128 results about "Gluconobacter oxydans" patented technology

The invention discloses a method for co-producing a plurality of saccharic acids from cellulose fuel ethanol. The method comprises the following steps: performing dilute acid hydrolysis pretreatment or dilute acid steam explosion pretreatment on plant fiber raw materials to obtain a hemicellulose pre-hydrolysate and cellulose-enriched solid material through solid-liquid separation, wherein an enzyme hydrolysate is obtained by neutralizing and performing cellulose enzyme hydrolysis on the solid material, and fuel ethanol is produced by anaerobically fermenting the enzyme hydrolysate through saccharomyces cerevisiae or zymomonas mobilis; and a plurality of saccharic acids are produced by whole-cell co-catalyzing through gluconobacter oxydans or pseudomonas fragi under an aerobiotic condition after the hemicellulose pre-hydrolysate is concentrated and neutralized. The plurality of saccharic acids can be co-produced from fuel ethanol prepared from the plant fiber raw materials, the inhibiting effect of hemicellulose pre-hydrolysate sugar fermentation can be effectively overcome, the utilization efficiency of various carbohydrate substances in the plant fiber raw material can be obviously improved, the pollution load in industrial wastewater is effectively reduced, thereby improving the economic benefit of bio-refinery of the plant fiber raw materials. The utilization rate of total carbohydrate in the raw materials is over 80%, and the carbohydrate conversion rate is more than 90%.

The invention discloses a method for improving gluconobacter oxydans to produce 2-keto-L-gulonic acid, which comprises the following steps: (1) seed culture, namely inoculating bevel gluconobacter oxydans into a seed culture medium to form seed culture solution; and (2) fermentation, namely inoculating the seed culture solution into a fermentation culture medium, oscillating the mixture in a shaking bed, carrying out fermentation culture for 48 to 96 hours, and adding a sulfhydryl compound into the mixture at any moment of the fermentation culture in a range of 0 to 36 hours to make the final concentration of sulfhydryl between 0.1 and 30mM. The method can improve the growth speed of the gluconobacter oxydans and the efficiency for producing the 2-keto-L-gulonic acid, and achieve the aim of partially replacing the companion effect of companion fungus so as to reduce the using amount of bacillus megaterium of the companion fungus, reduce the cost of the culture medium and reduce the pollution.

The invention relates to a method for producing bacterial cellulose with wheat straws / straws, comprising the following steps: (1) grinding the wheat straws or straws, soaking the ground wheat straws or straws in dilute sulfuric acid or hydrochloric acid to react, then separating the residues of the wheat straws or straws from the acid hydrolysate through pump filtration and collecting the hydrolysate for later use; (2) detoxicating the hydrolysate; and (3) taking the detoxicated hydrolysate as the carbon source in the culture medium and adding the nitrogen source to prepare the culture medium and inoculating acetobacter aceti or gluconobacter oxydans into a shaker at 25-30 DEG C and 160-250r / min to be cultured or an incubator at 25-30 DEG C to undergo static culture, thus obtaining the bacterial cellulose. The carbon source in the culture medium is good in quality, low in price and suitable for industrial production.

The invention discloses a method for producing pyrroloquinoline quinone by using gluconobacter oxydans. The method comprises the following steps: (1) fermenting the gluconobacter oxydans DSM2003 to obtain fermentation liquor; (2) carrying out high-speed centrifugation on the fermentation liquor, taking supernate and enriching by using an HP-20 type macroporous resin chromatographic column to obtain an eluted fraction which is rich in pyrroloquinoline quinone; and (3) concentrating the eluted fraction which is rich in pyrroloquinoline quinone under reduced pressure and purifying by using polyamide column chromatography to obtain a pyrroloquinoline quinone pure product. According to the method, under a common culture medium and common culture conditions, the PQQ (pyrroloquinoline quinone) yield of the gluconobacter oxydans strain can reach 125mg / L. The production and preparation method is simple in condition, fast in process and convenient for industrial production in a large scale and has important meaning for industrialization of PQQ.

