Industrial microbial technology is an important support for sustainable development, and it is the fundamental technical support for solving resource crisis, ecological environment crisis and transforming traditional industries. The development of industrial microorganisms has enabled modern biotechnology to penetrate into almost all industrial fields including medicine, agriculture, energy, chemicals, and environmental protection, and plays an important role. Countries such as Europe, the United States and Japan have formulated strategic plans to replace chemical processes with biological processes in the next few decades. It can be seen that industrial microbial technology plays an important role in the future social development process.
Microbial pharmaceutical technology is a major component of industrial microbial technology. The use of microbial drugs begins with well-known antibiotics, which are generally defined as: microbial products and their derivatives that selectively inhibit or affect other biological functions at low concentrations. (Some people have suggested that such substances with the same physiological activity as animal-derived sources such as fish, allicin, berberine, etc. are also classified as antibiotics, but most scholars believe that the traditional concept of antibiotics should still be limited to the secondary metabolism of microorganisms. Product.) In recent years, due to the development of basic life sciences and the application of various new biotechnologies, the reported microorganisms have increased the number of biologically active substances other than anti-infective and anti-tumor, such as specific enzyme inhibitors. Immunomodulators, receptor antagonists, and antioxidants, etc., have exceeded the range of activities that inhibit the life of certain microorganisms. However, these substances are secondary metabolites of microorganisms, which have common characteristics with antibiotics in biosynthesis mechanism, screening research procedures and production processes, but it is obviously inappropriate to refer to them as antibiotics, so many scholars These physiologically active (or pharmacologically active) secondary metabolites produced by microorganisms are collectively referred to as microbial drugs. Microbial drug production technology is microbial pharmaceutical technology. It can be considered to include five aspects:
*Access to the species
According to the data, it can be obtained or purchased directly from scientific research institutions, universities, factories or strain collection departments; new microbial strains can be isolated and screened from nature.
Separation ideas The separation of new strains is to select the required strains quickly and accurately by using various screening methods from various types of microorganisms according to the requirements of production and the characteristics of the strains. If the laboratory or production strains inadvertently contaminate the bacteria, they must be re-isolated. The specific separation operation is carried out from the following aspects.
Solution: First of all, you should consult the data to understand the growth and culture characteristics of the required strains.
Sampling: Samples are collected in a targeted manner.
Proliferation: artificially control the nutrient or culture conditions to make the desired strains proliferate and culture, and predominate in quantity.
Separation: Pure seeds are obtained using separation techniques.
Determination of fermentation performance: Determination of production performance. These characteristics include morphology, culture characteristics, nutritional requirements, physiological and biochemical characteristics, fermentation cycle, product variety and yield, tolerance to high temperature, growth and fermentation temperature, pH value of the Zui, extraction process, and the like.
Breeding of high-yield strains in the second aspect
Industrial production strains have been selected. Breeding of industrial strains is a multi-faceted transformation of a strain for specific biotechnological purposes using genetic principles and techniques. Through the transformation, the existing excellent traits can be strengthened, or the bad properties can be removed or new traits can be added.
Methods for industrial strain breeding: mutagenesis, gene transfer, and genetic recombination.
The breeding process includes the following three steps: (1) the introduction of beneficial genotypes without affecting the vigor of the strain. (2) It is hoped that the genotype will be selected. (3) Evaluation of improved strains (including experimental scale and industrial production scale).
Factors to be considered when selecting a breeding method (1) The nature of the trait to be improved and its relationship to the fermentation process (eg batch or continuous fermentation test); (2) Knowledge of the genetics and biochemistry of this particular species The degree of clarity; (3) economic costs. If the basic traits of specific strains and their processes are poorly known, most techniques such as random mutagenesis, screening and breeding are used; if there is a deep understanding of their genetic and biochemical traits, the genes can be selected. Orientation breeding by means of reorganization and other means.
Specific improvement ideas for industrial strains: (1) Lifting or bypassing the rate-limiting step in metabolic pathways (increasing the rate-limiting enzyme by increasing the copy number of a specific gene or increasing the expression capacity of the corresponding gene; A new metabolic step, thereby providing an alternative metabolic pathway.) (2) Increasing the concentration of the precursor. (3) Altering metabolic pathways, reducing the production of unwanted by-products and increasing the tolerance of strains to high concentrations of potentially toxic substrates, precursors or products. (4) inhibiting or eliminating product decomposition enzymes. (5) The ability to improve the exogenous products of the strain. (6) Eliminate feedback inhibition of metabolic products. Such as the induction of structural analog resistance of metabolic products.
