What are the methods of biopharmaceutical process extraction and separation

Microbial Pharmaceutical Technology

Industrial microbial technology is an important support for sustainable development, is the solution to the resource crisis, ecological and environmental crises and the transformation of the traditional industries of the fundamental technological dependence. The development of industrial microorganisms has enabled modern biotechnology to penetrate into almost all industrial fields, including medicine, agriculture, energy, chemical industry, environmental protection, etc., and play an important role. Europe, America, Japan and other countries have been developed to varying degrees in the next few decades to replace the chemical process with biological processes of the strategic plan, can be seen in the future of industrial microbial technology in the process of social development of an important position.

Microbial pharmaceutical technology is the most important component of industrial microbial technology. The utilization of microbial drugs began with the familiar antibiotics, which are generally defined as a microbial product and its derivatives that selectively inhibit or affect other biological functions at low concentrations. (It has been suggested that such substances of plant and animal origin with the same physiological activity, such as fisetin, allicin and safranin, should also be categorized as 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 sciences and the application of various new biotechnologies, an increasing number of bioactive substances other than anti-infective and anti-tumor produced by microorganisms have been reported, such as specific enzyme inhibitors, immunomodulators, receptor antagonists, and antioxidants, etc., whose activities have gone beyond inhibiting the life activities of certain microorganisms. However, these substances are microbial secondary metabolites, its biosynthetic mechanism, screening and research procedures and production processes and antibiotics have *** with the same characteristics, but they are commonly known as antibiotics is obviously inappropriate, so many scholars on the microorganisms produced by these physiological activity (or pharmacological activity) of the secondary metabolites collectively referred to as the microbial drugs. The production technology of microbial drugs is microbial pharmaceutical technology. It can be considered to include five aspects:

The first aspect of the acquisition of strains

Based on the information directly from the scientific research units, colleges and universities, factories, or strain preservation departments to request or purchase; from nature to isolate and screen new microbial strains.

Separation of the idea of isolation of new strains of microorganisms is to be separated from the mixed types of microorganisms in accordance with the requirements of the production, the characteristics of the strain, the use of a variety of screening methods, rapid and accurate selection of the required strains. Laboratory or production strains if inadvertently contaminated with stray bacteria, but also must be re-separated and purified. Specific isolation operations from the following aspects.

Determine the program: first of all, we must consult the information to understand the growth and culture characteristics of the required strains.

Sampling: targeted collection of samples.

Amplification: artificially by controlling the nutrients or cultivation conditions, so that the desired strains of bacteria after proliferation culture, in the number of dominant.

Isolation: the use of isolation techniques to obtain pure species.

FERMENTATION PROPERTIES MEASUREMENT: Production properties are measured. These properties include morphology, culture characteristics, nutritional requirements, physiological and biochemical properties, fermentation period, product variety and yield, tolerance of maximum temperature, optimum temperature for growth and fermentation, optimum pH, extraction process, etc.

The second aspect of high-yield strains of selective breeding

Industrial production strains are selected and bred. Breeding of industrial strains is a multifaceted modification of a strain for a specific biotechnological purpose using genetic principles and techniques. Through modification, existing good traits can be strengthened or undesirable properties can be removed or new traits can be added.

Methods of industrial strain breeding: mutagenesis, gene transfer, recombination.

The breeding process consists of the following 3 steps: (1) The introduction of beneficial genotypes without affecting the viability of the strain. (2)Selection of desired genotypes. (3) Evaluation of improved strains (including experimental scale and industrial production scale).

The factors to be considered when selecting a breeding method are (1) the nature of the trait to be improved and its relationship to the fermentation process (e.g., batch or continuous fermentation tests); (2) the degree of understanding of the genetic and biochemical aspects of the particular strain; and (3) the economic costs. If the basic traits of a particular strain and its process know very little, it is most likely to use random mutagenesis, screening and selection and other techniques; if the genetic and biochemical traits have a deeper understanding of its recombination and other means of directional breeding.