The invention discloses application of gluconobacter oxydans in preparing 1,3-dioxyacetone. The gluconobacter oxydans is named gluconobacter oxydans NH-10, and preserved in the China General Microbiological Culture Collection Center with the number of CGMCC No.2709 and the preservation data of October 14, 2008. The gluconobacter oxydans NH-10 can convert glycerol in high efficiency under a condition of high concentration glycerol to produce the 1,3-dioxyacetone. Under the technical condition of the application, the conversion rate of the glycerol can reach 99.7 percent (w / w), and the yield of the 1,3-dioxyacetone can reach 97 percent, the concentration of the 1,3-dioxyacetone in the conversion solution reaches 166.2g / L, and other components are scarcely contained in the conversion solution. The microorganism strain is applicable to industrial production of dioxyacetone.

The invention provides a Gluconobacter oxydans (G.oxydans) promoter and its application. The promoter has the following nucleotide sequence: 1) a nucleotide sequence shown as SEQ ID NO:1 in a sequence table; or 2) a nucleotide sequence hybridizing with the nucleotide sequence shown as SEQ ID NO:1 under rigorous conditions and having promoter functions; or 3) a nucleotide sequence which is obtained by subjecting the nucleotide sequence defined by 1) or 2) to substitution, deletion, insertion or adding by one or more basic groups, and has over 90% homology with the defined nucleotide sequence and promoter functions. The promoter provided by the invention has the function of effective expression of the endogenous or exogenous gene in Gluconobacter oxydans, and has higher promoter activity compared with common promoters. The Gluconobacter oxydans promoter can realize application to gene expression and functional gene screening research in G.oxydans, and also can be applied to construction of 2-KGA high-yield gene engineering bacteria.

The invention discloses a recombinant plasmid for expressing vitreoscilla hemoglobin in a cell and a construction method of the recombinant plasmid, a genetic engineering bacterium of Gluconobacter oxydans (G.oxydans) and a construction method and application of the genetic engineering bacterium. The recombinant plasmid comprises a vitreoscilla hemoglobin gene vgb, a PtufB promoter of the Gluconobacter oxydans (G.oxydans) and a suitable carrier, wherein the genetic engineering bacterium is obtained by transforming recombinant plasmid into a host bacterium. The genetic engineering bacterium can be used for expressing the vitreoscilla hemoglobin in the cell, can be used for improving the yield of biomass and catalysate 1, 3-dihydroxyacetone under the condition of not changing the existing equipment and energy consumption pressure and provides an effective path for solving the problem of in sufficient oxygen supply in the cell cultivation and catalytic process of the Gluconobacter oxydans (G.oxydans).

The invention discloses a strain in the field of genetic engineering and a method for producing dihydroxyacetone (DHA) by using the strain. The method is characterized in that: modified Gluconobacter oxydans is used to bioconvert glycerin into dihydroxyacetone; sldAB gene expression is added in the modified Gluconobacter oxydans through modification, and mgdh gene and madh gene are removed from the modified Gluconobacter oxydans; and the modified Gluconobacter oxydans is further subject to the adaptive evolution on a medium which uses glucose as the sole carbon source. The genetically engineered strain disclosed herein can grow well on the medium which uses glucose as the sole carbon source, overcomes the defects that wild type Gluconobacter oxydans can only use relatively expensive sorbitol and mannitol as the effective carbon source, and saves the production cost. In addition, compared with a genetically engineered strain GAN cultured on sorbitol, the genetically engineered strain GAN cultured on glucose has higher capability of producing DHA.

The invention relates to a method for producing bacterial cellulose with wheat straws / maize straws, comprising the following steps: (1) grinding the wheat straws or maize straws, soaking the ground wheat straws or maize straws in dilute sulfuric acid or hydrochloric acid to react, then separating the residues of the wheat straws or maize straws from the acid hydrolysate through pump filtration and collecting the hydrolysate for later use; (2) detoxicating the hydrolysate; and (3) taking the detoxicated hydrolysate as the carbon source in the culture medium and adding the nitrogen source to prepare the culture medium and inoculating acetobacter aceti or gluconobacter oxydans into a shaker at 25-30 DEG C and 160-250r / min to be cultured or an incubator at 25-30 DEG C to undergo static culture, thus obtaining the bacterial cellulose. The carbon source in the culture medium is good in quality, low in price and suitable for industrial production.

The invention discloses a Gluconobacter oxydans, which is classified and named as Gluconobacter oxydans NH-10, and preserved in China General Microbiological Culture Center of Microbial Culture Collection Management Committee with the preservation number of CGMCC No.2709. The invention further discloses a D-xylulose preparation method that uses the Gluconobacter oxydans. The strain obtained by screening can transform D-Arabitol so as to generate D-xylulose in high efficiency, the D-xylulose preparation method reaches the highest D-Arabitol transformation ratio of 99.5 percent (w / w) and the D-xylulose yield ratio of 95 percent, the D-xylulose concentration of a conversion fluid can reach 95g / L, and the conversion fluid hardly contains other ingredients.

