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Large-scale cultivation of microorganisms as a direct source of protein for human and animal nutrition was considered as a way to solve the problem of food shortages in Germany already during the First World War. Technological processes were developed for the cultivation of brewer's yeast, which, after processing and drying, was added to soups and sausages. During the Second World War, these processes were already well established.
The expression "proteins of unicellular organisms" arose in the 60s. in relation to bacterial biomass (mainly yeast), which is used as a food component of animals and humans. Particularly attractive is the fact that the nutrient medium for the cultivation of bacteria is often agricultural waste: sugar beet cake in the production of sugar, sunflower cake in the production of vegetable oil, whey in the production of cheese, wood chips and sawdust, etc.
Interest in this problem flared up after the publication of research results showing the possibility of producing such protein concentrates based on hydrocarbons. Oil companies funded the development of these studies not only because of the use of hydrocarbons, but also because of the favorable food test results and marketing prospects.
The first large-scale protein concentrate plant was developed by a joint venture between British Petroleum (UK) and Italprotein (Italy) in 1975, with a capacity of 100,000 t / year; the raw material was normal paraffins. Japan also took up this problem, 8 plants with a capacity of 1500 tons of protein / year were built. However, interest in the production of protein in single-celled organisms in the 70s. decreased slightly; partly due to the favorable agricultural situation of those years, but mainly due to imperfect technologies that do not remove some toxic substances from the final product.
In the 80s. The German company "Hoechst", which stands out on the market for its high technologies, has developed processes for obtaining high-quality protein concentrates. In the 80s. one of the world's leading protein producers was the USSR with its inexhaustible raw material base. A factory has been built in Finland using Paecilomyces in sulphite effluent from paper mills; factory capacity - 10,000 tons of protein / year.
In the EEC countries, about 25 million tons of protein concentrates are produced per year. These figures speak for the profitability of enterprises. Livestock feed is becoming expensive due to limited land holdings and for a number of other reasons. Proteins of unicellular organisms have huge advantages: high reproduction rate, availability of raw materials, solution of waste disposal problems of many enterprises, etc.
In addition, proteins have a constant and reproducible composition, it is easy to fortify them, add the necessary microelements; they are also easy to make as granules or tablets and are much easier to store than plants or other feed.
However, protein manufacturers do not consider their products as a substitute for protein in the diet of animals: protein concentrates serve as additives to feed, making them cheaper and improving their quality. It should be noted, however, that the production of protein supplements is not developing as rapidly as predicted in the 60s and 70s.The fact is that the requirements for the safety of technologies have become much more stringent, which must take into account the results of all the necessary toxicological and food tests.
You should be especially careful about the use of protein concentrates in human nutrition. However, their use to solve the problem of nutrition of the world's population has no alternative, since forecasts indicate that population growth does not match the growth of food products. It is safe to say that the development of microorganisms in human nutrition is just beginning.
Microorganisms began to be used in the production of protein products long before the emergence of microbiology. Suffice it to mention all kinds of cheese, as well as products obtained by fermentation of soybeans. In both the first and second cases, protein is the nutritional basis. During the development of these products, with the participation of microbes, a profound change in the properties of protein-containing raw materials occurs.
The result is food products that can be stored longer (cheese) or more convenient to consume (bean curd). Microbes play a role in the production of some meat products for storage. So, in the manufacture of some varieties of sausages, acidic fermentation is used, usually with the participation of a complex of lactic acid bacteria. The resulting acid contributes to the preservation of the product and contributes to the formation of its special taste.
This, perhaps, limits the use of microorganisms in the processing of proteins. The possibilities of modern biotechnology in these industries are small, with the exception of cheese making. Another thing is the cultivation and collection of microbial mass, processed into food: here biotechnology can manifest itself in its entirety.
Protein production of unicellular organisms
For many important indicators, the biomass of microorganisms can have a very high nutritional value. To a large extent, this value is determined by proteins: in most species, they make up a significant proportion of the dry mass of cells. For decades, the prospects for increasing the share of microbial protein in the total balance of protein produced worldwide have been actively discussed and investigated.
The production of such protein involves the large-scale cultivation of certain microorganisms that are collected and processed into food. To realize the fullest possible transformation of the substrate into microbial biomass, a multifaceted approach is required. Growing microbes for food is of interest for two reasons. First, they grow much faster than plants and animals: the time for doubling their numbers is measured in hours. This shortens the time it takes to produce a certain amount of food.
