15 results about "Shake-Flask Culture" patented technology

The application belongs to the technical field of microbial fermentation, and discloses soy sauce starter and a preparation method and application thereof. The preparation method of the soy sauce starter comprises the following steps: extracting a starter substrate, performing squeezing treatment, obtaining wet material and clear liquid; filtering the clear liquid to obtain filtered clear liquid; taking dry material and adding the wet material to obtain raw material; sterilizing the raw material to obtain starter material; inoculating spore powder of Aspergillus oryzae secondary strain into the starter material to obtain inoculated starter material; and culturing the inoculated starter material through starter culture including primary shake flask culture and secondary shake flask culture to obtain soy sauce starter. The preparation method innovatively constructs a synergistic regulation system of "physical structure improvement-nutrition precise supplement", solves the environmental protection treatment problem of the starter substrate, significantly improves the sensory quality and spore production effect of the soy sauce starter, and provides a sustainable development solution with economic and environmental protection benefits for the traditional fermentation industry.

This invention relates to the field of fungal strain cultivation technology, and particularly to a method for isolating and cultivating fungal strains from matsutake fruiting bodies. The method involves collecting matsutake mushrooms that are 70%–80% mature and free from insects, disinfecting them with alcohol, longitudinally splitting them in half, removing the flesh from the cap and stipe, and inoculating them onto a slant culture medium to obtain a primary strain. This primary strain is then inoculated into a culture medium and shaken to obtain a primary shake flask strain, which is then inoculated into a culture medium. The strain is then shaken and cultured, and the concentration of metabolites, dissolved oxygen, and pH in the culture medium are collected in real time to construct a strain growth time prediction model to predict the stage culture duration. The cell proliferation trend is determined based on the number of matsutake mycelial cells. Based on a comparison of the actual culture time during the initial adjustment period and the stage culture time, the adjustment method for the logarithmic growth phase of the current batch is determined. Based on a comparison of the shake flask culture time and the predicted culture time, the adjustment method for the initial culture conditions of the next batch is determined. This invention effectively optimizes shake flask culture conditions and improves strain quality.

The application discloses a method for improving cellulase activity by using a combination of an endogenous promoter and an associated 5' UTR of Trichoderma reesei and an engineering strain constructed by the method. TrAE The 5' UTR upstream of the TrAE gene TrAE Overexpressing a transcriptional regulator xyr1 The expression cassette of the gene is transformed into Trichoderma reesei Δ ku70 Rut‑C30 The application also discloses a high-yield cellulase Trichoderma reesei strain QEaeuX, and a method for producing high-activity cellulase by using the strain. ku70 Rut‑C30 The cellulase activity produced by the strain in a 96 h shake flask culture is 2.6 IU / mL, which is 101% higher than that of the original strain Δ The application has a good industrial development and application prospect.

The application discloses a kind of based on low salt culture and feeding optimization's high-efficiency fermentation method of schizochytrium, comprising: S1.low salt culture medium is used to carry out multiple rounds of screening and domestication to schizochytrium TKD-1, obtain the schizochytrium species of low salt high-yield DHA;S2.the schizochytrium species of good breeding is sequentially passed through shake flask culture, primary seed tank expansion culture and secondary seed tank expansion culture, obtain secondary seed liquid;S3.secondary seed liquid is connected into fermenter, carries out fermentation, obtains schizochytrium fermentation liquor;Wherein, in the fermentation process, the way of feeding is used to add glucose to fermenter, while detecting the glucose concentration in fermentation liquor on line to dynamically adjust the feeding rate of glucose;S4.after fermentation, enzyme hydrolysis method is used to break up bacteria, after centrifugation, extract DHA, obtain the oil rich in DHA.The method provided by the application can shorten the fermentation period, improve the biomass and DHA yield of schizochytrium, and reduce the production cost.

The invention discloses a bioengineering method for efficiently synthesizing lactoyl-N-tetrasaccharide, and belongs to the field of biotechnology and food fermentation engineering. The method comprises the following steps: firstly, carrying out combined regulation on lgtA, wbgO, galE, galT and galK genes in a lactoyl-N-tetrasaccharide synthesis pathway to optimize the expression dose of pathway genes, then, sequentially knocking out collateral metabolic pathway genes lacZ, wecB, ugd, nagB, pfkA, gloA and setA on a chromosome of escherichia coli BL21 (DE3) by utilizing a CRISPR-Cas9 technology, and respectively integrating a nucleotide glycometabolism key gene cluster galE-galT-galK at a yeeJ site, so as to obtain the lactoyl-N-tetrasaccharide synthesis pathway. A core glycosyl transferase gene lgtA-wbgO is integrated at a caiB site, and finally an engineering strain capable of efficiently synthesizing lactoyl-N-tetrasaccharide is constructed. The engineering bacterium can be used for realizing efficient synthesis of lactoyl-N-tetrasaccharide in a restrictive glycerol culture medium by taking lactose as a substrate. Under a shake-flask culture condition, the ability of the escherichia coli for synthesizing the lactoyl-N-tetrasaccharide is improved from 3.41 g / L to 11.15 g / L, and the yield of the lactoyl-N-tetrasaccharide reaches 125.35 g / L in fed-batch culture of a 3 L fermentation tank.