The invention discloses a soil remediation method for removing heavy metal lead. According to the method, the biological fermentation characteristic of gluconobacter oxydans is creatively utilized; lactonization reaction is conducted in the gluconobacter oxydans in presence of D-sorbitol and various sorts of nutritional ingredients inherent in soil, and finally L-ascorbic acid (vitamin C) is produced through fermentation; and then the L-ascorbic acid can be bonded with the heavy metal lead to produce non-toxic inorganic salt with the density being obviously larger than that of soil particles, and the heavy metal lead can be separated and removed from the soil through vibration sorting operation. Meanwhile, by the adoption of the L-ascorbic acid produced in the way that the D-sorbitol participates in biological fermentation reaction, the cost is far lower than that of adding L-ascorbic acid into the soil directly, and the material cost of soil remediation treatment can be greatly reduced.

The invention discloses a method for improving gluconobacter oxydans for producing 2-keto-L-gulconic acid, and belongs to the technical field of genetic engineering. On the basis of genetic engineering transformation, adaptor protein SH3 and ligand protein SH3lig of the adaptor protein, a CutA gene and a key enzyme gene in saccharic acid transformation are subjected to fusion expression, and G.oxydans is transformed to produce 2-KLG in a one-step fermentation manner, so that the yield of 2-KLG is finally increased to be 40.3 g / L, that is, the yield is increased by 24.4% when being compared with that of a one-step engineering bacterium of which CutA is not expressed; in addition, due to the thermal resistance of foreign protein, the thermal resistance of the engineering bacterium is further improved. Enzyme cross-linking inside cells can be achieved by expressing the foreign protein CutA, continuous catalytic reaction of a plurality of enzymes can be facilitated, and the method has significance in is one-step fermentation production of vitamin C.

The invention discloses a method utilizing mixed bacteria evolution subculturing to improve the 2-keto-L-gulonic acid yield, which comprises the following steps of: (1) solid culture; (2) seed culturing: the bacillus megaterium and gluconobacter oxydans are inoculated into a new seed culture medium, are shake cultured in a shaker of 200-280r / min at 28-35 DEG C, 24h-48h is taken as the passage period, the volume ratio of 1%-10% is taken as the passage ratio to inoculate into the new seed culture medium and passages for 50-80 days; (3) purification; and (4) fermentation. The method utilizes themixed bacteria evolution subculturing to culture for tens of generations, the growth rate of the bacillus megaterium and gluconobacter oxydans and the efficiency of utilizing the gluconobacter oxydans to produce 2-keto-L-gulonic acid can be obviously improved, accordingly, the utilization rate of the culture medium can be improved, and the L-sorbose transformation efficiency is improved about 10%.

The invention discloses a method of co-catalytically synthesizing various saccharic acids by virtue of the synergism of metal ions and a selective regulation whole-cell, and relates to the technical field of synthesizing the saccharic acids by biologically catalyzing sugar. The method is mainly characterized in that in a mixed sugar solution or lignocelluloses hydrolysate containing glucose and xylose, 1g / L to 10g / L of gluconobacter oxydans is used as a biological catalyst to co-catalyze the glucose and the xylose under an oxygen-supply condition so as to synthesize the saccharic acids. Metal salt of a given concentration containing zinc ions and trivalent iron ions is added so as to selectively inhibit the catabolism of a cell on the gluconic acid, but a dehydrogenation catalytic reaction of the xylose and the glucose is hardly affected, thus an effect of the cell for co-catalyzing and high-efficiently synthesizing a gluconic acid (salt) and xylonic acid (salt) product can be further achieved, and the reaction time is effectively shortened. By adopting the method, the utilization rate of the glucose and the xylose reaches 100 percent, and the yield of the gluconic acid is more than 70 percent, and the yield of the xylonic acid is more than 92 percent, and the total concentration (mass concentration) of the product gluconic acid (salt) and xylonic acid (salt) can be more than 30 percent.