Industrial strains of specific improvement ideas: (1) lifting or bypassing the rate-limiting step in the metabolic pathway (by increasing the copy number of specific genes or increasing the expression of the corresponding genes to increase the rate-limiting enzyme content; in the metabolic pathway to draw out the new metabolic step, thus providing a bypass metabolic pathway.) (2) Increase the concentration of precursors. (3) Altering metabolic pathways to reduce the production of unwanted byproducts as well as to increase the tolerance of the strain to high concentrations of potentially toxic substrates, precursors, or products. (4) Inhibition or elimination of product catabolic enzymes. (5) Improving the ability of the strain to exocytose products. (6) Elimination of feedback inhibition of metabolized products. Such as inducing structural analog resistance to the metabolized product.

Part III Strain Preservation Technology

Transfer culture or slant passaging preservation;

Ultra-low temperature or frozen preservation in liquid nitrogen;

Drying preservation of carriers such as soil or ceramic beads.

Part IV Determination of Fermentation Process Conditions

Nutrient Sources of Microorganisms

Energy sources, autotrophic bacteria: light; hydrogen, thiamine; nitrite, ferrous salts. Heterotrophs: organic matter such as carbohydrates, oil and gas and petrochemicals such as acetic acid.

Carbon source, carbonic acid gas; starch hydrolyzed sugar, molasses, sulfite pulp waste liquid, etc., petroleum, ortho-constructed paraffin, natural gas, petrochemicals such as acetic acid, methanol, ethanol, etc.

Nitrogen source, organic nitrogen such as hydrolyzed liquid of soybean cake or silkworm chrysalis, monosodium glutamate waste liquid, corn syrup, lees water, etc., inorganic nitrogen such as urea, ammonium sulphate, ammonia, nitrate, etc., gaseous nitrogen

Inorganic salts. Phosphates, potassium salts, magnesium salts, calcium salts and other mineral salts, iron, manganese, cobalt and other trace elements, etc.

Special growth factors, thiamine, biotin, p-aminobenzoic acid, inositol and so on

Determination of the culture medium

(1) First of all, it is necessary to do a good job of investigating research work, to understand the source of the strain, habits, physiological and biochemical characteristics and general nutritional requirements. Industrial production is mainly applied to bacteria, actinomycetes, yeasts and molds four major categories of microorganisms. Their nutritional requirements both ****, but also their own characteristics, should be based on the physiological characteristics of different types of microorganisms to consider the composition of the medium.

(2) Second, the production of strains of culture conditions, biosynthesis of metabolic pathways, metabolites, chemical properties, molecular structure, general extraction methods and product quality requirements, etc. also need to have a good understanding of the selection of culture media to do a good idea.

(3) It is best to choose a better chemical synthesis medium as a foundation, and do some shaking bottle experiments at the beginning; then further do a small fermentation tank culture, to find out the utilization of the strains of various major carbon and nitrogen sources and the ability to produce metabolites. Pay attention to the pH changes during the cultivation process, observe the two different pH suitable for the growth and propagation of the strain and the formation of metabolites, and constantly adjust the ratio to adapt to the above situations.

(4) Be careful to limit the change to one condition at a time. After the preliminary results are available, a medium ratio is determined first.

Secondly, the effect of various important metal and non-metal ions on fermentation is determined, i.e., the nutritional requirements of various inorganic elements are tested for their maximum, minimum, and optimum amounts. After certain results are obtained on the synthetic medium, a composite medium test is then done. Finally, the relationship between various fermentation conditions and the medium is tested. The pH within the medium can be adjusted by adding calcium carbonate, other such as sodium nitrate, ammonium sulfate can also be used to adjust.