The third part of the strain preservation technology
Transfer culture or slant passage preservation;
Cryopreservation or cryopreservation in liquid nitrogen;
The carrier such as soil or ceramic beads is dried and preserved.
The fourth part of the determination of fermentation process conditions
The source of microbial nutrients, autotrophic bacteria: light; hydrogen, thiamine; nitrite, ferrous salt. Heterotrophic bacteria: organic matter such as carbohydrates, oil and gas and petrochemical products such as acetic acid.
Carbon source, carbonic acid gas; starch hydrolyzed sugar, molasses, sulfite pulp waste liquid, etc., petroleum, normal paraffin, natural gas, acetic acid, methanol, ethanol and other petrochemical products nitrogen source, bean cake or silkworm cocoon hydrolysate, monosodium glutamate waste liquid, Corn syrup, distiller's grains water and other organic nitrogen, urea, ammonium sulfate, ammonia, nitrate and other inorganic nitrogen, gaseous nitrogen inorganic salts, phosphates, potassium salts, magnesium salts, calcium salts and other mineral salts, iron, manganese, cobalt and other traces Determination of special growth factors such as elements, thiamine, biotin, p-aminobenzoic acid, inositol and other media (1) First of all, we must do a good job in investigation and research to understand the source, living habits, physiological and biochemical characteristics and general Nutritional requirements. Industrial production mainly uses bacteria, actinomycetes, yeasts and molds. Their nutritional requirements are both common and have their own characteristics. The composition of the medium should be considered according to the physiological characteristics of different types of microorganisms.
(2) Secondly, it is necessary to understand the culture conditions of the production strains, the metabolic pathways of biosynthesis, the chemical properties of the metabolites, the molecular structure, the general extraction methods and the quality requirements of the products, so as to be There are counts in my heart.
(3) Zui is good to choose a better chemical synthesis medium as the basis, first do some shake flask experiments; then further do small fermenter culture, explore the use of various major carbon sources and nitrogen sources Situation and ability to produce metabolites. Pay attention to the pH change during the cultivation process, observe the two different pHs suitable for growth and reproduction of the strain and suitable for the formation of metabolites, and adjust the ratio to adapt to the above various conditions.
(4) Note that only one variation condition is allowed at a time. After the initial results, determine a medium ratio.
Secondly, we will determine the effects of various important metals and non-metal ions on the fermentation, that is, the nutritional requirements for various inorganic elements, and test the suitability of zui high, zui low and zui. After a certain result is obtained on the synthetic medium, a complex medium test is performed. The relationship between various fermentation conditions and medium was tested after zui. The pH in the medium can be adjusted by the addition of calcium carbonate, and others such as sodium nitrate or ammonium sulfate can also be used for adjustment.
(5) Some fermentation products, such as antibiotics, in addition to the preparation of the medium, through the intermediate feeding method, in the face of the appropriate control of carbon and nitrogen metabolism, while adding various nutrients and precursors, A pathway to direct fermentation to a synthetic product.
(6) According to the economic benefit, choose to consider the economical savings of the raw materials, and use the staple food as little as possible, and try to save food, or substitute other raw materials for food. Sugar is the main carbon source. Substitutes for carbon sources are mainly looking for plant starch, fiber hydrolysate, replacing molasses with dextrose, dextrin and glucose, and industrial glucose instead of edible glucose. Microbial fermentation of petroleum as a carbon source can also produce fermentation with food as a carbon source. product. The conservation and replacement of organic nitrogen sources is mainly aimed at reducing or replacing raw materials rich in protein such as soybean cake powder, peanut cake powder, edible peptone and yeast powder. The substitute raw materials may be cottonseed cake powder, corn syrup, silkworm pupa powder, miscellaneous Fish meal, yellow water or bran, feed yeast, petroleum yeast, bone glue, bacteria, vinasse, and various food industry scraps. Most of these substitutes are rich in protein and low in price, which is convenient for local materials and convenient for transportation.
Determination of the culture process:
Culture conditions: temperature, pH, oxygen, age, inoculum size, temperature Industrial microbial culture methods are divided into two types: static culture and aeration culture.
