid medium at 30 ° C for 48 hours; and then transferred to a seed medium at 30 ° C, 250 r / The shaker was shake-cultured for 48 hours to obtain the evolved Bacillus megaterium seed solution and the evolved Gluconobacter oxydans seed solution; (4) Fermentation:
  • the fermentation medium was prepared by weighing 80 g of L-xanthine, 20 g of corn syrup, 12 g of urea, 12 g of urea, KH 2 P0 4 lg, MgSO 4 0.5 g, CaC0 3 lg, adding water to 1 L, and adjusting the pH to 7.0, 121. C sterilized for 20 min to prepare a fermentation medium;
  • the original Bacillus megaterium and the evolved Gluconobacter oxydans were inoculated into the fermentation medium, and the density of Bacillus megaterium was 2 ⁇ 10 7 cfu/ml, and the density of the evolved Gluconobacter oxydans was 2 ⁇ 10 9 .
  • HPLC High performance liquid chromatography
  • Sample preparation Take 1 mL of fermentation broth at different times in a 1.5 mL centrifuge tube, centrifuge at 10000 r/min for 3 min, take the supernatant ⁇ in a 1.5 mL centrifuge tube, and add 900 ⁇ M ⁇ 2 phase (5 mM H 2 S0 4 ) A sample that is ten times dry is obtained. After oscillating and mixing, the sample was filtered through a 0.22 ⁇ m cellulose microporous membrane to obtain a sample to be tested.
  • High performance liquid chromatography conditions Color column: bio-rad HPX-87H, mobile phase: 5 mM H 2 S0 4 , flow rate: 0.6 mL/min, column temperature: 65. C, the differential detector.
  • ⁇ Original Gluconobacter oxydans and original Bacillus megaterium
  • ⁇ Passage 50 days of Gluconobacter oxydans and original Bacillus megaterium.
  • the yield of 2-keto-L-gulonic acid fermented by the 50-day subculture of Gluconobacter oxydans and the original Bacillus megaterium was 88.2%, with an increase of 10.2%.
  • Example 1-2 A method for improving the yield of 2-keto-L-gulonic acid by subculture of mixed bacteria comprises the following steps:
  • Gluconobacter oxydans and ⁇ stored in a 30% by volume aqueous solution of glycerol (Bacillus megaterium) in a 30% volume aqueous solution of glycerol Inoculated on solid medium, cultured at 35 ° C for 24 hours;
  • the Bacillus megaterium and Gluconobacter oxydans cultured in the step (1) were separately transferred to the seed medium at 35. C, 200 r / min shaker shaking culture for 24h, obtaining Bacillus megaterium seed solution and Gluconobacter oxydans seed solution;
  • the Bacillus megaterium and Gluconobacter oxydans was inoculated into a new seed medium, the density of the Bacillus megaterium is 2xl0 7 cfu / ml, the density of the Gluconobacter oxydans was 2xl0 8 cfu / ml, at 35. C, 200r / min shaker shaking culture, with 24h as the passage cycle, the volume ratio of 2% for the passage ratio into the new seed medium, passage 60 days;
  • the subcultured mixed strain obtained in the step (2) was streaked and purified, and then inoculated on a solid medium, and cultured at 35 C for 24 hours; respectively, transferred to a seed medium, and shaken at 35 ° C, 200 r / min.
  • the bed was cultured for 24 hours, and the evolved Bacillus megaterium seed solution and the evolved Gluconobacter oxydans seed solution were obtained;
  • the original Bacillus megaterium and the evolved Gluconobacter oxydans were inoculated into the fermentation medium, and the density of Bacillus megaterium was 2 ⁇ 10 7 cfu/ml, and the density of the evolved Gluconobacter oxydans was 2 ⁇ 10 9 .
  • the medium used in this example Preparation of solid medium: Weigh scale mountain Qian L- sugar 10g, corn steep liquor 10 g, beef bone 5 g, yeast extract 2 g, urea 5 g, peptone 5 g, agar 10 g, KH 2 P0 4 5 g, MgSO 4 0.5 g, CaCO 3 0.5 g, water was added to 1 L, pH was adjusted to 6.5, and sterilized at 121 ° C for 20 min to prepare a solid medium.
  • the seed medium was prepared as follows: 10 g of L-xanthose, 10 g of corn syrup, 8 g of beef bone, 8 g of beef bone, 2 g of yeast powder, 5 g of urea, 2 g of peptone, KH 2 P0 4 5 g, 0.5 g of MgSO 4 , CaCO 3 0.5 g, add water to 1L, adjust the pH to 6.5, 121. C was sterilized for 20 min to prepare a seed medium.
  • the fermentation medium was prepared as follows: 40 g of L-xanthine, 50 g of corn syrup, 20 g of urea, 20 g of urea, 0.5 g of KH 2 PO 4 , 1.2 g of MgS0 4 , 2 g of CaC0 3 2 g of water were added to 1 L, and the pH was adjusted to 6.5, 121. C was sterilized for 20 min to prepare a fermentation medium.
  • a method for improving the yield of 2-keto-L-gulonic acid by subculture of mixed bacteria comprising the following steps:
  • the Bacillus megaterium and Gluconobacter oxydans cultured in the step (1) were separately transferred to a seed medium at 30°C. C, 240 r / min shaker shaking culture for 36h, obtaining Bacillus megaterium seed solution and Gluconobacter oxydans seed solution;
  • Bacillus megaterium and Gluconobacter oxydans were inoculated into a new seed medium such that the density of Bacillus megaterium was 2 x 10 8 cfu/ml, and the density of Gluconobacter oxydans was 2 x 10 n cfu/ml at 30.
  • the subcultured mixed strain obtained in the step (2) was streaked and purified, and then inoculated separately on a solid medium, 30.
  • C was cultured for 36 hours; then transferred to seed culture medium, and cultured at 30 ° C, 240 r / min shaker for 36 h, to obtain an evolved Bacillus megaterium seed solution and an evolved Gluconobacter oxydans seed solution;
  • the original Bacillus megaterium and the evolved Gluconobacter oxydans were inoculated into the fermentation medium, and the density of Bacillus megaterium was 2 ⁇ 10 8 cfu/ml, and the density of the evolved Gluconobacter oxydans was 2 ⁇ 10 U. Cfu/ml, at 30. C, 240 r / min shaker shaking culture for 96h, to obtain 2-keto-L-gulonic acid.
  • the solid medium was prepared by weighing 30 g of L-xanthine, 2 g of corn syrup, 10 g of beef bone, 5 g of yeast extract, 0.5 g of urea, 12 g of peptone, 30 g of agar, and KH 2 PO 4 0.5. g, MgSO 4 0.7 g, CaCO 3 3 g, water was added to 1 L, and the pH was adjusted to 6.8, 121. C was sterilized for 20 min to prepare a solid medium.
  • the seed medium was prepared as follows: Weighed 30 g of L-wheat sugar, 2 g of corn syrup, 10 g of beef bone, 8 g of yeast extract, urea 0.5 g, peptone 8 g, KH 2 PO 4 0.5 g, MgSO 4 0.7 g, CaC0 3 3g, add water to 1L, adjust the pH to 6.8, 121. C was sterilized for 20 min to prepare a seed medium.
  • the fermentation medium was prepared by weighing 80 g of L-xanthine, 10 g of corn syrup, 25 g of urea, 25 g of KH 2 P0 4 lg, 0.2 g of MgSO 4 , and 5 g of CaC0 3 to 1 L, and adjusted to pH 6.8, 121. C was sterilized for 20 min to prepare a fermentation medium.
  • a method for improving the yield of 2-keto-L-gulonic acid by subculture of mixed bacteria comprises the following steps: (1) Solid culture:
  • the Bacillus megaterium and Gluconobacter oxydans cultured in step (1) were separately transferred to seed medium at 28. C, 280 r / min shaker shaking culture for 48h, obtaining Bacillus megaterium seed solution and Gluconobacter oxydans seed solution;
  • the Bacillus megaterium and Gluconobacter oxydans was inoculated into a new seed medium, so that the density of Bacillus megaterium 2xlO ie cfu / ml, the density of the Gluconobacter oxydans is 2xlO n cfu / ml, at 28. C, 280 r / min shaker shaking culture, 48h as the passage cycle, the volume ratio of 10% for the passage ratio into the new seed medium, passaging 80 days;
  • the subcultured mixed strain obtained in the step (2) was streaked and purified, and then inoculated on a solid medium, respectively.
  • C was cultured for 48 hours; then transferred to seed culture medium, and cultured at 28 °C, 280 r/min shaker for 48 hours, to obtain an evolved Bacillus megaterium seed solution and an evolved Gluconobacter oxydans seed solution;
  • the original Bacillus megaterium and the evolved Gluconobacter oxydans were inoculated into the fermentation medium, and the density of Bacillus megaterium was , the density of the evolved Gluconobacter oxydans is 2xlO u cfu / ml, at 28. C, 280 r / min shaker shaking culture for 72h, to obtain 2-keto-L-gulonic acid.
  • the solid medium was prepared by weighing: 50 g of L-wheat sugar, 5 g of corn syrup, 2 g of beef bone, 10 g of yeast extract, 3 g of urea, 2 g of peptone, 50 g of agar, KH 2 P0 4 3g, MgSO 4 0.1 g, CaC0 3 5 g, water was added to 1 L, and the pH was adjusted to 7.0, 121. C was sterilized for 20 min to prepare a solid medium.
  • the seed medium was prepared as follows: Proportioned L-mountain 50g, corn syrup 8g, beef bone 2g, yeast dipping powder 10g, urea 3g, peptone 12g, KH 2 P0 4 3g, MgSO 4 0.1g, CaCO 3 0.5 -5g, add water to 1L, adjust the pH to 7.0,121. C was sterilized for 20 min to prepare a seed medium.
  • the fermentation medium was prepared as follows: Weigh 120 g of L-wheat sugar, 30 g of corn syrup, 10 g of urea, 10 g of urea, KH 2 P0 4 3 g, 0.5 g of MgSO 4 , 0.5 g of CaCO 3 and 1 L of water, adjusted to pH 7.5, 121 . C was sterilized for 20 min to prepare a fermentation medium.
  • Example 2-1 Weigh 120 g of L-wheat sugar, 30 g of corn syrup, 10 g of urea, 10 g of urea, KH 2 P0 4 3 g, 0.5 g of MgSO 4 , 0.5 g of CaCO 3 and 1 L of water, adjusted to pH 7.5, 121 . C was sterilized for 20 min to prepare a fermentation medium.
  • a method for enhancing 2-bacterium interaction to increase 2-keto-L-gulonic acid production comprising the following steps:
  • the solid medium was prepared by weighing 20 g of L-xanthine, 3 g of corn syrup, 3 g of beef bone, 3 g of yeast dipping powder, urea lg, peptone 10 g, agar 20 g, KH 2 P0 4 lg, MgSO 4 0.2 g, CaC0 3 lg water to 1L, adjusted to pH 6.8, sterilized at 121 ° C for 20min, to make a solid medium;
  • the seed medium was prepared as follows: L-xanthine 20g, corn syrup 3g, beef bone 3g, yeast dip 3g, urea lg, peptone 10g, KH 2 P0 4 lg, MgSO 4 0.2 g, CaC0 3 lg Add water to 1 L and adjust the pH to 6.8, 121. C sterilization for 20min, made into seeds Medium
  • the Bacillus megaterium and Gluconobacter oxydans cultured in the step (1) were separately transferred to a seed medium at 30°C. C, 250 r / min shaker shaking culture, 48h, the Bacillus megaterium seed solution and Gluconobacter oxydans seed solution;
  • the Bacillus megaterium and Gluconobacter oxydans was inoculated into a new seed medium, the density of the Bacillus megaterium is 2xl0 7 cfu / ml, the density of the Gluconobacter oxydans is 2xl0 9 cfu / ml, at 30.