The invention relates to the technical field of fermentation, and discloses a method for fermentation production of vitamin C precursor 2-keto-L-ulonic acid. The method is that the mixing of Go fungus and Kv fungus forms one-step coversion from D-sorbitol to 2-keto-L-ulonic acid, simplifies the production technology, shortens the fermentation cycle, and reduces the production cost. Meanwhile, the mixed culture of the Go fungus and the Kv fungus is simple, is not easy to cause microbiological contamination, is stable, can utilize an original fermentation tank to perform fermentation, and is easy for realizing industrialization. Furthermore, the method for fermentation production of precursor 2-keto-L-ulonic acid adopts gluconobacter oxydans, with shunt metabolism-related genes of sorbose / sorbitol being knocked out, to be matched with the Kv to subjected to mixed fermentation, prevents the Go fungus and the Kv fungus from competitively fighting for substrate sorbose to a certain degree, and improves the conversion efficiency.

The invention relates to a method for producing bacterial cellulose with wheat straws / spruces, comprising the following steps: (1) grinding the wheat straws or spruces, soaking the ground wheat straws or spruces in dilute sulfuric acid or hydrochloric acid to react at 90-240 DEG C, then separating the residues of the wheat straws or spruces from the acid hydrolysate through pump filtration and collecting the hydrolysate for later use; (2) detoxicating the hydrolysate; and (3) taking the detoxicated hydrolysate as the carbon source in the culture medium and adding the nitrogen source to prepare the culture medium and inoculating acetobacter aceti or gluconobacter oxydans into a shaker at 25-30 DEG C and 160-250r / min to be cultured or an incubator at 25-30 DEG C to undergo static culture, thus obtaining the bacterial cellulose. The carbon source in the culture medium is good in quality, low in price and suitable for industrial production.

The invention relates to a method for preparing bacterial cellulose by using bagasse. The method comprises steps of: (1) drying and crushing the bagasse, soaking the bagasse in acid with a solid-liquid ratio of 1:5-1:20, then reacting at 100 DEG C-240 DEG C for 50-90 min, filtering after the reaction, and collecting a hydrolysate; (2) detoxifying the hydrolysate; and (3) using the detoxified hydrolysate as a carbon source for a medium, adding 0.1-2% of a nitrogen source, and sterilizing at 121 DEG C for 15-20 min to obtain a culture medium; inoculating bacillus aceticus or Gluconobacter oxydans with an inoculation amount of 5%-10%; and conducting shaking culture at 25-30 DEG C with 160-250rpm or conducting static culture at 25-30 DEG C for 6-25 days, so as to obtain bacterial cellulose. The culture medium carbon source prepared by the invention has good quality and low price, and is suitable for industrialized production.

This invention relates to an novel Gluconobacter oxydans 2-ketoreductase useful for the synthesis of chiral alcohols. Also related are isolated nucleic acids encoding G. oxydans 2-ketoreductase enzymes, and enzyme fragments and variants thereof, as well as vectors and host cells comprising these nucleic acids. Further related are isolated G. oxydans 2-ketoreductase polypeptides, and fragments and variants thereof, and antibodies that specifically bind to G. oxydans 2-ketoreductase polypeptides, fragments, or variants. The invention also relates to methods of obtaining isolated G. oxydans 2-ketoreductase nucleic acids, polypeptides, and antibodies, and methods of using G. oxydans 2-ketoreductase in various reactions for industrial or pharmaceutical applications.

The invention discloses a gluconobacter oxydans nonresistant marker gene knockout system, and belongs to the technical field of genetic engineering. According to the invention, through a homologous recombination technique, upp genes in G.o xydans are knocked out, so that a upp genetic deficient strain is obtained; a recombinant integrating plasmid containing a upp gene which can be subjected to good transcribed expression in G.o xydans and has a conditional lethal function, a resistance maker kanamycin resistant marker gene for the transformant selection of G.o xydans and an MCS polycloning site is built. The plasmid is applicable to the knockout and replacement of G.o xydans chromosome genes; compared with gene knockout implemented by taking a resistant marker gene as a selection marker, the system disclosed by the invention has the following two advantages: no resistant gene is introduced to a G.o xydans genome, so that a polar effect of the resistant gene on upstream and downstream genes is avoided; by using the knockout system, the multiple knockout and replacement of chromosome genes can be performed in a same strain.