In the static culture method, the culture medium is contained in the fermentation vessel, and after the inoculation, the air is not subjected to fermentation, which is also called anaerobic fermentation. The aerobic and amphoteric aerobic bacteria are produced in the aeration culture method. The environment in which they are grown must supply air to maintain a certain level of dissolved oxygen, so that the cells grow and ferment rapidly, which is also called aerobic fermentation.
In the two methods of standing and aeration culture, there are two types of liquid culture and solid culture, each of which has surface culture and deep culture.
About liquid submerged culture:
The liquid deep fermenter is ventilated from the bottom of the tank, and the supplied air is dispersed into fine bubbles by the stirring blade to promote the dissolution of oxygen. This culture method of agitating and stirring the bottom of the tank is called a submerged culture method with respect to the surface culture method in which oxygen is dissolved by natural diffusion from the gas-liquid interface. It is characterized in that it is easy to select the culture conditions of the Zui according to the nutrient requirements of the production strain for metabolism and the conditions of aeration, agitation, temperature, and hydrogen ion concentration in the medium at different physiological periods.
Deep medium Three control points for this operation 1 Sterilization: The fermentation industry requires pure culture, so the medium must be heat sterilized before the start of fermentation. Therefore, the fermenter has a steam jacket for heat sterilization of the medium and the fermentor, or the medium is sterilized by a continuous heating sterilizer and continuously conveyed to the fermenter. 2 Temperature control: After the medium is sterilized, it is cooled to the culture temperature and fermented. Since the microorganisms are proliferated and fermented, heat is generated, and heat is generated by stirring, so that the temperature is kept constant, and it is necessary to circulate through the jacket in the cooling water. 3 Ventilation, agitation: Before the air enters the fermenter, the bacteria are removed by an air filter to make sterile air, and then the bottom of the tank enters the person, and then the air is dispersed into fine bubbles by stirring. In order to prolong the bubble retention time, a baffle can be placed in the tank to generate eddy currents. The purpose of the agitation is to disperse the microorganisms in the culture solution uniformly in the fermenter in addition to the dissolved oxygen, to promote heat transfer, and to uniformly disperse the added acid and base for adjusting the pH.
Part 5: Separation and extraction of fermentation products
Extraction Method:
Filtration centrifugation and sedimentation cell disruption extraction adsorption and ion exchange chromatography separation and precipitation (salting out, organic solvent precipitation, isoelectric point, etc.)
Several concerns for membrane separation, crystallisation, drying, separation and extraction:
Water quality heat source removal (asbestos suction filtration, activated carbon adsorption, ion exchange column)
Solvent recovery waste treatment biosafety
Microbial pharmaceutical technology is a major component of industrial microbial technology. The use of microbial drugs begins with well-known antibiotics, which are generally defined as: microbial products and their derivatives that selectively inhibit or affect other biological functions at low concentrations. (Some people have suggested that such substances with the same physiological activity as animal-derived sources such as fish, allicin, berberine, etc. are also classified as antibiotics, but most scholars believe that the traditional concept of antibiotics should still be limited to the secondary metabolism of microorganisms. Product.) In recent years, due to the development of basic life sciences and the application of various new biotechnologies, the reported microorganisms have increased the number of biologically active substances other than anti-infective and anti-tumor, such as specific enzyme inhibitors. Immunomodulators, receptor antagonists, and antioxidants, etc., have exceeded the range of activities that inhibit the life of certain microorganisms. However, these substances are secondary metabolites of microorganisms, which have common characteristics with antibiotics in biosynthesis mechanism, screening research procedures and production processes, but it is obviously inappropriate to refer to them as antibiotics, so many scholars These physiologically active (or pharmacologically active) secondary metabolites produced by microorganisms are collectively referred to as microbial drugs. Microbial drug production technology is microbial pharmaceutical technology. It can be considered to include five aspects:
*Access to the species
According to the data, it can be obtained or purchased directly from scientific research institutions, universities, factories or strain collection departments; new microbial strains can be isolated and screened from nature.
Separation ideas The separation of new strains is to select the required strains quickly and accurately by using various screening methods from various types of microorganisms according to the requirements of production and the characteristics of the strains. If the laboratory or production strains inadvertently contaminate the bacteria, they must be re-isolated. The specific separation operation is carried out from the following aspects.
Solution: First of all, you should consult the data to understand the growth and culture characteristics of the required strains.
Sampling: Samples are collected in a targeted manner.
Proliferation: artificially control the nutrient or culture conditions to make the desired strains proliferate and culture, and predominate in quantity.
Separation: Pure seeds are obtained using separation techniques.