The application discloses a TteUPO mutant and application thereof, and belongs to the technical field of bioengineering and enzyme engineering.The TteUPO mutant disclosed by the application connects the nucleotide sequence of a short-chain non-specific peroxidase with a SUMO and 8HIS tag to a pET28a vector, obtains a recombinant vector, transforms E.coli BL21 (DE3), and obtains a genetically engineered bacterium; and the mutant is obtained through unit point mutation.The application uses an E.coli autoinduction system, can make shake flask culture grow to high density in a short time, contains rich nutrients in the culture medium, and does not need to monitor cell density and add IPTG in the expression process.Meanwhile, a hematin precursor 5ALA is added in the E.coli autoinduction culture medium, and finally, a high-activity short-chain non-specific peroxidase is obtained, and the enzyme after purification is reacted with anisole under the condition of hydrogen peroxide flow addition to obtain a product.

The application discloses an expression system suitable for short-chain non-specific peroxidase and application thereof, and belongs to the technical field of enzyme engineering and genetic engineering. The expression system disclosed by the application connects the nucleotide sequence of short-chain non-specific peroxidase with a SUMO and 8HIS label to a pET28a vector to obtain a recombinant vector, and the recombinant vector is used to transform Escherichia coli BL21 (DE3) to obtain a genetically engineered bacterium; the genetically engineered bacterium is cultured in a ZYM5052 liquid culture medium by using a two-stage temperature control method, and 5ALA is added to the culture medium. The application uses an Escherichia coli self-induction system, can make shake flask culture grow to a high density in a short time, and the culture medium contains rich nutrients, and the cell density does not need to be monitored and IPTG does not need to be added during the expression process. In addition, a hemin precursor 5ALA is added to the Escherichia coli self-induction culture medium, and finally, short-chain non-specific peroxidase with high activity is obtained.

This invention discloses the application of isocitric acid in the control of bacterial wilt in plants, wherein the isocitric acid is derived from tomato root exudates. The inhibitory effect of isocitric acid on *Ralstonia solanacearum* was evaluated through shake-flask culture experiments and plate inhibition zone experiments, and the control effect of isocitric acid on bacterial wilt was determined using greenhouse pot experiments. The results showed that within the concentration range of 10–200 μM, isocitric acid significantly inhibited the growth of *Ralstonia solanacearum*, forming a distinct inhibition zone. Under greenhouse pot conditions, applying 0.2–0.5 mg / kg of isocitric acid to the soil effectively reduced the incidence of bacterial wilt in tomatoes, with a disease control rate of 21.43%–52.38%. Furthermore, isocitric acid can indirectly inhibit the occurrence of bacterial wilt by influencing the soil bacterial community. The isocitric acid used in this invention is derived from natural plant root exudates, possessing advantages such as environmental friendliness and high safety, and can be used as a novel inhibitor of tomato bacterial wilt, showing promising application prospects in the field of green control of plant diseases.

The present application relates to the field of biotechnology, and more particularly to a reagent and method for high-throughput plate screening of 3-sialyltransferase mutants. The present application realizes efficient screening of 3SL high-yield mutants in large-scale mutant libraries by adopting solid plate color screening technology combined with pH indicator. The method quickly identifies the target mutant by directly observing the color change on the plate, thereby omitting the cumbersome steps of strain construction, sequencing, transfer and shake flask culture in the traditional screening process, significantly improving the screening speed and efficiency. Compared with the prior art, the present application not only greatly shortens the experimental time and simplifies the operation process, but also reduces the requirements for human resources and equipment, providing great convenience for screening of enzyme mutant libraries. The technical advantage of the present application is that it is fast, efficient and low-cost, and is expected to be widely applied in the fields of biomedicine and food science.

The invention relates to a method for efficiently synthesizing purple membrane protein by using photosensitive halophilic microorganisms. The preparation method comprises the following steps: (1) adding casein amino acid, a yeast extract, sodium citrate, MgSO4. 7H2O, KCl, FeSO4 and NaCl into ultrapure water to prepare a high-salt culture medium, and adjusting the pH value to 7.0-7.2 by using NaOH; (2) inoculating the high-salt culture medium with a halobacterium salina flora, and carrying out enrichment culture in a shake flask; (3) MgSO4. 7H2O, KCl, FeSO4 and NaCl are added into ultrapure water, sodium acetate and ammonium chloride are supplemented at the same time, a synthetic culture medium is prepared in the ultrapure water, and the pH is adjusted to 7.0-7.2 with NaOH; (4) A, introducing the synthetic culture medium in the step (3) into a shake flask inoculated with halobacterium salina groups, and carrying out enrichment culture in a constant-temperature shaking table: B, dividing the culture conditions in the step A into an illumination group and a dark group according to whether illumination is provided or not, and keeping the operation at a high-speed rotating speed without changing other conditions; and C, carrying out stable operation of shake-flask culture in the step B, and finally synthesizing the purple membrane protein. The method effectively improves the yield of purple membrane protein.