  • C 250 r / min shaker shaking culture, 24h as the passage cycle, the volume ratio of 4% for the passage ratio into the new seed medium, passaging for 100 days;
  • the subcultured mixed strain obtained in the step (2) is streaked and purified, and then inoculated on a solid medium at 30 ° C for 48 hours; and then transferred to a seed medium at 30 ° C, 250 r / The shaker was shake-cultured for 48 hours to obtain the evolved Bacillus megaterium seed solution and the evolved Gluconobacter oxydans seed solution;
  • the fermentation medium was prepared by weighing 80 g of L-xanthine, 20 g of corn syrup, 12 g of urea, 12 g of urea, KH 2 P0 4 lg, MgSO 4 0.5 g, CaC0 3 lg, adding water to 1 L, and adjusting the pH to 7.0, 121. C sterilized for 20 min to prepare a fermentation medium;
  • a method for enhancing 2-bacterium interaction to increase 2-keto-L-gulonic acid production comprising the following steps:
  • Steps (1), (2), and (3) are the same as the embodiment 2-1
  • HPLC High performance liquid chromatography
  • Sample preparation Take 1 mL of fermentation broth at different times in a 1.5 mL centrifuge tube, centrifuge at 10000 r/min for 3 min, take the supernatant ⁇ in a 1.5 mL centrifuge tube, and add 900 ⁇ M ⁇ 2 phase (5 mM H 2 S0 4 ) A sample that is ten times dry is obtained. After oscillating and mixing, the sample was filtered through a 0.22 ⁇ m cellulose microporous membrane to obtain a sample to be tested.
  • High performance liquid chromatography conditions Color column: bio-rad HPX-87H, mobile phase: 5 mM H 2 S0 4 , flow rate: 0.6 mL/min, column temperature: 65. C, the differential detector.
  • ⁇ Original Gluconobacter oxydans and B. megaterium; ⁇ : Passage for 100 days of Gluconobacter oxydans and passage of 100-day Bacillus megaterium; ⁇ : Passage for 100 days of Gluconobacter oxydans and Bacillus megaterium; The yield of 2-keto-L-gulonic acid of the original Gluconobacter oxydans and the original Bacillus megaterium using the fermentation conditions of the step (4) of Example 2-1 was 78%; using Example 2-1 The method of subculture for 100 days of evolution of Gluconobacter oxydans and subculture of 100 days of evolution of Bacillus megaterium mixed fermentation of 2-keto-L-gulonic acid yielded 91%-92%, it The yield of 2-keto-L-gulonic acid fermented by the mixed bacteria of the original Gluconobacter oxydans and the original Bacillus megaterium was increased by 13% -14%; the G.
  • Example 2-3 oxydans subcultured for 100 days was matched with the original Bacillus megaterium.
  • Example 2-3 The mixed fermentation (Example 2-2) fermentation cycle and 2-keto-L-gulonic acid conversion were similar to the G. oxydans subcultured for 100 days and the mixed Bacillus megaterium fermentation for 100 days.
  • Example 2-3
  • a method for enhancing 2-bacterium interaction to increase 2-keto-L-gulonic acid production comprising the following steps:
  • Gluconobacter oxydans and ⁇ stored in a 30% by volume aqueous solution of glycerol (Bacillus megaterium) in a 30% volume aqueous solution of glycerol Inoculated on solid medium, cultured at 35 ° C for 24 hours;
  • the Bacillus megaterium and Gluconobacter oxydans cultured in the step (1) were separately transferred to the seed medium at 35. C, 200 r / min shaker shaking culture for 24h, obtaining Bacillus megaterium seed solution and Gluconobacter oxydans seed solution;
  • the Bacillus megaterium and Gluconobacter oxydans was inoculated into a new seed medium, the density of the Bacillus megaterium is 2xl0 7 cfu / ml, the density of the Gluconobacter oxydans was 2xl0 8 cfu / ml, at 35.
  • C 200r/min shaker shaking culture, with 24h as the passage cycle, with a volume ratio of 2% as the passage ratio into the new seed medium, passaging for 150 days;
  • the subcultured mixed strain obtained in the step (2) was streaked and purified, and then inoculated on a solid medium, and cultured at 35 C for 24 hours; respectively, transferred to a seed medium, and shaken at 35 ° C, 200 r / min.
  • the bed was cultured for 24 hours, and the evolved Bacillus megaterium seed solution and the evolved Gluconobacter oxydans seed solution were obtained;
  • the evolved Bacillus megaterium and the evolved Gluconobacter oxydans were inoculated into a fermentation medium, and the density of the evolved Bacillus megaterium was 2 ⁇ 10 7 cfu/ml, and the density of the evolved Gluconobacter oxydans was increased. It is 2xl0 8 cfu/ml, at 35. C, 200r/min shake flask was cultured for 96h to obtain 2-keto-L-gulonic acid.
  • the seed medium was prepared as follows: 10 g of L-xanthose, 10 g of corn syrup, 8 g of beef bone, 8 g of beef bone, 2 g of yeast powder, 5 g of urea, 2 g of peptone, KH 2 P0 4 5 g, 0.5 g of MgSO 4 , CaCO 3 0.5 g, add water to 1L, adjust the pH to 6.5, 121. C was sterilized for 20 min to prepare a seed medium.
  • the fermentation medium was prepared as follows: 40 g of L-xanthine, 50 g of corn syrup, 20 g of urea, 20 g of urea, 0.5 g of KH 2 PO 4 , 1.2 g of MgS0 4 , 2 g of CaC0 3 2 g of water were added to 1 L, and the pH was adjusted to 6.5, 121. C was sterilized for 20 min to prepare a fermentation medium.
  • a method for enhancing 2-bacterium interaction to increase 2-keto-L-gulonic acid production comprising the following steps:
  • Steps (1), (2), and (3) are the same as Examples 2-3
  • the original Bacillus megaterium and the evolved Gluconobacter oxydans were inoculated into the fermentation medium, and the density of Bacillus megaterium was 2 ⁇ 10 7 cfu/ml, and the density of the evolved Gluconobacter oxydans was 2 ⁇ 10 9 .
  • Each medium was the same as in Example 2-2.
  • ⁇ Original Gluconobacter oxydans and original Bacillus megaterium
  • mouth Passage 150 days of Gluconobacter oxydans and passaging 150 days of Bacillus megaterium
  • ⁇ Passage 150 days of Gluconobacter oxydans and original Bacillus megaterium
  • o raw oxidized glucose rod The bacteria were mixed with a 150-day Bacillus megaterium.
  • the yield of 2-keto-L-gulonic acid of the original Gluconobacter oxydans and the original Bacillus megaterium using the fermentation conditions of the step (4) of Example 2-3 was 78%; using Example 2-3 The method of subculture of 150 days of evolution of Gluconobacter oxydans and subculture of 150 days of evolution of the Bacillus megaterium mixed fermentation of 2-keto-L-gulonic acid yield reached 95% at the end of the fermentation, than the original The 2-keto-L-gulonic acid yield mixed bacteria fermented by Gluconobacter oxydans and B. megaterium increased by 17%, and the fermentation cycle was shortened by 9% compared with the original mixed bacteria. Subculture for 150 days.
  • the 2-keto-L-gulonic acid conversion rate of the mixed fermentation system of the Gluconobacter oxydans and the original Bacillus megaterium was 16% higher than that of the original mixed bacteria, and the fermentation cycle and subculture 150 The days of mixed bacteria are similar.
  • a method for enhancing 2-bacterium interaction to increase 2-keto-L-gulonic acid production comprising the following steps:
  • the Bacillus megaterium and Gluconobacter oxydans cultured in the step (1) were separately transferred to a seed medium at 30°C. C, 240 r / min shaker shaking culture for 36h, obtaining Bacillus megaterium seed solution and Gluconobacter oxydans seed solution;
  • Bacillus megaterium and Gluconobacter oxydans were inoculated into a new seed medium such that the density of Bacillus megaterium was 2 x 10 8 cfu/ml, and the density of Gluconobacter oxydans was 2 x 10 n cfu/ml at 30.
  • the subcultured mixed strain obtained in the step (2) was streaked and purified, and then inoculated separately on a solid medium, 30.
  • C was cultured for 36 hours; then transferred to seed culture medium, and cultured at 30 ° C, 240 r / min shaker for 36 h, to obtain an evolved Bacillus megaterium seed solution and an evolved Gluconobacter oxydans seed solution;
  • the evolved Bacillus megaterium and the evolved Gluconobacter oxydans were inoculated into a fermentation medium, and the density of the evolved Bacillus megaterium was 2 ⁇ 10 8 cfu/ml, and the density of the evolved Gluconobacter oxydans was increased.
  • ZxloUcfu/ml at 30. C, 240 r / min shaker shaking culture for 96h, to obtain 2-keto-L-gulonic acid.
  • the solid medium was prepared by weighing 30 g of L-xanthine, 2 g of corn syrup, 10 g of beef bone, 5 g of yeast extract, 0.5 g of urea, 12 g of peptone, 30 g of agar, and KH 2 PO 4 0.5. g, MgSO 4 0.7 g, CaCO 3 3 g, water was added to 1 L, and the pH was adjusted to 6.8, 121. C was sterilized for 20 min to prepare a solid medium.
  • the seed medium was prepared as follows: Weighed 30 g of L-wheat sugar, 2 g of corn syrup, 10 g of beef bone, 8 g of yeast extract, urea 0.5 g, peptone 8 g, KH 2 PO 4 0.5 g, MgSO 4 0.7 g, CaC0 3 3g, add water to 1L, adjust the pH to 6.8, 121. C was sterilized for 20 min to prepare a seed medium.
  • the fermentation medium was prepared by weighing 80 g of L-xanthine, 10 g of corn syrup, 25 g of urea, 25 g of KH 2 P0 4 lg, 0.2 g of MgSO 4 , and 5 g of CaC0 3 to 1 L, and adjusted to pH 6.8, 121. C was sterilized for 20 min to prepare a fermentation medium.
  • a method for enhancing the interaction of two bacteria to increase the yield of 2-keto-L-gulonic acid comprising the following steps: (1) Solid culture:
  • the Bacillus megaterium and Gluconobacter oxydans cultured in step (1) were separately transferred to seed medium at 28. C, 280 r / min shaker shaking culture for 48h, obtaining Bacillus megaterium seed solution and Gluconobacter oxydans seed solution;
  • the Bacillus megaterium and Gluconobacter oxydans was inoculated into a new seed medium, so that the density of Bacillus megaterium 2xlO ie cfu / ml, the density of the Gluconobacter oxydans is 2xlO n cfu / ml, at 28. C, 280 r / min shaker shaking culture, 48h as the passage cycle, the volume ratio of 10% for the passage ratio into the new seed medium, passage for 180 days;
  • the subcultured mixed strain obtained in the step (2) was streaked and purified, and then inoculated on a solid medium, respectively.
  • C was cultured for 48 hours; then transferred to seed culture medium, and cultured at 28 °C, 280 r/min shaker for 48 hours, to obtain an evolved Bacillus megaterium seed solution and an evolved Gluconobacter oxydans seed solution;
  • the evolved Bacillus megaterium and the evolved Gluconobacter oxydans are inoculated into a fermentation medium to make the density of the evolved Bacillus megaterium
  • the density of the evolved Gluconobacter oxydans was 2xl u u cfu / ml, at 28. C, 280 r / min shaker shaking culture for 72hh, to obtain 2-keto-L-gulonic acid.