Determination of fermentation performance: Determination of production performance. These characteristics include morphology, culture characteristics, nutritional requirements, physiological and biochemical characteristics, fermentation cycle, product variety and yield, tolerance to high temperature, growth and fermentation temperature, pH value of the Zui, extraction process, and the like.
Breeding of high-yield strains in the second aspect
Industrial production strains have been selected. Breeding of industrial strains is a multi-faceted transformation of a strain for specific biotechnological purposes using genetic principles and techniques. Through the transformation, the existing excellent traits can be strengthened, or the bad properties can be removed or new traits can be added.
Methods for industrial strain breeding: mutagenesis, gene transfer, and genetic recombination.
The breeding process includes the following three steps: (1) the introduction of beneficial genotypes without affecting the vigor of the strain. (2) It is hoped that the genotype will be selected. (3) Evaluation of improved strains (including experimental scale and industrial production scale).
Factors to be considered when selecting a breeding method (1) The nature of the trait to be improved and its relationship to the fermentation process (eg batch or continuous fermentation test); (2) Knowledge of the genetics and biochemistry of this particular species The degree of clarity; (3) economic costs. If the basic traits of specific strains and their processes are poorly known, most techniques such as random mutagenesis, screening and breeding are used; if there is a deep understanding of their genetic and biochemical traits, the genes can be selected. Orientation breeding by means of reorganization and other means.
Specific improvement ideas for industrial strains: (1) Lifting or bypassing the rate-limiting step in metabolic pathways (increasing the rate-limiting enzyme by increasing the copy number of a specific gene or increasing the expression capacity of the corresponding gene; A new metabolic step, thereby providing an alternative metabolic pathway.) (2) Increasing the concentration of the precursor. (3) Altering metabolic pathways, reducing the production of unwanted by-products and increasing the tolerance of strains to high concentrations of potentially toxic substrates, precursors or products. (4) inhibiting or eliminating product decomposition enzymes. (5) The ability to improve the exogenous products of the strain. (6) Eliminate feedback inhibition of metabolic products. Such as the induction of structural analog resistance of metabolic products.
The third part of the strain preservation technology
Transfer culture or slant passage preservation;
Cryopreservation or cryopreservation in liquid nitrogen;
The carrier such as soil or ceramic beads is dried and preserved.
The fourth part of the determination of fermentation process conditions
The source of microbial nutrients, autotrophic bacteria: light; hydrogen, thiamine; nitrite, ferrous salt. Heterotrophic bacteria: organic matter such as carbohydrates, oil and gas and petrochemical products such as acetic acid.
Carbon source, carbonic acid gas; starch hydrolyzed sugar, molasses, sulfite pulp waste liquid, etc., petroleum, normal paraffin, natural gas, acetic acid, methanol, ethanol and other petrochemical products nitrogen source, bean cake or silkworm cocoon hydrolysate, monosodium glutamate waste liquid, Corn syrup, distiller's grains water and other organic nitrogen, urea, ammonium sulfate, ammonia, nitrate and other inorganic nitrogen, gaseous nitrogen inorganic salts, phosphates, potassium salts, magnesium salts, calcium salts and other mineral salts, iron, manganese, cobalt and other traces Determination of special growth factors such as elements, thiamine, biotin, p-aminobenzoic acid, inositol and other media (1) First of all, we must do a good job in investigation and research to understand the source, living habits, physiological and biochemical characteristics and general Nutritional requirements. Industrial production mainly uses bacteria, actinomycetes, yeasts and molds. Their nutritional requirements are both common and have their own characteristics. The composition of the medium should be considered according to the physiological characteristics of different types of microorganisms.
(2) Secondly, it is necessary to understand the culture conditions of the production strains, the metabolic pathways of biosynthesis, the chemical properties of the metabolites, the molecular structure, the general extraction methods and the quality requirements of the products, so as to be There are counts in my heart.
(3) Zui is good to choose a better chemical synthesis medium as the basis, first do some shake flask experiments; then further do small fermenter culture, explore the use of various major carbon sources and nitrogen sources Situation and ability to produce metabolites. Pay attention to the pH change during the cultivation process, observe the two different pHs suitable for growth and reproduction of the strain and suitable for the formation of metabolites, and adjust the ratio to adapt to the above various conditions.
(4) Note that only one variation condition is allowed at a time. After the initial results, determine a medium ratio.