The application discloses a high-yield resveratrol Yarrowia lipolytica engineering strain and construction and application thereof, and belongs to the technical field of synthetic biology and metabolic engineering. The Yarrowia lipolytica engineering strain provided by the application can realize high-yield resveratrol by using glucose as a substrate, and the yield of resveratrol reaches 3.157 g / L in shake flask fermentation, and the yield of resveratrol is increased to 30.7 g / L in 5L scale batch feeding fermentation, which is the highest value reported at present.

The invention discloses a preparation method of stropharia rugoso-annulata cultivated species. The preparation method comprises the following steps: selecting pollution-free stropharia rugoso-annulata slant mother species with white and robust hyphae and a PDA (potato dextrose agar) substrate; preparing a shake flask liquid culture medium, sterilizing and inoculating; carrying out shake-flask culture and hypha scattering; preparing a cultivation bag and sterilizing; and carrying out sterile inoculation and spawn running culture and the like. According to the method, by optimizing a culture medium formula, an inoculation process and culture conditions, the seed production time of the stropharia rugoso-annulata is effectively shortened, efficient preparation is achieved, the quality of the obtained cultivated seeds is good, and the efficient and high-quality requirements of modern agriculture are met.

The invention discloses a solid waste aerobic fluidized bed biological treatment method, and belongs to the technical field of waste biological engineering treatment.The solid waste aerobic fluidized bed biological treatment method comprises the steps that firstly, a high-activity aspergillus oryzae strain is obtained through test tube slant culture, liquid shake-flask culture and three-stage seed tank amplification culture; a special culture medium containing peanut straw powder, a composite nitrogen source and vitamins is adopted in the culture process, and the proper temperature, rotating speed and ventilation volume are regulated and controlled to guarantee the activity of the strains; mixing the solid waste with a bulking agent and aspergillus oryzae liquid, building a heap on an aerobic fluidized bed for reaction, driving air circulation to supply oxygen through biological heat, controlling the heap height, humidity, temperature and material turning period, and reacting for 7-25 days to obtain the aspergillus oryzae converter. The method disclosed by the invention can be suitable for various solid wastes in industry, agriculture, forestry and the like, and diversified resource conversion of the wastes is realized. The method is controllable in process, simple and convenient to operate, free of additional forced oxygen supply equipment, low in operation cost, environment-friendly and economical.

This invention provides a sodium hyaluronate seed culture and its preparation method. It uses *Streptococcus vesicae* as the production strain and glucose as the carbon source. The culture medium contains 0.5%–1% monosodium glutamate (MSG), and the glucose is separately sterilized at a temperature lower than that of the culture medium. The sodium hyaluronate seed culture is prepared using the following steps: strain resuscitation, strain transfer, and seed tank culture, without any shake-flask culture. By omitting the shake-flask culture step, optimizing the culture medium ratio, and using glucose for separate low-temperature sterilization, the preparation cycle is shortened, the risk of contamination and carbon source loss is reduced, the cell concentration is increased, and the fermentation cost is reduced. This significantly improves the cell concentration of the seed culture and the subsequent fermentation yield, adapting to different scale production needs and effectively reducing the production cost of sodium hyaluronate fermentation.

The invention discloses a method for improving the fermentation yield of human milk oligosaccharides, belongs to the technical field of human milk oligosaccharides, and solves the problems of how to optimize a fermentation process and realize effective synthesis of 2 '-FL by taking glucose and lactose as substrates due to the fact that a microbial whole-cell synthesis method of 2'-FL has the characteristics of mild conditions, low cost and the like and is the most common 2 '-FL synthesis method. Comprising the following steps: S1, transferring activated glycerol tube seeds into a slant culture dish for culture, and then inoculating the seeds into a seed shake flask for culture; s2, inoculating the seeds in the step S1 into a fermentation container for fermentation; s3, fermentation is conducted for 48 h, sucrose laurate is added, and fermentation is continued. In the later period of fermentation, the surfactant is added, so that the permeability of a cell membrane structure is improved, absorption of glucose and lactose and excretion of human milk oligosaccharide are promoted, the fermentation period is prolonged, the fermentation yield is increased, sucrose laurate has low toxicity and biodegradability, and the sucrose laurate has the advantages of being environmentally friendly and free of pollution. The pollution to the environment can be reduced, and the safety of the product can be ensured.