The invention discloses a gradient intensity promoter of gluconobacter oxydans and belongs to the field of genetic and metabolic engineering. Sequences between constitutively expressed genes and previous genes are selected from the gluconobacter oxydans and preliminarily screened by promoter prediction software, an obtained high-score potential strong promoter is subjected to qualitative and quantitative detection through a fluorescent microscope and a fluorescence microplate reader respectively by using enhanced green fluorescence protein (EGFP) as a reporter gene, and finally promoters with an intensity sequence of P[tuf-WSH003]>P[tuf-621H]>P[sldh]-P[psldh]-P[sod]>P[aldh] are selected from G.oxydans WSH-003. A series of gradient promoters and a novel strong promoter are screened out, and great help is provided for genetic and metabolic engineering reform of the industrial production strain G.oxydans WSH-003, especially adjustment of gene transcription level.

The invention discloses a pyrroloquiniline quinone (PQQ) synthetic gene cluster and a coded protein system thereof. The protein system consists of the following proteins: 1) proteins shown in SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9 and SEQ ID No.11; or 2) proteins which are derived from the proteins 1) with the amino acid sequences shown in SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9 and SEQ ID No.11 through replacing, deleting or adding one or more of amino acid residues, and have the same activities. The invention also provides the gene cluster for coding the protein system, wherein the sequence of the gene cluster is shown in SEQ ID No.1. The PQQ synthetic gene cluster and coded protein system provided by the invention play important roles in the PQQ biosynthesis, the improvement and construction of PQQ producing strains, the increase of small bacterium saccharic acid conversion efficiency and the construction of alcohol saccharic acid one-step conversion engineering bacteria.

The invention relates to the reuse of shrimp and crab waste, specifically a method for simultaneously producing shrimp and crab xanthin and chitosan by using shrimp and crab waste, using the synergistic effect of Bacillus subtilis and Gluconobacter oxydans to add glucose to ferment Shrimp and crab skin and crab head are desalinated and deproteinized, and then fermented by Streptococcus thermophilus, Lactobacillus acidophilus, and Lactobacillus bulgaricus for one week to decalcify and decolorize, use chitin deacetylase to treat chitin, and use leather Glucosamine can be obtained by processing Plasmomonas Shigella, a lambs negative bacteria. During the fermentation process, there is no need to add a large amount of acid and alkali, which reduces environmental pollution, and microbial fermentation saves costs compared with enzyme fermentation, and the content of active ingredients obtained is higher, which realizes the complete utilization of active ingredients of raw materials, and is economically feasible.

The invention provides a method of fementing shrimp heads to prepare active substances, chitin and organic acidity calcium, wherein Bacillus licheniformis and Gluconobacter oxydans are utilized to ferment the shrimp heads to prepare the active substances, the chitin and the organic acidity calcium. The method utilizes synergistic effects of the Bacillus licheniformis and the Gluconobacter oxydans to ferment the shrimp head, is free of adding any acid and alkali, and meanwhile uses tap water to ferment, thereby greatly reducing production cost. The obtained broth is rich in shrimp incense, free of fishy smell and bitter taste, and simultaneously rich in amino acids, polyphenol, compound enzymes and a plurality of beneficial components, and can be used for producing flavourings, hydrolyzed protein products, functional foods, health products and other fields. By using the method provided by the invention, a plurality of active substances can be simultaneously separated and prepared from a batch of shrimp heads, such as hydrolyzed proteins rich in the amino acids, the polyphenol and a plurality of active substances; food-grade chitin; and calcium gluconate relatively appropriate for the body to absorb, thereby realizing comprehensive utilization of effective components in the raw materials.

This invention relates to Gluconobacter oxydans mutant (CGMCC No. 1184) and its application in preparing 6-(2-hydroxyethyl) amino-6-deoxy-alpha-L-sorbfuranose by microbe conversion. The Gluconobacter oxydans mutant has such advantages as high stability, mild and controllable culture conditions, rapid propagation and short conversion time.

The invention relates to a method for increasing Gluconobacter oxydans biomass by fermenting with an oxygen-carrier-added culture medium. An appropriate oxygen carrier is added into a culture medium to provide an oxygen carrier fermenting system, so that the concentration of dissolved oxygen in the culture medium is greatly increased without adding other substances under the condition of no extra energy consumption, thereby greatly increasing the fermentation biomass.