(5) some fermentation products, such as antibiotics, in addition to the preparation of the medium, but also through the intermediate replenishment method, one face of carbon and nitrogen metabolism to be controlled appropriately, and the other side of the intermittent addition of a variety of nutrients and precursors to guide the fermentation to the synthesis of the product pathway.

(6) According to the economic benefits of the selection of peppermint-based raw materials

Considering the economic savings, as little as possible or do not use staple grains, and strive to save the use of grain, or other raw materials instead of grain. Sugar is the main source of carbon. Carbon source of substitution is mainly looking for plant starch, fiber hydrolysate, waste molasses instead of starch, dextrin and glucose, industrial glucose instead of edible glucose; oil as a carbon source of microbial fermentation can also be produced to grain as a carbon source of fermentation products. Organic nitrogen source saving and substitution mainly for the reduction or replacement of soybean cake flour, peanut cake flour, edible peptone and yeast powder and other protein-rich raw materials as the goal, the substitute raw materials can be cottonseed cake flour, corn syrup, silkworm chrysalis powder, miscellaneous fish meal, yellow slurry water or bran juice, feed yeast, petroleum yeast, bone gelatin, fungus, lees, and a variety of food industry scraps and so on. Most of these substitutes are protein-rich, inexpensive, locally sourced, and easy to transport.

Determination of the culture process:

Culture conditions: temperature, pH, oxygen, seed age, inoculum, temperature

The culture method of industrial microorganisms is divided into two major types of static culture and aeration culture.

The static culture method is the culture medium in the fermentation container, after inoculation, do not pass the air for fermentation, also known as anaerobic fermentation. Aerated culture method of production strains of aerobic bacteria and parthenogenetic aerobic bacteria, the environment in which they grow must be supplied with air to maintain a certain level of dissolved oxygen, so that the rapid growth of bacteria and fermentation, also known as aerobic fermentation.

In the two types of static and aerated culture methods can be divided into two major types of liquid culture and solid culture, each of which has a surface culture and deep culture.

On the liquid deep culture:

The liquid deep fermentation tank is aerated from the bottom of the tank, and the incoming air is dispersed into tiny bubbles by the stirring paddles in order to promote the dissolution of oxygen. This kind of cultivation method by the bottom of the tank ventilation and stirring, compared with the surface cultivation method by the gas-liquid interface by natural diffusion of oxygen dissolution, is called the deep culture method. It is easy to choose the best culture conditions according to the nutritional requirements of the production strains for metabolism and the conditions of aeration, stirring, temperature, and hydrogen ion concentration in the culture medium in different physiological periods.

Three control points for the basic operation of deep culture

①Sterilization: The fermentation industry requires pure culture, so the medium must be heated and sterilized before fermentation begins. So the fermenter has a steam jacket, so that the medium and the fermenter for heating and sterilization, or the medium by the continuous heating sterilizer sterilization, and continuously transported in the fermenter. ② Temperature control: after sterilization of the medium, cooled to the culture temperature for fermentation, due to the proliferation of microorganisms and fermentation will heat, stirring heat, etc., so in order to maintain a constant temperature, must be in the jacket to the cooling water circulating through. ③Aeration, stirring: air into the fermenter before the air filter to remove bacteria, made of sterile air, and then from the bottom of the tank into the people, and then through the stirring of the air dispersed into tiny bubbles. In order to extend the bubble retention time, can be installed in the tank baffle vortex. The purpose of stirring in addition to dissolved oxygen, can make the culture solution of microorganisms evenly dispersed in the fermenter, to promote heat transfer, as well as for the adjustment of pH and make the addition of acid and alkali evenly dispersed.

Part V. Separation and Extraction of Fermentation Products

Extraction Methods:

Filtration

Centrifugation and Sedimentation

Cell Fragmentation

Extraction

Adsorption and Ion Exchange

Color Chromatographic Separation

Precipitation (Salt Analysis, Organic Solvent Precipitation, Isoelectrolysis etc.)

Membrane Separation

Crystallization

Drying