  • the solid medium was prepared by weighing: 50 g of L-wheat sugar, 5 g of corn syrup, 2 g of beef bone, 10 g of yeast extract, 3 g of urea, 2 g of peptone, 50 g of agar, KH 2 P0 4 3g, MgSO 4 0.1 g, CaC0 3 5 g, water was added to 1 L, and the pH was adjusted to 7.0, 121. C was sterilized for 20 min to prepare a solid medium.
  • the seed medium was prepared as follows: Proportioned L-mountain 50g, corn syrup 8g, beef bone 2g, yeast dipping powder 10g, urea 3g, peptone 12g, KH 2 P0 4 3g, MgSO 4 0.1g, CaCO 3 0.5 -5g, add water to 1L, adjust the pH to 7.0,121. C was sterilized for 20 min to prepare a seed medium.
  • the fermentation medium was prepared as follows: Weigh 120 g of L-wheat sugar, 30 g of corn syrup, 10 g of urea, 10 g of urea, KH 2 P0 4 3 g, 0.5 g of MgSO 4 , 0.5 g of CaCO 3 and 1 L of water, adjusted to pH 7.5, 121 . C was sterilized for 20 min to prepare a fermentation medium.
  • Example 3-1 Weigh 120 g of L-wheat sugar, 30 g of corn syrup, 10 g of urea, 10 g of urea, KH 2 P0 4 3 g, 0.5 g of MgSO 4 , 0.5 g of CaCO 3 and 1 L of water, adjusted to pH 7.5, 121 . C was sterilized for 20 min to prepare a fermentation medium.
  • Example 3-1 Example 3-1
  • a method for detecting vitamin C industrial mixed bacteria to pass on different algebraic protein changes comprising the following steps:
  • the Bacillus megaterium and Gluconobacter oxydans cultured in the step (1) 1 were separately transferred to a seed medium at 28. C, 200r / min shaker shaking culture for 24h, obtaining Bacillus megaterium seed solution and Gluconobacter oxydans seed solution;
  • Bacillus megaterium and Gluconobacter oxydans were inoculated into a new seed medium such that the density of Bacillus megaterium was 2 x 10 7 CFU/mL, and the density of Gluconobacter oxydans was 2 x 10 8 CFU/mL, at 28. C, 200 r/min shaking shake culture, with 24h as the passage week Period, the volume ratio of 1% is used as the passage ratio to access the new seed culture medium, passaging for 150 days, and selecting 4 sampling times to take 4 samples, respectively, are 0 days, 50 days, 100 days, 150 days;
  • the four mixed cells obtained in the step (1) 2 were streaked and purified, and then inoculated on a solid medium, respectively.
  • C was cultured for 24 h; then transferred to seed medium separately at 28.
  • C 200 rpm shaker shaking culture for 24 h, the evolved Bacillus megaterium seed solution and the evolved Gluconobacter oxydans seed solution; preserved in a 15% volume aqueous solution of glycerol;
  • the evolved Bacillus megaterium and the evolved Gluconobacter oxydans obtained in the step (1) 3 were inoculated into the seed medium, respectively, so that the density of the evolved Bacillus megaterium was 2 ⁇ 10 7 CFU/mL, and the evolution was oxidized.
  • the density of Gluconobacter is
  • the Bacillus megaterium culture and the oxidized Glucose oxidase culture obtained in step (1) 4, respectively, are at 4. Centrifuge at 4000 rpm, collect the lower layer of cells, wash with phosphate buffer of pH 7.2, quench with liquid nitrogen, terminate the metabolic reaction; crush the cells with liquid nitrogen grinding;
  • the broken cells were placed in a centrifuge tube, each of which was placed in a centrifuge tube, 0.5 mL of cell lysate was added to each tube, mixed, and sonicated on ice for 20 s; 5 L of DNase I / RNaseA enzyme at a mass ratio of 2:1 was added. Mix the solution and mix well, 4. C. The reaction was allowed to stand for 10 min; 5 L of 80 mM phenylmethylsulfonyl fluoride isopropanol solution was added, 4. C is allowed to stand lh; centrifuged at 15000 rpm for 25 min; the supernatant is taken to obtain a protein solution;
  • the cell lysate is: 8 mol-L 1 urea, a concentration of 4% 3-[(3) -cholestylpropyl)-diethylamine]-propanesulfonic acid, 40 mM Tris, the balance being water;
  • the Bradford kit was used to convert bovine serum albumin from a low concentration to a high concentration into Coomassie Brilliant Blue G-250 solution.
  • the absorbance of each protein solution obtained in step (2) 2 at 595 nm was determined to establish a standard curve; each protein was determined.
  • Each protein solution obtained in the step (2) 2 containing 50 proteins was added, and 4 volumes of -20 were added per portion.
  • the candidate differential protein content obtained in step (3) is plotted according to time series, and the regularity of these protein changes is observed and analyzed, and then the mixed culture evolution culture is found to improve the production of 2-keto-L-gulonic acid by Gluconobacter oxydans. The role.
  • Gluconobacter oxidans produced a variety of evolutionary directions, showing that intracellular proteins exhibit different expression patterns in different algebras, as shown in Figure 5.
  • category 1, category 3 and category 5 showed little change in the expression of each generation of proteins; category 2 and category 4 showed different degrees of evolutionary protein sheets.
  • Raise including the hawthorn/bean ketone dehydrogenase responsible for the conversion of hawthorn sugar to 2-keto-L-gulonic acid.
  • the expression of this enzyme in the generation is shown in Fig. 6. It was slightly down-regulated in the 50th generation, and gradually increased in the 100th and 150th generations.
  • the reason for this phenomenon may be that during the 50th generation, the violent interaction between the mixed bacteria occurs, and the adaptive selection is carried out. With the synergistic effect, the Gluconobacter oxidans is more and more adapted to the mixed environment and has a stronger Production capacity.
  • Immune inhibitor A is a protein that degrades antibacterial peptides in Islam, and plays an important role in the resistance of Bacillus to the external immune system. In addition, it is an important component of the outer wall of Bacillus spores. The expression level increased with the subculture algebra and showed an up-regulation, indicating that the resistance of Bacillus megaterium spores was improved. At the same time, the ability to transport oligopeptides has also increased, which is conducive to their own growth needs.
  • a method for detecting vitamin C industrial mixed bacteria to pass on different algebraic protein changes comprising the following steps:
  • Gluconobacter oxydans stored in a volumetric 20% glycerol aqueous solution and Bacillus megaterium deposited in a 20% by volume aqueous solution of glycerol in a volume of 20% glycerol solution were inoculated separately. On a solid medium, cultured at 30 ° C for 36 hours;
  • the Bacillus megaterium and Gluconobacter oxydans cultured in the step (1) 1 were separately transferred to a seed medium at 30°C. C, 250 r / min shaker shaking culture for 36h, obtaining Bacillus megaterium seed solution and Gluconobacter oxydans seed solution;
  • the three mixed cells obtained in the step (1) 2 were streaked and purified, and then inoculated separately on a solid medium, 30.
  • C shaking culture at 250 rpm for 36 h, obtaining an evolved Bacillus megaterus seed solution and an evolved Gluconobacter oxydans seed solution; stored in a 20% by volume aqueous solution of glycerin;
  • the evolved Bacillus megaterium and the evolved Gluconobacter oxydans obtained in the step (1) 3 were inoculated separately into the seed medium, and the evolved Bacillus megaterium had a density of 2 ⁇ 10 8 CFU/mL, and the evolved oxidation.
  • the density of Gluconobacter is
  • the Bacillus megaterium culture and the oxidized Glucose oxidase culture obtained in step (1) 4, respectively, are at 4. After centrifugation at 5000 rpm, the lower layer of cells was collected, washed with a phosphate buffer solution of pH 7.3, quenched with liquid nitrogen, and the metabolic reaction was terminated; the cells were disrupted by liquid nitrogen milling;
  • the crushed cells were placed in a centrifuge tube, 90 mL of each was placed in a centrifuge tube, 0.8 mL of cell lysate was added to each tube, mixed, and sonicated on ice for 40 s; DNase I / RNaseA enzyme with a mass ratio of 3:1 was added. Mix the solution and mix well, 4. C was allowed to stand for 20 min; 10 L OO mM of a phenylmethylsulfonyl isopropanol solution was added, 4. C is allowed to stand for 2h; Centrifuge at 15000 rpm for 30 min; take the supernatant to obtain a protein solution;
  • the cell lysate is: 8 mol-L 1 urea, 4% by mass of 3-[(3-cholamidopropyl)-diethylamine]-propanesulfonic acid, 40 mM Tris, the balance being water ;
  • the Bradford kit was used to convert bovine serum albumin from a low concentration to a high concentration into Coomassie Brilliant Blue G-250 solution.
  • the absorbance of each protein solution obtained in step (2) 2 at 595 nm was determined to establish a standard curve; each protein was determined.
  • the candidate differential protein content obtained in step (3) is plotted according to time series, and the regularity of these protein changes is observed and analyzed, and then the mixed culture evolution culture is found to improve the production of 2-keto-L-gulonic acid by Gluconobacter oxydans. The role.
  • a method for detecting vitamin C industrial mixed bacteria to pass on different algebraic protein changes comprising the following steps:
  • the Bacillus megaterium and Gluconobacter oxydans cultured in the step (1) 1 were separately transferred to a seed medium at 35°C. C, 280 r / min shaker shaking culture for 48h, obtaining Bacillus megaterium seed solution and Gluconobacter oxydans seed solution;
  • Bacillus megaterium and Gluconobacter oxydans were inoculated into a new seed medium such that the density of Bacillus megaterium was 2 x 10 1 G CFU/mL, and the density of Gluconobacter oxydans was 2 x 10 n CFU/mL at 35.
  • C, 280 r/min shaker shaking culture with 48h as passage Cycle, with a volume ratio of 10% as the passage ratio into the new seed medium, pass through 150 days, select 4 sampling times to take 4 samples, respectively 0 days, 50 days, 100 days, 150 days;
  • the four mixed cells obtained in the step (1) 2 were streaked and purified, and then inoculated separately on a solid medium, 35.
  • C was cultured for 48 h; and then transferred to seed medium separately at 35.
  • C shaking culture at 280 rpm for 48 h, obtaining an evolved Bacillus megaterus seed solution and an evolved Gluconobacter oxydans seed solution; deposited in a 30% volume aqueous solution of glycerin;
  • the evolved Bacillus megaterium and the evolved Gluconobacter oxydans obtained in the step (1) 3 were inoculated separately into the seed medium, so that the density of the evolved Bacillus megaterium was 2xlO ie CFU/mL, and the evolution was oxidized.
  • the density of Gluconobacter is
  • the Bacillus megaterium culture and the oxidized Glucose oxidase culture obtained in the step (1) 4, respectively, are at 4.
  • C centrifuge at 6000 rpm, collect the lower layer of cells, and wash with phosphate buffer of pH 7.4, quench with liquid nitrogen to terminate the metabolic reaction; crush the cells with liquid nitrogen grinding;
  • each 100 mg of each is placed in a centrifuge tube, add 1 mL of cell lysate to each tube, mix, and acoustically disrupte on ice for 50 s; add 15 L of DNase l / RNaseA enzyme with a mass ratio of 4:1. Solution, mix, 4. C was allowed to stand for 30 min; 15 L of 120 mM phenylmethylsulfonyl isopropanol solution was added, 4. C is allowed to stand for 3 hours;
  • the cell lysate is: 8 mol-L 1 urea, a concentration of 4% 3-[(3) -cholestylpropyl)-diethylamine]-propanesulfonic acid, 40 mM Tris, the balance being water;
  • the Bradford kit was used to convert bovine serum albumin from a low concentration to a high concentration into Coomassie Brilliant Blue G-250 solution.
  • the absorbance of each protein solution obtained in step (2) 2 at 595 nm was determined to establish a standard curve; each protein was determined.
  • Each of the protein solutions obtained in the step (2) 2 containing 100 proteins was added to each of 6 volumes of -40.