The invention provides a method for rapidly and quantitatively detecting a mutant strain for a miglitol key intermediate, and the strain thereof. The method comprises the following steps: performing fermentation culture on a mutagenized strain obtained by mutagenizing wild Gluconobacter oxydans, taking obtained fermentation-cultured mutated strain wet cells into a substrate reaction solution, completing a conversion reaction at 15 DEG C, centrifuging the obtained reaction solution, measuring the content of 6NSL in the obtained supernatant, and performing screening according to the level of the6NSL content to obtain the high-vitality mutated strain for synthesizing the miglitol key intermediate. The detection method has the characteristics of simplicity, fastness, high specificity and goodrepeatability, and can be further applied to the screening of the high-vitality Gluconobacter oxydans and the effective acceleration of the breeding process, and the screened mutated strain has a significantly improved catalysis effect on the conversion synthesis of the miglitol key intermediate 6NSL, and is suitable for industrial large-scale production of miglitol.

The invention provides a method for producing dihydroxyacetone by using gluconobacter sp. CGMCC No. 5146. The method comprises the following steps of: performing initial fermentation culture by using a culture medium using sorbitol as a substrate till the thalli of the gluconobacter sp. are grown to a stable stage, putting the obtained solution containing the thalli and the dihydroxyacetone product into a membrane bioreactor, performing continuous fermentation by using a culture medium using glycerol as a substrate, continuously filtering the fermentation solution containing no thalli throughthe membrane bioreactor, meanwhile, constantly supplementing fresh culture medium using glycerol as the substrate to the membrane bioreactor, separating the dihydroxyacetone in the fermentation solution, and purifying the dihydroxyacetone. When the glycerol concentration of the substrate is 60g / L, the concentration of the dihydroxyacetone (DHA) is 56.75g / L, the volume yield of the DHA is 9.02 g / (L. h), the conversion rate of the glycerol reaches over 90 percent, and the yield of the DHA reaches over 80 percent; and the technical bottlenecks of low yield and high cost in a microbial fermentation method for preparing the dihydroxyacetone are broken. The method has the characteristics of simplicity and convenience in operation, energy conservation, high production efficiency and the like, and is suitable for industrialized large-scale production.

The invention discloses a method for improving the yield and production intensity of gluconobacter oxydans sorbose and belongs to the technical field of fermentation engineering. A related gene is formed by knocking out D-sorbitol or an L-sorbose metabolism by-product in gluconobacter oxydans, the formation of the by-products is reduced, the efficiency of converting the D-sorbitol into the L-sorbose is improved, and accordingly the yield and production intensity of the L-sorbose are improved. The L-sorbose conversion rate of a constructed recombinant strain G.oxydan-11 reaches 96.12% comparedwith control bacteria and is improved by 4.47% compared with that of wild bacteria, tthe production intensity is 14g / L h, and the fructose by-product content is only 5.6g / L is decreased by 45.6% compared with that of the wild bacteria.

The invention discloses a method for detecting intracellular protein changes in a process of acting on gluconobacter oxydans by glutathione. The method comprises the following steps of: 1, determination of an intracellular protein: (1) collecting and quenching the cell, (2) extracting the intracellular protein, (3) determining protein concentration, (4) precipitating the protein, (5) subjecting the protein to reduction and enzymolysis, (6) labeling the protein, (7) mixing solutions, (8) identifying the protein through differential expression; 2, main component analysis; and 3, process analysis. By utilization of the method disclosed by the invention, the important proteins in a fermentation process can be found out by revealing the change rule of the intracellular protein after the gluconobacter oxydans is acted by the glutathione, the change rule of the content of the proteins provides a basis for learning the internal mechanism of the fermentation process, so that the fermentation process is further optimized, the yield of Vitamin C is increased, and a theoretic basis is provided for carrying out molecular modification on the gluconobacter oxydans. Meanwhile, new concept and method are provided for researching the industrial mixed fermentation process.

The invention discloses a method for detecting nutrition environment change in a subculture process of a vitamin C production strain. The method comprises the following steps of: subculture of mixed strains, detection on matters in a nutrition environment, analysis on main ingredients and analysis on the process. The method can be used for finding important matters influencing the subculture nutrient environment from revelation of the influence of the mixed strain subculture on bacillus megaterium, gluconobacter oxydans and mixed strain systems, and the change rules of the contents of the matters provide fundamental basis for understanding the mechanism of action of the subculture to promote the growth of gluconobacter oxydans and the production of 2-keto-L-gulonic acid hydrate, thus providing fundamental basis for further optimizing the fermentation process and improving the yield of vitamin C.