Significance of microbial technology application to modern microbial pharmaceutical industry

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Microbial pharmaceutical technology is an important part of industrial microbial technology. The use of microbial drugs began 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. It has been suggested that such substances with the same physiological activity come from animals and plants, such as fish essence, allicin, berberine, etc. , should also belong to the category of antibiotics, but most scholars believe that the traditional concept of antibiotics should still be limited to the secondary metabolites of microorganisms. In recent years, due to the development of basic life science and the application of various new biotechnology, it is reported that other bioactive substances produced by microorganisms, such as specific enzyme inhibitors, immunomodulators, receptor antagonists and antioxidants, are increasing day by day, and their activities have exceeded the scope of inhibiting some microbial life activities. However, these substances are secondary metabolites of microorganisms, which have the same characteristics as antibiotics in biosynthesis mechanism, screening research procedures and production technology. But it is obviously inappropriate to call them antibiotics, so many scholars call these secondary metabolites with physiological activity (or pharmacological activity) produced by microorganisms microbial drugs. The production technology of microbial drugs is microbial pharmaceutical technology. It can be considered as including five aspects:

The first aspect is the technology of obtaining strains.

According to the information, directly ask for or buy from scientific research units, universities, factories or strain preservation departments; Isolation and screening of new microbial strains from nature.

The idea of separation The separation of new strains is to select the required strains quickly and accurately through various screening methods according to the requirements of production and the characteristics of strains. If bacteria used in laboratory or production accidentally pollute miscellaneous bacteria, they must be separated and purified again. The specific separation operation is carried out from the following aspects.

Scheme: First of all, we should consult the information to understand the growth and culture characteristics of the required strains.

Sampling: collect samples in a targeted manner.

Proliferation: By artificially controlling nutrients or culture conditions, the required strains will proliferate and dominate in number.

Separation: using separation technology to obtain purebred.

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, maximum tolerance temperature, optimum temperature for growth and fermentation, optimum pH value, extraction technology and so on.

The second aspect is the breeding technology of high-yield strains.

The strains used in industrial production are all screened. The cultivation of industrial strains is the multi-directional transformation of strains used for specific biotechnology purposes by using genetic principles and technologies. Through transformation, we can not only strengthen the existing excellent traits, but also remove the unhealthy symptoms or add new traits.

Breeding methods of industrial strains: mutation, gene transfer and gene recombination.

The breeding process includes the following three steps: (1) Introducing beneficial genotypes without affecting the viability of the strain. (2) Select the required genotype. (3) Evaluation of improved strains (including experimental scale and industrial production scale).

Factors to be comprehensively considered in selecting breeding methods (1) The properties of the characters to be improved and their relationship with fermentation technology (such as batch or continuous fermentation experiments); (2) Understand clearly the genetics and biochemistry of this particular strain; (3) Economic expenses. If we don't know much about the basic characteristics and techniques of a particular strain, we usually adopt techniques such as random mutation, screening and breeding. If we have a deep understanding of its genetic and biochemical characters, we can choose gene recombination and other means for directional breeding.

The specific improvement ideas of industrial strains: (1) removing or bypassing the rate-limiting step in metabolic pathway (increasing the copy number of specific genes or improving the expression ability of corresponding genes to increase the content of rate-limiting enzymes; A new metabolic step is introduced into the metabolic pathway, thus providing a bypass metabolic pathway. (2) Increase the concentration of the precursor. (3) Change the metabolic pathway, reduce the generation of useless by-products, and improve the tolerance of the strain to high concentration of potentially toxic substrates, precursors or products. (4) Inhibiting or eliminating product-decomposing enzymes. (5) Improving the exocrine product ability of the strain. (6) Eliminating the feedback inhibition of metabolites. Such as inducing structural analogue resistance of metabolites.

The third part is the preservation technology of strains.

Transfer culture or slant passage preservation;

Cryopreservation or cryopreservation in liquid nitrogen;

Dry preservation of carriers such as soil or ceramic beads.

The fourth part is the determination of fermentation conditions.

Nutrient sources of microorganisms

Energy, autotrophic bacteria: light; Hydrogen and thiamine; Nitrite, ferrous salt. Heterotrophic bacteria: organic substances 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, petroleum, normal alkanes, natural gas, petrochemical products such as acetic acid, methanol and ethanol.

Nitrogen source, organic nitrogen such as bean cake or silkworm chrysalis hydrolysate, monosodium glutamate waste liquid, corn steep liquor and distiller's grains water, inorganic nitrogen such as urea, ammonium sulfate, ammonia water and nitrate, and gaseous nitrogen.

Inorganic salts, phosphates, potassium salts, magnesium salts, calcium salts and other mineral salts, and trace elements such as iron, manganese and cobalt.

Special growth factors, thiamine, biotin, p-aminobenzoic acid, inositol, etc.

Determination of culture medium

(1) First of all, we should do a good job of investigation and study to understand the source, living habits, physiological and biochemical characteristics and general nutritional requirements of strains. Four kinds of microorganisms are mainly used in industrial production: bacteria, actinomycetes, yeast and mold. Their nutritional requirements have their own characteristics, so the composition of culture medium should be considered according to the physiological characteristics of different types of microorganisms.