  • C acetone, at -40. Place C under ⁇ for 20h; centrifuge, discard the supernatant, and use -40 for precipitation. Washing with a volumetric concentration of C of 85% aqueous acetone; drying to obtain a dry protein powder;
  • the candidate differential protein content obtained in step (3) is plotted according to time series, and the regularity of these protein changes is observed and analyzed, and then the mixed culture evolution culture is found to improve the production of 2-keto-L-gulonic acid by Gluconobacter oxydans. The role.
  • Example 3-2 and Example 3-3 are similar to those of Example 3-1.
  • the composition of the solid medium and the seed medium used in the present invention is selected from the medium disclosed in Chinese Patent Application No. 201110314740.9, for example:
  • Example 4-1
  • a method for analyzing changes in phospholipid groups during the passage of vitamin C production strains the characteristics of which include the following steps:
  • Gluconobacter oxydans and ⁇ stored in a 20% volume aqueous solution of glycerol were stored at a volume concentration of 20%.
  • Bacillus megaterium in an aqueous solution was inoculated on a solid medium, and cultured at 30 ° C for 36 hours;
  • the Bacillus megaterium and Gluconobacter oxydans cultured in the step (1) were separately transferred to a seed culture medium, and shake cultured at 30 ° C, shaking at 240 rpm for 36 hours to obtain a Bacillus megaterus seed solution and a Gluconobacter oxydans seed solution. ;
  • the four subcultured mixed strains obtained in the step (2) were streaked and purified, and then inoculated on a solid medium, and cultured at 30 ° C for 36 hours; and then transferred to a seed medium at 30 ° C, 240 rpm. Shake the shaker for 36h to obtain the evolved Bacillus megaterium seed solution and the evolved Gluconobacter oxydans seed solution;
  • the evolved Bacillus megaterium, the evolved Gluconobacter oxydans, and the mixed evolved Bacillus megaterium and the evolved Gluconobacter oxydans were inoculated separately into the fermentation medium, and the density of the evolved Bacillus megaterium was 2xl0 8 cfu/mL, the density of the evolved Gluconobacter oxydans is Incubate at 30 ° C, shaking at 240 rpm for 13 h;
  • the three cells obtained in step 1 were separately ground in a mortar using liquid nitrogen to prepare a dry powder.
  • Each of the 250 mg dry cell powder was weighed and placed in three centrifuge tubes A, and 0.6 mL of ultrapure water was added separately, and thoroughly mixed. ;
  • step 7 The organic solvent in the mixture obtained in step 6 is distilled under reduced pressure at 30 ° C to obtain three total phospholipid extraction mixtures;
  • phospholipid standards were added to the three samples.
  • the phospholipid standards were bis-dodecanoylphosphatidylglycerol, bis-dodecanoylphosphatidylethanolamine, dodecanoyl hemolytic phosphatidylethanolamine and double twelve. Alkanoyl phosphatidic acid, the final concentration of these four phospholipid standards is 1.0 g / mL;
  • the extract is a chloroform/methanol solution containing dibutylhydroxytoluene, the volume ratio of the chloroform/methanol is 1.5:1, and the mass fraction of the dibutylhydroxytoluene is 0.007%;
  • the storage solution is a chloroform/methanol solution containing dibutylhydroxytoluene, the volume ratio of the chloroform/methanol is 2:1, and the mass fraction of the dibutylhydroxytoluene is 0.007%;
  • the LC-MS detection conditions are:
  • Injection volume 10 ⁇ ;
  • Mobile phase A (%): chloroform (89.5), methanol (10), ammonium hydroxide (0.5); mobile phase B (%): chloroform (55), methanol (39), ammonium hydroxide (0.5), water ( 5.5);
  • Ionization mode ESI (negative ion mode);
  • Ion source temperature 100 ° C;
  • Desolvent gas flow 400 L/Hr;
  • Inlet/outlet energy 50;
  • HM1/LM1/HM2/LM2 15.0; Ion Energy 1: 1.0;
  • the molecular content data of the phospholipid group in the process of passage of the vitamin C production strain obtained in the step (6) were subjected to multivariate statistical analysis to obtain differential phospholipid molecular markers for distinguishing the vitamin C producing strains in different passage times; the multivariate statistical analysis method was Pareto pretreatment. After the principal component analysis; see Figure 10, Figure 13 and Figure 16;
  • the content of the differential phospholipid molecular markers obtained in the step (7) was plotted according to different passage times in Fig. 11, Fig. 14 and Fig. 17, and the regularity of the changes of these phospholipid molecules was observed and analyzed, and it was found that the mixed bacteria were in the process of subculture.
  • Table 4-1 Molecular identification table of Bacillus megaterium phospholipids
  • PA32:1, PA32:0, PA33:1, PA33:0 As shown in Table 4-1, the vitamin C producing strain was extracted and detected by the method of the present invention.
  • PA28 PA28:0 as shown in Table 4-2, extracted by the method of the present invention A total of 22 species of Gluconobacter oxydans phospholipids were detected in the vitamin C production strain, including 21 phosphatidylglycerol molecules and 1 phosphatidic acid molecule.
  • PA34:1 PA35:3
  • FIG. 11 shows the change in the content of outliers (molecular markers) in the load map of the main component analysis.
  • PE34:2, PE34:1 and LPE16:0 had the highest content in 50 days of cells; a series of PG molecules had the lowest content in 50 days, then gradually increased; a series of PA molecules decreased significantly in 50 days, and surged again in 100 days. .
  • the above changes indicate that the phospholipid metabolism process undergoes significant changes as the time between the two passages increases.
  • FIG. 13 Principal component analysis was performed as a matrix of all phospholipid molecules in the 0, 50, 100, and 150 days of Gluconobacter oxydans phospholipid samples (Figure 13).
  • the score plot shows a clear distinction between the 0 and 50 day cell phospholipid components, while 100 There was little difference between cells in 150 days and cells in 50 days; the load map showed that the content of PA molecules increased after 50 days of passage.
  • Figure 14 is a graph showing the percentage change of molecular markers for different passage times, in which the content of PA28:0 increased with the passage time.
  • the total cell phospholipid content of the mixed cells at 0, 50, 100, and 150 days was 8.39, 6.33, 6.62, and 7.91 nmol/mg dry cell weight, respectively, indicating that the total cell phospholipid content was significant at 50 days of passage. Decline, and then increase the trend.
  • Principal component analysis was performed using a matrix of all phospholipid molecules in 0, 50, 100, and 150 days of mixed phospholipid samples (Figure 16). The scores show significant differences in cell phospholipids at 0, 50, 100, and 150 days.
  • FIG. 17 is a phospholipid molecular marker that distinguishes mixed passages of different passage numbers.
  • a series of PG and LPE molecules decreased with the passage, while PE and PA molecules increased. This indicates that as the passage time of the two bacteria increases, the interaction time prolongs and the phospholipid metabolism changes.
  • the decrease of LPE and the increase of PA suggest different roles of different phospholipid metabolism-related enzymes in the common passage and cell membrane signal transduction of the two bacteria.
  • a method for analyzing changes in phospholipid groups during the passage of vitamin C production strains the characteristics of which include the following steps:
  • Solid culture Gluconobacter oxydans stored in a 15% volume aqueous glycerol solution and Bacillus megaterium stored in a 15% glycerol aqueous solution were inoculated on a solid medium. , 28 ° C, cultured for 48 hours;
  • the Bacillus megaterium and Gluconobacter oxydans cultured in the step (1) were separately transferred to a seed culture medium, and cultured at 28 ° C, shaking at 200 rpm for 48 hours to obtain a Bacillus megaterus seed solution and a Gluconobacter oxydans seed solution. ;
  • the culture was shaken at 200 rpm, and the passage time was 24 h. The volume ratio was 1% for the passage ratio.
  • the new seed culture medium was used for 130 days, on the 0th, 20th, 40th, and 80th. Take 5 samples on the day of the day and the 130th day;
  • the five subcultured mixed strains obtained in the step (2) were streaked and purified, and then inoculated on a solid medium, and cultured at 28 ° C for 24 hours; respectively, and then transferred to a seed medium at 28 ° C, 200 rpm.
  • the shaker was shake-cultured for 48 hours to obtain an evolved Bacillus megaterus seed solution and an evolved Gluconobacter oxydans seed solution;
  • the evolved Bacillus megaterium, the evolved Gluconobacter oxydans, and the mixed evolved Bacillus megaterium and the evolved Gluconobacter oxydans were respectively inoculated into a fermentation medium to make the evolved Bacillus megaterium
  • the density is 2 ⁇ 10 7 cfu/mL
  • the density of the evolved Gluconobacter oxydans is 2 ⁇ 10 8 cfu/mL, and cultured at 28° C., shaking at 200 rpm for 15 h;
  • step 2 The cells obtained in step 1 were ground in a mortar using liquid nitrogen to prepare a dry powder, and 200 mg of dry powder was weighed, placed in a centrifuge tube A, and 0.5 mL of ultrapure water was added thereto, and thoroughly mixed;
  • the total phospholipid extraction mixture is dissolved in 0.2 mL of the stock solution to prepare a sample, which is stored at -40 ° C or below.
  • a phospholipid standard was added to the sample, and the phospholipid standard was m-icosyl phosphatidylglycerol and diicosyl phosphatidylethanolamine.
  • the final concentration of the two phospholipid standards was 0.5 g/mL. ;
  • the extract is a chloroform/methanol solution containing dibutylhydroxytoluene, the volume ratio of the chloroform/methanol is 1:1, and the mass fraction of the dibutylhydroxytoluene is 0.005%;
  • the storage solution is a chloroform/methanol solution containing dibutylhydroxytoluene, the volume ratio of the chloroform/methanol is 1:1, and the mass fraction of the dibutylhydroxytoluene is 0.005%;
  • the sample added with the phospholipid standard obtained in the step (5) was detected by LC-MS, and the molecular structure and content data of the phospholipid group in the passage process of the vitamin C producing strain were obtained; LC-MS detection conditions are the same as in Example 4-1;
  • the molecular content data of the phospholipid group in the process of passage of the vitamin C production strain obtained in the step (6) were subjected to multivariate statistical analysis to obtain differential phospholipid molecular markers for distinguishing the vitamin C producing strains in different passage times; the multivariate statistical analysis method was Pareto pretreatment. Principal component analysis
  • the content of the differential phospholipid molecular markers obtained in step (7) is plotted according to different passage times, and the changes of these brick lipid molecules are observed and analyzed, and the phospholipid molecules which play a key role in the subculture process of mixed bacteria and related are found.
  • the metabolic pathway provides direction for strain modification and optimization of culture conditions for the purpose of improving 2-keto-L-gulonic acid.
  • a method for analyzing changes in phospholipid groups during the passage of vitamin C production strains the characteristics of which include the following steps:
  • the Bacillus megaterium and Gluconobacter oxydans cultured in the step (1) were separately transferred to a seed culture medium, and cultured at 35 ° C, shaking at 280 rpm for 24 hours to obtain a Bacillus megaterus seed solution and a Gluconobacter oxydans seed solution. ;
  • the three subculture strains obtained in the step (2) were streaked and purified, and then inoculated on a solid medium, and cultured at 35 ° C for 48 hours; and then transferred to a seed medium at 35 ° C, 280 rpm. Shake the shaker for 24h to obtain the evolved Bacillus megaterium seed solution and the evolved Gluconobacter oxydans seed solution;
  • the evolved Bacillus megaterium, the evolved Gluconobacter oxydans, and the mixed evolved Bacillus megaterium and the evolved Gluconobacter oxydans were respectively inoculated into a fermentation medium to make the evolved Bacillus megaterium
  • the density is 2xl0 1G cfu / mL
  • the density of the evolved Gluconobacter oxydans is 2xlO u cfu / mL
  • step 2 The cells obtained in step 1 were ground in a mortar using liquid nitrogen to prepare a dry powder, and 300 mg of the cell dry powder was weighed, placed in a centrifuge tube A, and 0.8 mL of ultrapure water was added thereto, and thoroughly mixed;
  • step 3 Repeat step 3 4 times until the cells are completely settled
  • the total phospholipid extraction mixture is dissolved in 2 mL of the stock solution to prepare a sample, and stored at -40 ° C or below;
  • a phospholipid standard was added to the sample, and the phospholipid standards were mercaptoacylphosphatidylglycerol, bis-decanoylphosphatidylethanolamine and bis-dodecanoylphosphatidic acid, so that the three phospholipid standards were finalized.