Secondly, we will determine the effects of various important metals and non-metal ions on the fermentation, that is, the nutritional requirements for various inorganic elements, and test the suitability of zui high, zui low and zui. After a certain result is obtained on the synthetic medium, a complex medium test is performed. The relationship between various fermentation conditions and medium was tested after zui. The pH in the medium can be adjusted by the addition of calcium carbonate, and others such as sodium nitrate or ammonium sulfate can also be used for adjustment.
(5) Some fermentation products, such as antibiotics, in addition to the preparation of the medium, through the intermediate feeding method, in the face of the appropriate control of carbon and nitrogen metabolism, while adding various nutrients and precursors, A pathway to direct fermentation to a synthetic product.
(6) According to the economic benefit, choose to consider the economical savings of the raw materials, and use the staple food as little as possible, and try to save food, or substitute other raw materials for food. Sugar is the main carbon source. Substitutes for carbon sources are mainly looking for plant starch, fiber hydrolysate, replacing molasses with dextrose, dextrin and glucose, and industrial glucose instead of edible glucose. Microbial fermentation of petroleum as a carbon source can also produce fermentation with food as a carbon source. product. The conservation and replacement of organic nitrogen sources is mainly aimed at reducing or replacing raw materials rich in protein such as soybean cake powder, peanut cake powder, edible peptone and yeast powder. The substitute raw materials may be cottonseed cake powder, corn syrup, silkworm pupa powder, miscellaneous Fish meal, yellow water or bran, feed yeast, petroleum yeast, bone glue, bacteria, vinasse, and various food industry scraps. Most of these substitutes are rich in protein and low in price, which is convenient for local materials and convenient for transportation.
Determination of the culture process:
Culture conditions: temperature, pH, oxygen, age, inoculum size, temperature Industrial microbial culture methods are divided into two types: static culture and aeration culture.
In the static culture method, the culture medium is contained in the fermentation vessel, and after the inoculation, the air is not subjected to fermentation, which is also called anaerobic fermentation. The aerobic and amphoteric aerobic bacteria are produced in the aeration culture method. The environment in which they are grown must supply air to maintain a certain level of dissolved oxygen, so that the cells grow and ferment rapidly, which is also called aerobic fermentation.
In the two methods of standing and aeration culture, there are two types of liquid culture and solid culture, each of which has surface culture and deep culture.
About liquid submerged culture:
The liquid deep fermenter is ventilated from the bottom of the tank, and the supplied air is dispersed into fine bubbles by the stirring blade to promote the dissolution of oxygen. This culture method of agitating and stirring the bottom of the tank is called a submerged culture method with respect to the surface culture method in which oxygen is dissolved by natural diffusion from the gas-liquid interface. It is characterized in that it is easy to select the culture conditions of the Zui according to the nutrient requirements of the production strain for metabolism and the conditions of aeration, agitation, temperature, and hydrogen ion concentration in the medium at different physiological periods.
Deep medium Three control points for this operation 1 Sterilization: The fermentation industry requires pure culture, so the medium must be heat sterilized before the start of fermentation. Therefore, the fermenter has a steam jacket for heat sterilization of the medium and the fermentor, or the medium is sterilized by a continuous heating sterilizer and continuously conveyed to the fermenter. 2 Temperature control: After the medium is sterilized, it is cooled to the culture temperature and fermented. Since the microorganisms are proliferated and fermented, heat is generated, and heat is generated by stirring, so that the temperature is kept constant, and it is necessary to circulate through the jacket in the cooling water. 3 Ventilation, agitation: Before the air enters the fermenter, the bacteria are removed by an air filter to make sterile air, and then the bottom of the tank enters the person, and then the air is dispersed into fine bubbles by stirring. In order to prolong the bubble retention time, a baffle can be placed in the tank to generate eddy currents. The purpose of the agitation is to disperse the microorganisms in the culture solution uniformly in the fermenter in addition to the dissolved oxygen, to promote heat transfer, and to uniformly disperse the added acid and base for adjusting the pH.
Part 5: Separation and extraction of fermentation products
Extraction Method:
Filtration centrifugation and sedimentation cell disruption extraction adsorption and ion exchange chromatography separation and precipitation (salting out, organic solvent precipitation, isoelectric point, etc.)
Several concerns for membrane separation, crystallisation, drying, separation and extraction:
Water quality heat source removal (asbestos suction filtration, activated carbon adsorption, ion exchange column)
Solvent recovery waste treatment biosafety
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