Modern technologies of probiotics, amino acids, vitamins and feed antibiotics.
Individual microorganisms are of great interest and as probiotics. Probiotics are biologics containing living microorganisms - symbionts of humans and animals that have the ability to restore disturbed microecology of organisms. These include bifidobacteria, lactobacilli, streptococci, etc., present in the body since birth. In veterinary practice, in the development of probiotics, in addition to these genera of bacteria, yeasts and fungi are used (Saccharomyces cerevisiae, Candida pintolonesi, Aspergillus niger, Asp.oryzae). Probiotics are used to correct microecology.
The raw material used to prepare the culture medium must be harmless to humans and animals. The main substrate is skimmed milk, hydrolyzed milk. Biomass of symbiotic microbes is concentrated on the separators, then components of the supporting medium are stored during storage (gelatin, sucrose, skim milk), poured into ampoules or vials, and lyophilized. In the technology of creating probiotic drugs, mainly lactic acid bacteria are used, rarely bacilli, escherichia and others. For example, the composition of bactolactate (Japan) includes Lactobacillus rhamnosum, Lactobacillus casei, Lactobacillus faecium, linex (Russia) -Lactobacillus, Bifidobacterium, Streptococcus, biosuprin (Ukraine) -Bacillus sibtilis, Bacillus cereus.
In biotechnology an important role is played not only by microorganisms themselves, but also by their metabolites, namely amino acids, vitamins (primary metabolites), antibiotics, etc.
Amino acids. In the world, 700-800 thousand tons of amino acids are produced annually (more than half are glutamine and lysine). About 300 different amino acids are known, 20 are used in living nature, 8 of which are indispensable for humans (isoleicin, leucine, lysine, methionine, threonine, valine, phenylalanine, tryptophan). The latter enter the body with an animal and vegetable protein. Amino acid composition, their number in cells of animal, plant and microbial origin have some differences. Thus, in plant proteins, lysine, methionine, tryptophan, and threonine are not sufficient. Most microorganisms, unlike higher organisms, are able to synthesize all 20 amino acids, except for lactic acid bacteria and some other groups.
On an industrial scale, protein amino acids are obtained: by hydrolysis of natural protein-containing raw materials; chemical synthesis; microbiological synthesis; biotransformation of amino acids precursors with the help of microorganisms or enzymes isolated from them. Microbiological synthesis of amino acids is the most promising and economically beneficial. Industrial production of amino acids became possible after the discovery of the ability in some microorganisms to release into the culture medium significant amounts of any one amino acid. It was observed that most of the several thousand analyzed wild strains of microorganisms produced amino acids in the external environment, but in very small quantities. There is no fixed relationship between the taxonomic position of microorganisms and the ability to produce one or another amino acid. So, among the possible producers of glutamic acid, organisms are noted, of which 30% are yeast, 30% are streptomycetes, 20% are bacteria and 10% are microscopic fungi. And only one of the investigated strains of microorganisms - Corynebacterium glutamicum was able to supersynthesize glutamate. This strain was used in the organization of the world's first large-scale production of glutamic acid by a microbiological method in Tokyo (1956). This amino acid has found wide application in the food industry in order to improve the taste of the product.
Promising producers constantly improve by selecting mutants with a modified genetic program and regulatory properties. To them, in addition to Corynebacterium, it is possible to classify strains of such microorganisms as Brevibacterium, Micrococcus, Arthrobacter and others.
The source of carbon for Corynebacterium glutamicum strains is glucose, sucrose, less often fructose, maltose (in the composition of molasses, whey, hydrolyzate of casein, etc.). The source of nitrogen can be urea, ammonium sulfate or phosphate, corn extract, yeast hydrolyzate. Growth promoters use corn extract, yeast hydrolyzate, B vitamins, macro- and microelements (Ca, Mg, Mn, Fe, P).
Lysine is synthesized on an industrial scale, primarily as a feed additive. Under conditions of production of lysine, deep-seated periodic fermentation of Brevibacterium flavum or Corynebacterium glutamicum is obtained at 30-33 ° C, pH 7.0-7.2 for 2-3 days. Lysine accumulates in the culture liquid at the end of the exponential growth phase. At the end of the fermentation, the culture liquid is separated from the cell mass. Lysine is isolated from the culture liquid, mixed with filler (wheat bran, etc.), granulated or in liquid form used as a feed concentrate. Granular lysine contains 7-10% lysine. In cells of microorganisms, lysine is synthesized from aspartic acid and serves as the final product of a branched metabolic pathway of biosynthesis, common to three amino acids - lysine, methionine and threonine.