  • the concentration is 1.5 g/mL;
  • the extract is a chloroform/methanol solution containing dibutylhydroxytoluene, the volume ratio of the chloroform/methanol is 2:1, and the mass fraction of the dibutylhydroxytoluene is 0.01%;
  • the storage solution is a chloroform/methanol solution containing dibutylhydroxytoluene, the volume ratio of the chloroform/methanol is 4:1, and the mass fraction of the dibutylhydroxytoluene is 0.01%;
  • the sample obtained by the step (5) and added with the phospholipid standard was detected by LC-MS, and the molecular structure and content data of the phospholipid group in the passage process of the vitamin C producing strain were obtained;
  • the molecular content data of the phospholipid group in the process of passage of the vitamin C production strain obtained in the step (6) were subjected to multivariate statistical analysis to obtain differential phospholipid molecular markers for distinguishing the vitamin C producing strains in different passage times; the multivariate statistical analysis method was Pareto pretreatment. Principal component analysis
  • the content of the differential phospholipid molecular markers obtained in step (7) is plotted according to different passage times, and the changes of these brick lipid molecules are observed and analyzed, and the phospholipid molecules which play a key role in the subculture process of mixed bacteria and related are found.
  • Metabolic pathway It provides direction for strain modification and optimization of culture conditions for the purpose of improving 2-keto-L-gulonic acid.
  • Examples 4-2 and 4-3 are similar to the results of Example 4-1.
  • the composition of the solid medium, the seed medium, and the fermentation medium used in the present invention is selected from the medium disclosed in Chinese Patent Application No. 201110314740.9, for example:
  • Solid medium weigh 20 g of L-xanthine, 3 g of corn syrup, 3 g of beef bone, 3 g of yeast dipping powder, 1 g of urea, 10 g of peptone, 20 g of agar, 20 g of agar, KH 2 P0 4 1 g , MgS0 4
  • a method for detecting a change in a nutrient environment during the passage of a vitamin C producing strain comprises the following steps:
  • Gluconobacter oxydans stored in a volumetric 15% glycerol aqueous solution and 500 Bacillus megaterium deposited in a 15% by volume aqueous solution of glycerol in liquid nitrogen were inoculated separately. On the medium, 28 ° C, culture for 24 h; 2 seed culture:
  • the Bacillus megaterium and Gluconobacter oxydans cultured in the step (1) 1 were separately transferred to a seed medium at 28. C, 200 r / min shaker shaking culture for 24h, obtaining Bacillus megaterium seed solution and Gluconobacter oxydans seed solution;
  • Bacillus megaterium and Gluconobacter oxydans were inoculated into a new seed medium such that the density of Bacillus megaterium was 2 x 10 7 CFU/mL, and the density of Gluconobacter oxydans was 2 x 10 8 CFU/mL, at 28.
  • C 200 r / min shaker shaking culture, 24h as the passage cycle, the volume ratio of 1% for the passage ratio into the new seed medium, passaging 150 days to obtain mixed cells, in the passage of 0-150 days
  • Four sampling times were selected for 0 days, 50 days, 100 days, and 150 days, respectively.
  • the four mixed cells obtained in the step (1) 2 were streaked and purified, and then inoculated on a solid medium, and cultured at 28 C for 24 h; and then transferred to a new seed medium at 28 ° C, respectively.
  • the Bacillus megaterium seed solution and the Gluconobacter oxydans seed solution were respectively obtained; and the solution was stored in a glycerin aqueous solution having a volume concentration of 15%;
  • the Bacillus megaterium and Gluconobacter oxydans obtained in the step (1) 3 and the mixed bacteria in which the two bacteria are mixed together are inoculated into a new seed medium to make the density of Bacillus megaterium 2x10 7 CFU/mL.
  • the density of Gluconobacter oxydans is 2x10 8 CFU/mL, at 28. C, 200 r / min shaker shaking culture for 10 h;
  • the color column has a specification of 30 mx0.25 mm i.d. and an inlet temperature of 250.
  • the carrier gas is high purity helium
  • the flow rate is 0.6 ml/min
  • the split ratio is 3: 1
  • the column oven temperature rise procedure is: initial 50.
  • C keep 2 min, to 4.
  • the speed of C/min rose to 260.
  • the score map and load map for expressing the similarity and difference are obtained.
  • the closer the sample points are to each other the larger the similarity of the samples is, and the farther the distance is, the larger the sample difference is.
  • each point represents the nutrient composition, the farther away from the center point
  • the difference in the process of subculture is greater, and it can be used as a subculture to cultivate nutrient environment changes. Markers; see Figure 18, Figure 20 and Figure 22.
  • Glycerol asparagine uric acid inositol glycerol As shown in Table 5-1, 74 kinds of nutrient environmental substances in the process of passage of vitamin C production strain (Gluconobacter oxydans) were detected by the method of the present invention, among which 11 kinds of sugar and 20 kinds of amino acid There are 9 kinds of sugar derivatives, 21 kinds of organic acids, 2 kinds of fatty acids, 11 kinds of other substances such as amines and nitrogen-containing compounds.
  • Glycerol asparagine uric acid inositol hypoxanthine as shown in Table 5-2 by the method of the present invention, a total of 74 kinds of nutrient environmental substances were detected in the process of passage of the vitamin C producing strain (Bacillus megaterium), among which 11 kinds of sugar, amino acid 20 kinds, 8 kinds of sugar derivatives, 22 kinds of organic acids, 2 kinds of fatty acids, 11 kinds of other substances such as amines and nitrogen compounds.
  • Glycerol asparagine uric acid inositol hypoxanthine as shown in Table 5-3, by the method of the present invention, a total of 77 kinds of nutrient environmental substances in the process of mixing and mixing vitamin C production strains were detected, among which 11 kinds of sugars and 20 kinds of amino acids.
  • Sugar derivation There are 9 kinds of substances, 24 kinds of organic acids, 2 kinds of fatty acids, 11 kinds of other substances such as amines and nitrogen-containing compounds.
  • FIG. 18 The relative content of nutrient environmental substances subcultured by 0, 50, 100 and 150 generations of Gluconobacter oxydans was sample matrix, and principal component analysis was performed (Fig. 18).
  • the score maps can be clearly divided into four categories, and the load maps are displayed.
  • Figure 19 is a graph showing the relative content changes of molecular markers for different passage times, wherein valine, isoleucine, valine, acetyl acid, alanine, serine, 5-ketoproline, butyric acid And tryptophan increased with the passage of time, and higher than the blank medium, which is the result of the degradation of proteins by Gluconobacter oxydans.
  • the accumulation of these substances provided sufficient nutrition for the growth of large bacteria.
  • the relative content of nutrient environmental substances subcultured by Bacillus megaterium at 0, 50, 100 and 150 generations is a sample matrix, and principal component analysis is performed.
  • the score maps can be effectively divided into four categories, in the load map.
  • the five molecular markers are sequentially distributed in the direction of the first principal component, indicating a tendency of these markers to gradually change as the algebra increases.
  • Figure 21 shows the trend of the relative content of molecular markers in the culture environment of Bacillus megaterium in different passage times. Except for erythrose and 4-hydroxyproline, the content of other marker molecules is lower than that of blank medium. We can think that they can be used by big bacteria for growth and synthesis of their own substances.
  • the content of proline and glycine decreased gradually, especially in the culture environment of 150 generations of large bacteria, which was significantly lower than that of the original strain. Accumulation of more proline in the large bacteria helps to resist environmental stress; glycine plays an important role in the cell membrane, and high concentration of glycine helps to increase the permeability of the cell membrane. As the passage increases, the large bacteria on the scent The acid utilization ability is enhanced, which facilitates the dry release of the intracellular metabolites of the large bacteria in the mixed culture, thereby promoting the synthesis of 2-KLG.
  • the concentration of erythrose and 4-hydroxyproline in the culture environment was higher than that in the blank medium, which showed that they were the extracellular accumulation of large bacteria, and the concentration of these two marker molecules gradually increased with the passage time. It reached its maximum in the passage of 150 generations of large bacteria.
  • Akasaka Sugar is a synthetic precursor of aromatic amino acids and vitamin B 6 , which can help oxidize Gluconobacter to synthesize acid and improve carbon center metabolism.
  • the mixed cells were subjected to principal component analysis based on the relative content of the substances in the mixed nutrient environment at the passage time of 0, 50, 100 and 150 days.
  • the score chart shows that the mixed bacteria of 50 and 100 generations were compared. Close, the 0-generation mixed bacteria is clearly distinguished from the evolved mixed bacteria.
  • a method for detecting a change in a nutrient environment during the passage of a vitamin C producing strain comprises the following steps:
  • Gluconobacter oxydans deposited in a 10% volume aqueous solution of glycerol and 10 Bacillus megaterium deposited in a 20% by volume aqueous solution of glycerol in liquid nitrogen were inoculated separately. On the medium, 30 ° C, culture for 36 h;
  • the Bacillus megaterium and Gluconobacter oxydans cultured in the step (1) 1 were separately transferred to a seed medium at 30°C. C, 250 r / min shaker shaking culture for 36h, respectively, to obtain Bacillus megaterium seed solution and Gluconobacter oxydans seed solution; Bacillus megaterium and Gluconobacter oxydans were inoculated into a new seed medium such that the density of Bacillus megaterium was 2 x 10 8 CFU/mL, and the density of Gluconobacter oxydans was 2 x 10 9 CFU/mL at 30.
  • the three mixed cells obtained in the step (1) 2 were streaked and purified, and then inoculated on a solid medium, and cultured at 30 C for 36 h; and then transferred to a new seed medium at 30 ° C, respectively. After shaking for 36 h at 250 rpm shaker, the Bacillus megaterium seed solution and the Gluconobacter oxydans seed solution were respectively obtained; and stored in a 20% volume aqueous solution of glycerin;
  • the Bacillus megaterium and Gluconobacter oxydans obtained in the step (1) 3 and the mixed bacteria in which the two bacteria are mixed together are inoculated into a new seed medium to make the density of Bacillus megaterium 2x10 8 CFU/mL. , the density of Gluconobacter oxydans is
  • the filtrate 20 obtained in the step (2) 1 is placed in a centrifuge tube, and 100 of 0.10 mg/ml yttrium-labeled succinic acid methanol solution is added as an internal standard, and lyophilized; 80 is added at a concentration of 20 mg/mL.
  • a solution of oxyamine hydrochloride in pyridine was at 37. "Chemical reaction 90 mill in C water bath; add 80 ⁇ -methyl-N-trimethylsilane trifluoroacetamide At 37. Silanization reaction in a C water bath for 40 min;
  • the color column has a specification of 30 mx0.25 mm i.d. and an inlet temperature of 270.
  • the carrier gas is high purity helium
  • the flow rate is 0.8 ml/min
  • the split ratio is 20: 1
  • the column temperature riser is: initial 60. C, keep 3 min, to 6.
  • the speed of C/min rose to 280.