(2) Secondly, it is necessary to know the culture conditions of producing strains, metabolic pathways of biosynthesis, chemical properties, molecular structure, general extraction methods and product quality requirements of metabolites, so as to know fairly well when selecting culture medium.

(3) It's best to choose a better chemical synthesis medium as the basis and start doing some shake-flask experiments; Then further do small fermentor culture to explore the utilization of various main carbon and nitrogen sources and the ability to produce metabolites. Pay attention to the change of pH in the process of culture, observe two different pH suitable for the growth and reproduction of the strain and the formation of metabolites, and constantly adjust the ratio to adapt to the above situation.

(4) Note that only one change condition is allowed at a time. With the preliminary results, first determine a medium proportion.

Secondly, the effects of various important metal and nonmetal ions on fermentation were determined, that is, the nutritional requirements of various inorganic elements were tested, and their maximum, minimum and optimum amounts were tested. After obtaining certain results on synthetic medium, the compound medium experiment was carried out. Finally, the relationship between fermentation conditions and culture medium was tested. The pH value in the culture medium can be adjusted by adding calcium carbonate, or by using other substances such as sodium nitrate and ammonium sulfate.

(5) Some fermentation products, such as antibiotics, should be supplemented in addition to preparing culture medium, and at the same time, carbon and nitrogen metabolism should be properly controlled, and various nutrients and precursors should be added intermittently to guide the fermentation to the direction of synthetic products.

(6) Select raw materials of blending agent according to economic benefits.

Considering economy, we should use less or no staple food as far as possible, save food as much as possible, or replace food with other raw materials. Sugar is the main carbon source. The substitutes of carbon source are mainly looking for plant starch and fiber hydrolysate, using waste molasses to replace starch, dextrin and glucose, and using industrial glucose to replace edible glucose; Microbial fermentation with petroleum as carbon source can also produce fermented products with grain as carbon source. The main purpose of saving and replacing organic nitrogen sources is to reduce or replace raw materials rich in protein, such as bean cake powder, peanut cake powder, edible peptone, yeast powder, etc. The substitute materials can be cottonseed cake powder, corn syrup, silkworm pupa powder, miscellaneous fish meal, yellow syrup or bran juice, feed yeast, petroleum yeast, bone glue, bacteria, distiller's grains and various food industry leftovers. Most of these substitutes are rich in protein, so they are cheap and easy to get local materials and transport.

Determination of culture technology:

Culture conditions: temperature, pH value, oxygen, seed age, inoculation amount and temperature.

The culture methods of industrial microorganisms can be divided into static culture and aeration culture.

Static culture means that the culture medium is put into a fermentation container and fermented without air after inoculation, which is also called anaerobic fermentation. Aerobic bacteria and facultative aerobic bacteria are the main strains produced by aeration culture. The environment in which they grow must be provided with air to maintain a certain level of dissolved oxygen, so that bacteria can grow and ferment rapidly, which is also called aerobic fermentation.

In static culture and aeration culture, it can be divided into liquid culture and solid culture, each of which can be divided into surface culture and deep culture.

About liquid submerged culture:

The liquid submerged fermentation tank is ventilated from the bottom of the tank, and the air is dispersed into tiny bubbles by stirring blades to promote the dissolution of oxygen. This culture method of aeration and stirring at the bottom of the tank is called submerged culture, which is compared with the surface culture method of natural diffusion of dissolved oxygen at the gas-liquid interface. Its characteristic is that it is easy to choose the best culture conditions according to the nutritional needs of the production strain metabolism and the conditions of ventilation, stirring, temperature and hydrogen ion concentration in the culture medium at different physiological stages.

Three control points of the basic operation of deep culture

① Sterilization: The fermentation industry requires pure culture, so the culture medium must be heated and sterilized before fermentation. Therefore, the fermentation tank has a steam jacket to heat and sterilize the culture medium and the fermentation tank, or the culture medium is sterilized by a continuous heating sterilizer and continuously transported to the fermentation tank. ② Temperature control: after sterilization, the culture medium is cooled to the culture temperature for fermentation. Because microbial proliferation and fermentation will generate heat, cooling water must be circulated in the jacket in order to keep the temperature constant. (3) aeration and stirring: before the air enters the fermentation tank, it is removed by an air filter to make sterile air, then it enters from the bottom of the tank and is dispersed into tiny bubbles through stirring. In order to prolong the residence time of bubbles, baffles can be installed in the tank to generate eddy currents. The purpose of stirring is not only to dissolve oxygen, but also to make the microorganisms in the culture solution evenly dispersed in the fermentor, promote heat transfer, and evenly disperse the added acid and alkali to adjust the pH value.

The fifth part is the separation and extraction technology of fermentation products.

Extraction method:

filter

Centrifugation and precipitation

Cell rupture

Extract, lineage

Adsorption and ion exchange

chromatography

Precipitation (salting out, organic solvent precipitation, isoelectric point, etc.). )

Barrier separation

transparent

dry