The fermentation industry is both old and new, and its formation has taken place over a long period of time. With the development of science and technology, the fermentation industry has been continuously developed and enriched. Modern fermentation industry is the combination of traditional fermentation technology and modern DNA recombination, cell fusion and other new technologies, and the development of modern biotechnology, and through the modernization of engineering technology for the production of useful substances or directly used in industrial production of a large industrial system, is an important part of biotechnology.
Operation method
High density fermentation
Principle
The fermentation industry is both old and new, and its formation has taken place over a long period of time. With the development of science and technology, the fermentation industry has been continuously developed and enriched. Modern fermentation industry is the combination of traditional fermentation technology and modern DNA recombination, cell fusion and other new technologies, and the development of modern biotechnology, and through the modernization of engineering technology to produce useful substances or directly used in industrial production of a large industrial system, is an important part of biotechnology. Fermentation industry plays an irreplaceable role in the development of genetic engineering drugs. The organic combination of recombinant DNA technology and large-scale cultivation technology makes it possible to produce large quantities of natural proteins that cannot be obtained in large quantities, especially genetically engineered drugs, and the market for clinically applied genetically engineered drugs is growing at an annual rate of 5~15%. The use of high-density fermentation technology can improve the density of the bacteria, and ultimately increase the specific productivity of the product (product yield per unit volume per unit time) can not only reduce the volume of culture, strengthen the downstream separation and extraction, but also shorten the production cycle, reduce the investment in equipment and thus reduce the cost of production, improve market competitiveness. Fermentation engineering bacteria in addition to high concentration, high yield, high yield should also meet: the use of readily available cheap raw materials; not pathogenic, does not produce endotoxin; easy to metabolic regulation; easy to DNA recombination technology. Currently the most used is E. coli (clear genetic background, easy to operate, easy to control the culture conditions, low cost). The conditions needed for the growth and reproduction of engineering bacteria are: good physical environment - fermentation temperature, pH, dissolved oxygen, etc.; suitable chemical environment - suitable for the growth and metabolism of engineering bacteria required by the concentration of various nutrients, and limit the concentration of harmful substances that impede the growth and metabolism. During the fermentation process, many control parameters affect the growth of engineering bacteria and need to be constantly adjusted (see the following table), so as to achieve the purpose of optimal control.
Materials and Instruments
Fermentation Strain Move I. Preparation of reagents For more product details, please visit Aladdin Scientific website.
Tryptone Yeast Extract Sodium Chloride Ammonium Chloride Ammonium Sulfate Dipotassium Phosphate Dipotassium Phosphate Disodium Phosphate Ammonia Antifoamer
Biostat 5L Automatic Fermenter Flasks Petri dishes Test tubes Centrifuge
1. Seed solution (1 000 ml, pH 7.0):Trypton 16 gYeast Extract 10 gGlycerol 20 mlNaCl 5 g2. Fermentation solution (600 ml):Trypton 50 gYeast Extract 50 gGlycerol 100 ml MgSO4-7H2O 3 g3. Replenishment (2400 ml):Trypton 200gYeast Extract 100gGlycerol 1000ml MgSO4-7H2O 12 g4. Salt (1,000 ml, pH 7.2): KH2PO4 20 g K2HPO4 40 g Na2HPO4-12H4O 70 g( NH4)2SO4 12 gNH4Cl 0.2 gII. Instruments
Biostat 5 L automatic fermenter, B. Braun, Germany.III. Operational steps
1. Sterilize all reagents, piping, etc.
2. Sterilization of fermenters.
3. Screening of strains for fermentation Take the glycerol strains stored at -70℃ and draw a plate, incubate at 37℃ overnight, pick single colonies in 5ml test tubes, oscillate and incubate them until the A600 is about 0.6, inoculate them at 20 ml/L into 500 ml shake flasks containing the seed solution, and incubate them with oscillations until the mid-logarithmic stage, which will be used as seed solution for the fermentation tanks. The above link should be prepared before the experiment is carried out.
4. Turn on the instrument and adjust the appropriate parameters.
5. Add fermentation liquid and salt to the fermenter.
6. The seed solution was inoculated into the fermenter at 40 ml/L, controlling the appropriate range of parameters: dissolved oxygen at ≥30%; temperature at 37°C; pH set at 7.0, and flow-through addition of 300 ml/L of ammonia to maintain constancy.
7. When the fermentation density reaches a certain range (dissolved oxygen starts to rise).
8. Inducing the expression of target proteins.
9. Collection of bacteriophage.
10. Clean the fermenter and turn off the apparatus.