  • the relative content of the differential nutrient environmental markers is plotted according to different passage times, and the laws of the changes of these substances are observed and analyzed, and then the nutrient environment components that play a key role in the subculture process of the mixed bacteria are found, thereby revealing the mixed culture of the mixed bacteria.
  • the interaction mechanism and optimization of culture conditions between the two bacteria provide the direction.
  • a method for detecting a change in a nutrient environment during the passage of a vitamin C producing strain comprises the following steps:
  • Gluconobacter oxydans stored in liquid nitrogen at a concentration of 30% in glycerol aqueous solution and 200 Bacillus megaterium deposited in a 30% by volume aqueous solution of glycerol were inoculated separately. On a solid medium, cultured at 35 ° C for 48 h;
  • the Bacillus megaterium and Gluconobacter oxydans cultured in the step (1) 1 were separately transferred to a seed medium at 35°C. C, 280 r / min shaker shaking culture for 48 h, obtaining Bacillus megaterium seed solution and Gluconobacter oxydans seed solution;
  • the Bacillus megaterium and Gluconobacter oxydans was inoculated into a new seed medium, the density of the Bacillus megaterium is 2xlO ie CFU / mL, so that the density is Gluconobacter oxydans 2xlO u CFU / mL, at 35. C, 280 r / min shaker shaking culture, 48h as the passage cycle, with a volume ratio of 10% for the passage ratio into the new seed medium, selected 4 sampling time in the 0-150 days of passage to take 4 The same, respectively, 0 days, 50 days, 100 days, 150 days;
  • the four mixed cells obtained in the step (1) 2 were streaked and purified, and then inoculated separately on a solid medium, 35. C culture for 48 h; then transfer to new seed culture medium, shake culture at 280 rpm for 48 h at 35 ° C, respectively, to obtain Bacillus megaterium seed solution and Gluconobacter oxydans seed solution; preserved in a volume concentration of 30 % in glycerol in water;
  • the mixing step (1) 3 obtained from Bacillus megaterium and Gluconobacter oxydans strain as well as two strains were mixed together inoculated into seed medium, so that the density of Bacillus megaterium 2xl0 1G CFU / mL, Gluconobacter The density of the bacillus is 2x10 U CFU/mL at 35. C, 280 r / min shaker shaking culture for 15h;
  • the filtrate 50 obtained in the step (2) 1 was placed in a centrifuge tube, and 200 0.14 mg/ml yttrium-labeled succinic acid methanol solution was added as an internal standard, and lyophilized; 100 was added at a concentration of 20 mg/mL.
  • the pyridine solution of oxyamine hydrochloride is at 40. In the C water bath, the reaction was carried out for 120 min; then 100 ⁇ -methyl-N-trimethylsulfonyltrifluoroacetamide was added to the silylation reaction in a water bath at 40 ° C for 60 min;
  • the color column has a specification of 30 mx 0.25 mm id and an inlet temperature of 280.
  • the carrier gas is high purity helium
  • the flow rate is 0.7 ml/min
  • the split ratio is 5: 1
  • the column temperature rise program is: initial 80. C, keep 5 min, to 8. The speed of C/min rose to 300.
  • Example 5-2 and Example 5-3 are similar to the results of Example 5-1.
  • the composition of the solid medium and the seed medium used in the present invention is selected from the medium disclosed in Chinese Patent Application No. 201110314740.9, for example:
  • Solid medium weigh 20 g of L-xanthine, 3 g of corn syrup, 3 g of beef bone, 3 g of yeast dipping powder, 1 g of urea, 10 g of peptone, 20 g of agar, 20 g of agar, KH 2 P0 4 1 g , MgS0 4
  • Example 6-1
  • a method for analyzing changes in small molecule metabolites during the passage of a vitamin C producing strain which is characterized by the following steps:
  • the Bacillus megaterium and Gluconobacter oxydans cultured in the step (1) 1 were separately transferred to a seed culture medium, and shake cultured at 30, 240 rpm for 36 h, respectively, to obtain a Bacillus megaterus seed solution and a Gluconobacter oxydans seed solution. ;
  • the four mixed cells obtained in the step (1) 2 were streaked and purified, and then inoculated on a solid medium, 30 cells for 36 hours; and then transferred to a new seed medium, respectively, at 30* ⁇ , 240
  • the cultured Bacillus megaterium seed solution and Gluconobacter oxydans seed solution were respectively obtained by shaking with a rpm shaker for 36 h; and stored in an aqueous solution of glycerin having a volume concentration of 20%; 4 fermentation:
  • the evolved Bacillus megaterium and Gluconobacter oxydans obtained in the step (1) 3 and the mixed bacteria in which the two bacteria are mixed together are inoculated into a new seed medium to increase the density of the evolved Bacillus megaterium.
  • the density of the evolved Gluconobacter oxydans was 2xl0 1G CFU/mL, and cultured at 30* ⁇ , shaking at 240 rpm for 13 h;
  • the cells obtained in the step (2) 1 were prepared into a dry powder by liquid nitrogen grinding, and 50 mg of the cell dry powder were weighed and placed in three centrifuge tubes, and then 1.0 mL of the extract was added thereto, and the concentration of 50 ⁇ was 0.040 mg/ The mL ⁇ labeled succinic acid methanol solution was used as an internal standard, and mixed; 2000 111 was centrifuged 5 111111, and the supernatant was placed in three new centrifuge tubes and lyophilized;
  • Step (2) 2 to obtain three centrifugation tubes, respectively, add 50 methamine hydrochloride pyridine solution in a concentration of 20 m g / mL in a 30 water bath for 90 min; add 80 L N- Methyl-N-trimethylsilane trifluoroacetamide was subjected to a silylation reaction in a 37 water bath for 30 min;
  • the extract is an aqueous methanol solution having a volume fraction of 50;
  • the sample obtained in the step (2) 3 is introduced into the gas phase color, the column is DB-5MS, and the color column is 3 111 ⁇ 30 111 > 0.25 111111 1.
  • the inlet temperature is 280
  • the carrier gas is high-purity helium, constant pressure 91KPa, split ratio 10: 1
  • column oven temperature program is: initial 70* ⁇ , hold for 5 min, rise to 280 at 5/min for 5 min, use EI ionization Source, source temperature 250, detector voltage 2500V, ionization voltage 70 eV, Current 40 ⁇ ; mass transfer detection range 50-800 m/z; identification of small molecule metabolites using NIST 2005 database, mass spectrometry data processing and metabolite relative content determination using Masslynx 4.1 software; and by color peak area integration Treating, and comparing with the peak area of the internal standard, obtaining the relative content of small molecule metabolites;
  • the content of small molecule metabolite markers was graphed according to different passage times, and the changes of small molecule metabolite markers were observed and analyzed, and the changes of intracellular small molecule metabolites during the passage of vitamin C producing strains were detected. Furthermore, metabolite molecules and related metabolic pathways that play a key role in the subculture of mixed bacteria are discovered, in order to reveal the interaction mechanism of the two bacteria during the subculture of the mixed bacteria and to improve 2-keto-L-gulonic acid. The strains are modified and the culture conditions are optimized to provide direction.
  • FIG. 24 The relative content of small molecule metabolites subcultured with Gluconobacter oxydans of 0, 50, 100 and 150 generations was a sample matrix and principal component analysis was performed (Fig. 24).
  • the score map (Fig. 24-1) can be clearly divided into four categories, of which 150 generations are obviously far from the metabolite spectrum of 0, 50 and 100 generations, and 0 generations are close to 50 generations, indicating that the evolutionary differences are not Very big.
  • Figure 25 is a graph showing the relative content changes of molecular markers for different passage times of Gluconobacter oxydans.
  • the content of palmitic acid, oleic acid and octadecanoic acid decreased with the passage time, indicating that the cells were consumed during growth. More fatty acids produce cell membrane phospholipids, so free fatty acids are significantly reduced after 100 passages.
  • FIG. 26 is a graph showing the trend of the change in the relative content of small molecule metabolic markers of Bacillus megaterium over time in different passage times. Among them, palmitic acid and octadecanoic acid decreased significantly with the prolongation of the passage time of Bacillus megaterium, indicating that cell growth consumes more fatty acids to produce cell membrane phospholipids, so free fat is reduced. In addition, most of the amino acids, such as proline and 4-hydroxyproline, are significantly reduced, 5-oxo It also shows that Bacillus megaterium consumes a large amount of acid to maintain its own growth and synthesize intracellular proteins.
  • FIG. 28 Principal component analysis was performed on the sample matrix using the relative content of small molecule metabolites at 0, 50, 100, and 150 days of the mixed cells (Fig. 28).
  • the score map (Fig. 28-1) showed 0 generations and 50s.
  • the mixed samples of 100 and 150 generations were clearly distinguished, indicating that the mixed evolution of the strain system was significantly different from that of the original starting bacteria system during the subculture of the mixed bacteria, but the 50th and 100th generations were closer.
  • the first principal component cannot be clearly distinguished.
  • Figure 29 is a graph showing the trend of the relative content of small molecule metabolite markers mixed in different passage times.
  • a method for analyzing changes in small molecule metabolites during the passage of a vitamin C producing strain comprising the following steps:
  • the Bacillus megaterium and Gluconobacter oxydans cultured in the step (1) 1 were separately transferred to a seed culture medium, and shake cultured at 28, 200 rpm for 48 hours, respectively, to obtain a Bacillus megaterus seed solution and a Gluconobacter oxydans seed solution. ;
  • the three mixed cells obtained in the step (1) 2 were streaked and purified, and then inoculated on a solid medium, respectively, for 28 hours; then transferred to a new seed medium, and shaken at 28, 200 rpm. After shaking for 48 h, the evolved Bacillus megaterium seed solution and Gluconobacter oxydans seed solution were separately obtained; they were stored in a glycerin aqueous solution having a volume concentration of 15%;
  • the evolved Bacillus megaterium and Gluconobacter oxydans obtained in the step (1) 3 and the mixed bacteria in which the two bacteria are mixed together are inoculated into a new seed medium to increase the density of the evolved Bacillus megaterium.
  • the density of the evolved Gluconobacter oxydans was 2 ⁇ 10 8 CFU/mL, and cultured at 28* ⁇ , 200 rpm shaker for 15 h;
  • the cells obtained in the step (2) 1 were made into a dry powder by grinding the cells with liquid nitrogen, and 30 mg of the cell dry powder were weighed and placed in three centrifuge tubes, and 0.5 mL of the extract was added thereto, and the concentration of 30 ⁇ was 0.020 mg. /mL ⁇ labeled succinic acid methanol solution as internal standard, mix; 1000 111 centrifugation 10 111111, take the supernatant in three new centrifuge tubes and freeze-dry;
  • step (2) 2 to the three centrifuge tubes to add 40 to the concentration
  • the extract is an aqueous methanol solution having a volume fraction of 70%
  • the sample obtained in the step (2) 3 is introduced into the gas phase color, the column is DB-5MS, and the color column is 3 111 ⁇ 30 111 > 0.25 111111 1.
  • the inlet temperature is 250
  • the carrier gas is high-purity helium, constant pressure 80KPa, split ratio 3: 1
  • the column temperature rise program is: initial 50* ⁇ , hold for 3 min, rise to 260 at 4/min, hold for 3 min, use EI ionization Source, source temperature detector voltage 2300 V, ionization voltage 60 eV, current 30 ⁇ ; mass transfer detection range 50-800 m/z; identification of small molecule metabolites using NIST 2005 database, mass spectrometry data processing and relative metabolite content
  • the measurement was performed using Masslynx 4.1 software; and the relative content of small molecule metabolites was obtained by integrating the peak area of the color peak and comparing with the peak area of the internal standard;
  • the content of small molecule metabolite markers was graphed according to different passage times, and the changes of small molecule metabolite markers were observed and analyzed, and the changes of intracellular small molecule metabolites during the passage of vitamin C producing strains were detected.