Vitamins.The biological activity of vitamins is determined by the fact that they are part of the active centers of enzymes as cofactors. Therefore, the lack of vitamins lowers the biocatalytic activity of enzymes, affects metabolic processes, growth and development of the body. Biosynthesis of vitamins under natural conditions is carried out by plants and microorganisms. When processing plant foods, vitamin loss is often observed. So when receiving flour of the highest grade, 80-90% of vitamins are lost.
Bioconcentrated microorganisms, actinomycetes, methane-forming, photosynthesizing bacteria, including more than 10 kinds of propionic acid bacteria are used as bio-producers. A strain of Propionibacterium ari, capable of actively isolating B12 from the cell, is selected, in contrast to other producers of this genus accumulating the vitamin within the cell. The product is obtained by deep cultivation of producer strains under anaerobic conditions on a substrate containing corn extract, glucose, cobalt salts and ammonium sulfate. Riboflavin (vitamin B2) synthesizes higher plants, yeast, mycelial fungi and bacteria. From 1 ton of carrots one can get 1 g of riboflavin, of 1 ton of liver - 6 grams, and cultivation of production strains - Eremothecium ashbyii or Ashbya gossipii in 1 ton of nutrient medium accumulates 25 kg of vitamin. Also mutant strains of B. subtilis and Asp.niger are used as producers. Cultivation of strains is carried out in fermenters, with constant aeration. Soy flour, molasses, whey, fish and corn flour are used as substrates. As producers of ergosterol (the precursor of vitamin D2 - calciferol), Saccharomyces carlsbergensis and S. cerevisiae are used. Fermentation of yeast is carried out under aeration conditions. The resulting biomass is hydrolyzed with a solution of hydrochloric acid, then purified with alcohol, concentrated and irradiated with UV at a wavelength of 280-300 nm. Radiation excites individual chemical bonds in the carbon cycles, causes the conversion of ergosterol into a vitamin.
Large-scale production of another, no less important for the human body and animals, vitamin C-L-ascorbic acid, is a laborious process involving one microbiological stage and several chemical ones. The initial substrate for it is D-glucose. At the last stage of this process, 2-keto-L-gulonic acid (2-KLG) is chemically converted to L-ascorbic acid. Biochemical studies of the metabolism of various microorganisms have shown that 2- KLG can be obtained, including the co-cultivation of the microorganisms Corynebacterium and Erwinica herbicola for the conversion of glucose to 2- KLG. However, the conditions of cultivation, optimal for one organism, are unacceptable for another, which entails a spontaneous "washout" from the environment of one of them. In such cases, it is possible to cultivate microorganisms sequentially, but this process is difficult to make continuous, since for the growth of microorganisms, essentially different media are needed.
The simplest method - the creation of a single microorganism capable of converting D-glucose into 2-KLG, consists in isolating the 2-KLG-reductase gene of Corynebacterium and introducing it into Erwinica herbicola.
Transformed Erwinica herbicola cells actively convert D-glucose directly into 2 KLG. In this case, the native Erwinica herbicola enzymes, localized in the internal membrane of the bacterial cell, convert glucose to 2,5-DKG (2,5-diketoglucanic acid), and the 2,5-DKG reductase localized in the cytoplasm catalyzes the conversion process 2.5 - DKG in 2 KLG. Consequently, with the help of genetic manipulations, it was possible to carry out metabolic reactions in one organism, proceeding in so different microorganisms. This hybrid acquired the ability to synthesize the end product of the combined metabolic pathway. Such an organism is used as a factory for the production of 2-KLG, replacing three stages in the process of obtaining L-ascorbic acid, which is currently dominant.
Antibiotics, like pigments and toxins, belong to the secondary metabolites of microorganisms, i.e. The substance is not mandatory for the growth or functioning of the cell, but synthesized in a stationary phase. They are used in livestock not only as a medicinal product, but also as a feed additive. The properties of stimulating the growth of animals have more than 20 antibiotics, synthesized by mycelial fungi (biomycin and terramycin) and streptomycins (grisin, flavomycin, monensin, tylosin). Filler feed is most often used soybean flour.
The biosynthesis of antibiotics, as well as of any other secondary metabolites, increases in the phase of slow growth of the cell population (end of trophophase) and reaches a maximum in the stationary phase (idiophase). It is believed that at the end of the trophophase the enzymatic status of the cells changes, secondary metabolism inducers appear that release the genes of secondary metabolism from under the influence of catabolic repression. Therefore, any mechanisms that inhibit cellular proliferation and active growth, stressful situations, activate the formation of antibiotics.
The process of cultivation of idylites goes through two phases. In the first phase, a sufficient amount of biomass is accumulated, which is grown on a growth medium for the microorganism. This phase should be fast, and the nutrient medium is cheap. In the second phase, an active antibiotic synthesis is initiated and active. At this phase, fermentation is conducted on a productive medium.
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