  • a method for analyzing changes in small molecule metabolites during the passage of a vitamin C producing strain comprising the following steps:
  • Gluconobacter oxydans stored in liquid nitrogen at a concentration of 30% in glycerol aqueous solution and 500 Bacillus megaterium deposited in a 30% by volume aqueous solution of glycerol were inoculated separately. On a solid medium, culture 24;
  • the Bacillus megaterium and the Gluconobacter oxydans cultured in the step (1) 1 were respectively transferred into a seed culture medium, and shake cultured at 35, 280 rpm for 24, respectively, to obtain a Bacillus megaterium seed liquid and a Gluconobacter oxydans seed liquid;
  • the four mixed cells obtained in the step (1) 2 were streaked and purified, and then inoculated on a solid medium, respectively, and cultured for 35 hours; and then transferred to a new seed medium at 35* ⁇ , 280, respectively.
  • the cultured Bacillus megaterium seed solution and Gluconobacter oxydans seed solution were respectively obtained by shaking with a rpm shaker for 24 h; and stored in a 30% volume aqueous solution of glycerin;
  • the evolved Bacillus megaterium and Gluconobacter oxydans obtained in the step (1) 3 and the mixed bacteria in which the two bacteria are mixed together are inoculated into a new seed medium to increase the density of the evolved Bacillus megaterium.
  • the density of the evolved Gluconobacter oxydans is 2 ⁇ 10 U CFU/mL, and shaken at 35* ⁇ , shaking at 280 rpm for 10;
  • the cells obtained in the step (2) 1 were ground into a dry powder by grinding the cells with liquid nitrogen, and 60 mg of the cell dry powder were weighed and placed in three centrifuge tubes, and 1.5 mL of the extract was added thereto, and the concentration of 70 ⁇ was 0.060 mg. /mL ⁇ labeled succinic acid methanol solution as internal standard, mix; 3000 111 centrifugation 3 111111, take the supernatant in three new centrifuge tubes and freeze-dry;
  • Step (2) 2 to obtain three centrifugation tubes, respectively, add 100 methamine hydrochloride pyridine solution at a concentration of 30 m g / mL in a 40 water bath for 120 min; then add ⁇ ⁇ - ⁇ The silylation reaction of hydrazinyl-trimethylacetate in a 40 water bath for 60 min;
  • the extract is an aqueous methanol solution having a volume fraction of 60%; 4 GC-TOF/MS detection:
  • the sample obtained in the step (2) 3 is introduced into the gas phase color, the column is DB-5MS, and the color column is 3 111 ⁇ 30 111 > 0.25 111111 1.
  • the inlet temperature is 280
  • the carrier gas is high-purity helium, constant pressure 100KPa, split ratio 20: 1
  • the column temperature rise program is: initial 80* ⁇ , hold for 6 min, increase to ⁇ / ⁇ to 300* ⁇ , hold for 8 min, use EI ionization source, source temperature 260* ⁇ , detector voltage 2700V, ionization voltage 80 eV, current 50 ⁇ ; mass transfer detection range 50-800 m/z; identification of small molecule metabolites using NIST 2005 database, mass spectrometry data processing
  • the relative content of metabolites was determined using Masslynx 4.1 software; and the relative content of small molecule metabolites was obtained by integrating the peak area of the color peaks and comparing with the peak area of the internal standard;
  • the content of small molecule metabolite markers was graphed according to different passage times, and the changes of small molecule metabolite markers were observed and analyzed, and the changes of intracellular small molecule metabolites during the passage of vitamin C producing strains were detected.
  • Example 6-2 and Example 6-3 are similar to those of Example 6-1.
  • the composition of the solid medium and the seed medium used in the present invention is selected from the medium disclosed in Chinese Patent Application No. 201110314740.9, for example:
  • the strain Bacillus megaterium CGMCC No 1.459 and Gluconobacter oxydans used in the present invention are used.
  • CGMCC No 1.110 is for illustrative purposes only, but is not intended to limit the invention. It is to be understood that other strains of Bacillus megaterium and Gluconobacter oxydans can also be used in the present invention.
  • Example 7
  • Gluconobacter oxydans containing a gene encoding a kaempferol dehydrogenase gene to improve the fermentation performance of 2-keto-L-gulonic acid mixed bacteria. It is characterized by the following steps:
  • the bacterial genome extraction kit was used to extract the genome of Gluconobacter oxydans; the genomic solution 1 ⁇ 1, 5xFastPfu buffer 4 ⁇ 1, 20mM dNTP 2 ⁇ 1, 20 ⁇ primer 1 0.4 ⁇ 1, 20 ⁇ primer 2 0.4 ⁇ 1, sterile water 11.8 ⁇ 1, FastPfu Enzyme 0.4 ⁇ 1, mixed evenly in the PCR tube; PCR cycle; ⁇ PCR instrument for amplification cycle, the amplification procedure is: 95. C 2min; 95 ° C 20s, 55 ° C 35s, 72 ° C 2min, a total of 25 cycles; 72 ° C 5min; C 30min.
  • the PCR product was subjected to agarose gel electrophoresis, and the PCR product strip was excised, and the PCR amplification product was purified by an agarose gel recovery kit.
  • the sequence of the primer 1 is: 5,-CCCAAGCTTGACTGGCAGCAGCGCAAC-3'
  • the sequence of the primer 2 is: 5,-
  • the ligation product was transformed into Escherichia coli DH5 (x, chemically transformed, coated on LB solid medium containing antibiotics, and cultured at 37 C for 12 h.
  • the single colony obtained after the culture was in LB liquid medium 37.
  • the plasmid was extracted with a plasmid miniprep kit to obtain a gene encoding the kaempferol dehydrogenase gene.
  • Gluconobacter oxidans was coated on solid medium at 30. C culture for 48 hours; wash the cells with 2 ml of sterile water, water bath for 10 min, 4. C, centrifuge at 5 rpm for 5 min, collect the cells, wash once with pre-cooled sterile water, wash twice with 10% glycerol, resuspend the cells with ⁇ 10% glycerol; and obtain the kaempferol dehydrogenase gene vector obtained from step (2).
  • the aqueous solution was uniformly mixed in an electric rotor, and the ice bath was placed for 5 minutes.
  • the electric rotor was placed in a 1800V electric shock in the electrorotation apparatus; the electroshock product was uniformly mixed with the 900 ⁇ l seed medium at 30.
  • the solid medium is: L-mountain 20 g , corn syrup 3 g , beef bone 3 g , yeast dip powder 3 g, urea lg, peptone 10 g, agar 20 g, KH 2 P0 4 lg, MgSO 4 0.2 g, CaC0 3 lg, add water to 1L, adjust pH to 6.8, 121. C sterilized for 20 min.
  • the seed medium is: L-mountain 20 g , corn syrup 3 g , beef bone 3 g , yeast dipping powder 3 g, urea lg, peptone 10 g, KH 2 P0 4 lg, MgSO 4 0.2 g, CaC0 3 lg Add water to 1L and adjust pH to 6.8, 121. C sterilized for 20 min.
  • the Gluconobacter oxydans and Bacillus megaterium containing the vector encoding the kaempferol dehydrogenase gene obtained in the step (3) are inoculated into a fermentation medium to obtain Gluconobacter oxydans containing a gene encoding a kaempferol dehydrogenase gene.
  • the density is 4x10 8 cfu/ml
  • the density of Bacillus megaterium is 4x10 8 cfu/ml at 30.
  • C cultured at 250 rpm for 120 h to obtain 2-keto-L-gulonic acid.
  • the fermentation medium is: Weigh 80g of L-wheat sugar, 20g of corn syrup, 12g of urea, 12g of urea, KH 2 P0 4 lg, 0.5g of MgSO 4 , CaC0 3 lg, add water to 1L, adjust the pH to 7.0, 121 . C sterilized for 20 min.
  • HPLC High performance liquid chromatography
  • Sample preparation Take 1 mL of fermentation broth at different times in a 1.5 mL centrifuge tube, centrifuge at 10000 r/min for 3 min, take the supernatant ⁇ in a 1.5 mL centrifuge tube, and add 900 ⁇ M ⁇ 2 phase (5 mM H 2 S0 4 ) A sample that is ten times dry is obtained. After oscillating and mixing, the sample was filtered through a 0.22 ⁇ m cellulose microporous membrane to obtain a sample to be tested.
  • High performance liquid chromatography conditions Color column: bio-rad HPX-87H, mobile phase: 5 mM H 2 S0 4 , flow rate: 0.6 mL/min, column temperature: 65. C, the differential detector.
  • Example 7-1 The test results are shown in Fig. 30.
  • ⁇ Original fermentation of Gluconobacter oxydans and Bacillus megaterium
  • ⁇ Mixed fermentation of Gluconobacter oxydans and Bacillus megaterium containing the gene encoding the swainone dehydrogenase gene.
  • Example 7-1 The original Gluconobacter oxydans and Bacillus megaterium using the steps (7-1) of Example 7-1
  • the yield of 2-keto-L-gulonic acid in the fermentation condition was 87% with the fermentation conditions; the Gluconobacter oxydans containing the gene encoding the kaempferone dehydrogenase gene obtained by the method of Example 7-1 and the huge The yield of 2-keto-L-gulonic acid fermented by Bacillus mixed bacteria reached 98%; the yield of 2-keto-L-gulonic acid was increased by 12.6%.
  • Example 8-1 The yield of 2-keto-L-gulonic acid in the fermentation condition was 87% with the fermentation conditions; the Gluconobacter oxydans containing the gene encoding the kaempferone dehydrogenase gene obtained by the method of Example 7-1 and the huge The yield of 2-keto-L-gulonic acid fermented by Bacillus mixed bacteria reached 98%; the yield of 2-keto-L-gulonic acid was increased by 12.
  • a method for detecting changes in intracellular proteins of glutathione during the action of Gluconobacter oxydans comprising the steps of:
  • 3 parts of Gluconobacter oxydans were inoculated into the fermentation medium A in a volume ratio of 8%, and 3 parts of the Gluconobacter oxydans were inoculated into the fermentation medium B at a volume ratio of 8%, respectively.
  • the medium after inoculation was at 28.
  • C the fermentation is carried out at a rotation speed of 200 rpm, and the fermentation broth sample is taken at 1 h and 15 h in the fermentation process, at 4.
  • Centrifuge at 4000 rpm collect the lower layer of cells, wash with phosphate buffer of pH 7.2, immediately quench with liquid nitrogen, terminate the metabolic reaction; use liquid nitrogen to grind the cells to obtain 3 parts.
  • the quenched and disrupted cells were collected by fermentation in medium A for 1 h, 3 parts of the crushed cells were collected from medium A for 15 h, and 3 parts were fermented from medium B for 1 h to collect the quenched broken cells, 3 parts from The quenched broken cells were collected by fermentation in medium B for 15 h;
  • A is the fermentation medium: SO gL- 1 Sugar Hill Qian O gL 1 corn steep liquor, 1 g * L 1 KH 2 P0 4, 0.2 g * L 1 MgS0 4, ⁇ 12 g * L 1 of urea, with the balance water;
  • the enzyme medium B is: 80 1 hawthorn O g'L 1 corn syrup, 1 gL 1 KH 2 P0 4 , 0.2 gL 1 MgS0 4 , and 12 g*L 1 urea, 0.8 ⁇ 1.5 mg*mL 1 glutathione, the balance is water;
  • the cell lysate is: 8 mol-L 1 urea, 4% by mass of (3-[(3-cholamidopropyl)-diethylamine]-propanesulfonic acid) (CHAPS), 40 mM Tris , the balance is water;
  • the bovine serum albumin was added from the low concentration to the high concentration into the Coomassie Brilliant Blue G-250 solution, and the absorbance at each protein obtained in step 2 was determined to establish a standard curve; the protein protein of each protein solution was determined. concentration;
  • the solution obtained in the step solution 6 is mixed to obtain 3 parts of the mixed solution, and each mixed solution includes the labeled protein which is obtained by the fermentation of the crushed cells from the medium A for 1 h, and the obtained step 2-6, from the culture.
  • the crushed cells in the base A were collected for 15 h, and the labeled proteins obtained after the steps 2-6 were collected, and the quenched broken cells were collected from the medium B for 1 h to collect the labeled proteins obtained after the steps 2-6.
  • the labeled protein was obtained by fermentation of the crushed cells from the medium B for 15 h and then after the steps 2-6; C save;
  • the mixed solution obtained in 7 was subjected to Q-Tof mass identification to obtain a protein spectrum, and differentially expressed proteins of each mixed group sample were obtained by quantitative determination;
  • step (1)8 Using matlab to normalize the data obtained in step (1)8, perform principal component analysis to obtain data categories with different changes ⁇ , and obtain candidate differential proteins;
  • the candidate differential protein content obtained in step (2) was plotted according to time series, and the regularity of these protein changes was observed and analyzed, and then glutathione was found to increase the production of 2-keto-L-gulonic acid by Gluconobacter oxydans. The role. After the addition of glutathione, the intracellularly identified proteins in the fermentation of Gluconobacter oxydans are shown in Table 1. Gluconobacter oxydans intracellular protein
  • Ribosomal protein S8 family protein single-stranded DNA-binding protein (SSB) S8 family protein single-stranded DNA-binding protein
  • NADH NADH-dependent enoyl-ACP reductase 1
  • ribosomal protein L5 BL6
  • cold shock protein cspB Major cold shock protein
  • Ribosom protein L24 sorbose/sorbosone dehydrogenase ribosomal protein S17 family protein trigger factor
  • BL4 ribosomal protein L4 (BL4) cyclophilin type peptidyl-prolyl cis-trans isomerase/CLD family protein
  • Double-strand break repair protein AddB bacterial extracellular solute-binding family protein
  • DNA-binding protein HU 1 DNA-binding cobaltochelatase, CobS subunit
  • NAD(P)H q uinone oxidoreductase transaldolase
  • Glutathione S-transferase C-terminal bacterial extracellular solute-binding domain protein proteins, family 5 Middle family protein binding-protein-dependent transport system H-NS histone family protein
  • acetyl-CoA carboxylase carboxyl N-6 DNA Methylase family protein transferase, beta subunit
  • Glutamine synthetase catalytic domain transketolase Cold-shock DNA-binding domain protein conserved hypothetical protein lysyl-tRNA synthetase insulinase (Peptidase family Ml 6) family protein
  • glutathione has a heavy effect on the growth of Gluconobacter oxydans and 2-keto-L-gulonic acid production, combined with related protein changes, growth curves and acid production, indicating that glutathione is beneficial.
  • This finding provides a basis for subsequent genetic modification of Gluconobacter oxydans and provides a basis for research and control of industrial mixed bacteria processes.
  • a method for detecting intracellular protein changes during the action of glutathione on Gluconobacter oxydans comprises the following steps:
  • the quenched broken cells were collected in the base A for 1 h, 4 parts of the crushed cells were collected from the medium A for 15 h, and 4 parts of the crushed cells were collected from the medium B for 1 h, and 4 parts were cultured.
  • the crushed cells were collected by fermentation in the base B for 15 h; the fermentation medium A and the fermentation medium B were the same as those in Example 8-1;
  • the bovine serum albumin was added from the low concentration to the high concentration into the Coomassie Brilliant Blue G-250 solution, and the absorbance at each protein obtained in step 2 was determined to establish a standard curve; the protein protein of each protein solution was determined. concentration;
  • each mixed solution includes the fermentation of the quenched broken cells from the medium A for 1 h, and the labeled protein obtained after the step 2-6, from the culture.
  • the crushed cells in the base A were collected for 15 h, and the labeled proteins obtained after the steps 2-6 were collected, and the quenched broken cells were collected from the medium B for 1 h to collect the labeled proteins obtained after the steps 2-6.
  • the labeled protein was obtained by fermentation of the crushed cells from the medium B for 15 h and then after the steps 2-6; C save;
  • the mixed solution obtained in 7 was subjected to Q-Tof mass identification to obtain a protein spectrum, and differentially expressed proteins of each mixed group sample were obtained by quantitative determination;
  • step (1) 8 After matricizing the data obtained in step (1) 8 with matlab, principal component analysis is performed to obtain data categories with different changes ⁇ , and candidate differential proteins are obtained;
  • the candidate differential protein content obtained in step (2) was plotted according to time series, and the regularity of these protein changes was observed and analyzed, and then glutathione was found to increase the production of 2-keto-L-gulonic acid by Gluconobacter oxydans. The role.
  • a method for detecting intracellular protein changes during the action of glutathione on Gluconobacter oxydans comprises the following steps:
  • Gluconobacter oxydans were inoculated into the fermentation medium A at a volume ratio of 16%, and another 5 parts of the glucobacteria oxidizing bacteria were inoculated into the fermentation medium B at a volume ratio of 16%, respectively.
  • the medium after inoculation was at 32.
  • C culture fermentation under the condition of rotation speed of 250 rpm, take lh, 15 h as the point to take the fermentation liquid sample during the fermentation process.
  • Centrifuge at 6000 rpm collect the lower layer of cells, wash with phosphate buffer of pH 7.4, quench with liquid nitrogen, terminate the metabolic reaction; crush the cells with liquid nitrogen to obtain 5 parts from medium A.
  • the quenched broken cells were collected by fermentation for 1 h, 5 parts of the crushed cells were collected from the medium A for 15 h, and 5 parts were fermented from the medium B for 1 h to collect the quenched broken cells, and 5 parts from the medium B.
  • the quenched broken cells were collected for 15 h in fermentation; the fermentation medium A and the fermentation medium B were the same as those in Example 8-1;

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Abstract

Fait l'objet de cette invention un procédé d'amélioration du rendement d'acide 2-céto-L-gulonique, un précurseur de vitamine C, comprenant un procédé d'amélioration du rendement de l'acide 2-céto-L-gulonique précurseur de la vitamine C par mélange de souches afin de créer une sous-culture et par amélioration de l'interaction entre les deux souches, ce procédé comprenant les étapes de culture en milieu solide, culture de semences, isolement et purification, puis fermentation. Fait aussi l'objet de cette invention un procédé d'utilisation de la sous-culture mixte afin de détecter des modifications de la protéine, du composant phospholipidique, de l'environnement nutritionnel et du métabolite micromoléculaire lors du passage de souches pour la production industrielle de la vitamine C. Fait également l'objet de cette invention une utilisation du Gluconobacter oxydans à vecteur codant pour un gène de sorbosone déshydrogénase, un procédé de détection de modification de la protéine dans des cellules lors de l'application de glutathione au Gluconobacter oxydans et un procédé d'indice de commande de la qualité de l'extrait soluble de maïs utilisé comme matière première pour la fermentation.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103045710A (zh) * 2011-10-17 2013-04-17 天津大学 强化两菌相互作用提高2-酮基-l-古龙酸产量的方法
CN111909971A (zh) * 2020-08-14 2020-11-10 山东鲁维制药有限公司 一种酸胁迫减弱发酵产酸能力基因的混合发酵培养方法
CN114292893A (zh) * 2022-01-05 2022-04-08 山东天力药业有限公司 一种在vc混菌种子罐中添加多种微量元素缩短发酵周期提高产酸收率的方法
CN117243885A (zh) * 2023-11-15 2023-12-19 北京青藤谷禧干细胞科技研究院有限公司 一种改善皮肤的干细胞外泌体组合物及其制备方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3194559A1 (fr) 2015-11-27 2017-07-26 Technische Universität Ilmenau Procédé et dispositif pour la fermentation

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1515669A (zh) * 1993-03-08 2004-07-28 ����ҩƷ��ҵ��ʽ���� 由氧化葡糖杆菌t-100得到的l-山梨酮脱氢酶
CN1621518A (zh) * 2003-11-28 2005-06-01 华北制药集团有限责任公司 一种l－山梨酮脱氢酶及其编码基因与应用
CN101603060A (zh) * 2009-07-10 2009-12-16 天津大学 提高氧化葡糖杆菌产2-酮-l-古龙酸的方法
CN102010885A (zh) * 2010-12-07 2011-04-13 江南大学 一种提升2-酮基-l-古龙酸生产强度的方法
CN102331474A (zh) * 2011-06-20 2012-01-25 天津大学 寻找发酵用原料玉米浆的质量控制指标的方法
CN102352403A (zh) * 2011-10-17 2012-02-15 天津大学 混菌进化传代培养提高2-酮基-l-古龙酸产量的方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101338335A (zh) 2008-08-07 2009-01-07 江南大学 一种提高2-酮基-l-古龙酸发酵生产稳定性的方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1515669A (zh) * 1993-03-08 2004-07-28 ����ҩƷ��ҵ��ʽ���� 由氧化葡糖杆菌t-100得到的l-山梨酮脱氢酶
CN1621518A (zh) * 2003-11-28 2005-06-01 华北制药集团有限责任公司 一种l－山梨酮脱氢酶及其编码基因与应用
CN101603060A (zh) * 2009-07-10 2009-12-16 天津大学 提高氧化葡糖杆菌产2-酮-l-古龙酸的方法
CN102010885A (zh) * 2010-12-07 2011-04-13 江南大学 一种提升2-酮基-l-古龙酸生产强度的方法
CN102331474A (zh) * 2011-06-20 2012-01-25 天津大学 寻找发酵用原料玉米浆的质量控制指标的方法
CN102352403A (zh) * 2011-10-17 2012-02-15 天津大学 混菌进化传代培养提高2-酮基-l-古龙酸产量的方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GAO, YUN ET AL.: "Comprehensive quality evaluation of corn steep liquor in 2-keto-L-gulonic acid fermentation", JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, vol. 59, no. 18, 27 July 2011 (2011-07-27), pages 9845 - 9853 *
MA, QIAN ET AL.: "Proteomic analysis of ketogulonicigenium vulgare under glutathione reveals high demand for thiamin transport and antioxidant protection", PLOS ONE, vol. 7, no. 2, 22 February 2012 (2012-02-22), pages 1 - 12 *
NIU, JIANSHUANG ET AL.: "Effect of Corn Steep Liquor on 2-Keto-L-Gulonic Acid in Two-Step VC Fermentation", JOURNAL OF MICROBIOLOGY, vol. 31, no. 2, March 2011 (2011-03-01), pages 29 - 31 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103045710A (zh) * 2011-10-17 2013-04-17 天津大学 强化两菌相互作用提高2-酮基-l-古龙酸产量的方法
CN111909971A (zh) * 2020-08-14 2020-11-10 山东鲁维制药有限公司 一种酸胁迫减弱发酵产酸能力基因的混合发酵培养方法
CN114292893A (zh) * 2022-01-05 2022-04-08 山东天力药业有限公司 一种在vc混菌种子罐中添加多种微量元素缩短发酵周期提高产酸收率的方法
CN114292893B (zh) * 2022-01-05 2023-08-29 山东天力药业有限公司 一种在vc混菌种子罐中添加多种微量元素缩短发酵周期提高产酸收率的方法
CN117243885A (zh) * 2023-11-15 2023-12-19 北京青藤谷禧干细胞科技研究院有限公司 一种改善皮肤的干细胞外泌体组合物及其制备方法
CN117243885B (zh) * 2023-11-15 2024-01-26 北京青藤谷禧干细胞科技研究院有限公司 一种改善皮肤的干细胞外泌体组合物及其制备方法

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