Secondly, depending on the grown microorganisms, various types of raw materials can be used as substrates. As for substrates, here you can go in two main directions: to process low-quality waste products or to focus on readily available carbohydrates and obtain microbial biomass containing high-quality protein from them.
Obtaining microbial protein on methanol
The main advantage of this substrate is its high purity and absence of carcinogenic impurities, good solubility in water, high volatility, which makes it easy to remove its residues from the finished product. The biomass obtained on methanol does not contain undesirable impurities, which makes it possible to exclude the purification stage from the technological scheme.
However, during the process, it is necessary to take into account such features of methanol as flammability and the possibility of the formation of explosive mixtures with air.
Both yeast and bacterial strains have been studied as producers using methanol in constructive metabolism.Of the yeast, Candida boidinii, Hansenula polymorpha and Piehia pastoris were recommended for production, the optimal conditions for which (temperature 34-37 ° C, pH 4.2-4.6) allow the process to be carried out with an economic coefficient of assimilation of the substrate up to 0.40 at a speed flow in the range of 0.12-0.16 h-1.
Among bacterial cultures, Methylomonas clara, Pseudomonas rosea and others are used, capable of developing at a temperature of 32-34 ° C, pH 6.0-6.4 with an economic coefficient of substrate assimilation up to 0.55 at a flow rate of up to 0.5 h-1.
Features of the cultivation process are largely due to the used producer strain (yeast or bacteria) and the conditions of asepsis. A number of foreign companies offer to use yeast strains and carry out cultivation in the absence of strict asepsis. In this case, the technological process takes place in an ejection-type fermenter with a productivity of 75 tons of protein per day, and the specific consumption of methanol is 2.5 tons / ton of protein.
When cultivating yeast under aseptic conditions, a column or airliphite type apparatus with a capacity of 75-100 tons of protein / day with a methanol consumption of up to 2.63 tons / ton of protein is recommended. In both cases, the cultivation process is carried out in one stage, without the stage of "ripening", with a low concentration of the substrate (8-10 g / l).
In a number of countries, bacterial strains are used as producers, the process is carried out under aseptic conditions in airliphite or jet fermenters with a capacity of 100-300 tons / day and a methanol consumption of up to 2, 3 tons / ton of protein. Fermentation is carried out in one step at low alcohol concentrations (up to 12 g / l), with a high degree of methanol utilization.
The most promising in terms of its design is the jet fermenter of the Institute of Technical Chemistry (Germany). The fermenter with a volume of 1000 m consists of sections located one above the other and interconnected by shaft overflows.
The fermentation medium from the lower section of the fermenter through a pressure pipeline is supplied by centrifugal circulation pumps to the upper shaft overflows, through which it passes to the lower section, while sucking air from the gas conduit. Thus, the medium flows from section to section, constantly sucking in new portions of air. Falling jets in mine overflows provide intensive aeration of the medium.
The nutrient medium is continuously supplied to the area of the upper shaft overflow, and the microbial suspension is removed from the remote circuits. At the stage of isolation for all types of producers, the separation of granulation is provided in order to obtain a finished product in granules.
Fodder yeast obtained on methanol has the following composition (in%): crude protein 56-62; lipids 5-6; ash 7-11; moisture 8-10; nucleic acids 5-6. Bacterial biomass is characterized by the following composition (in%): crude protein 70-74; lipids 7-9; ash 810; nucleic acids 10-1h; humidity 8-10.
In addition to methanol, ethanol is used as a high-quality raw material, which has low toxicity, good solubility in water, and a small amount of impurities.
Yeast (Candida utilis, Sacharomyces lambica, Hansenula anomala, Acinetobacter calcoaceticus) can be used as microorganisms - protein producers on ethyl alcohol as the only carbon source. The cultivation process is carried out in one stage in fermenters with high mass transfer characteristics at an ethanol concentration of no more than 15 g / l.
Ethanol-grown yeast contains (in%): crude protein - 60-62; lipids - 2-4; ash - 8-10; moisture - up to 10.
Obtaining protein substances from carbohydrate raw materials
Historically, one of the first substrates used to obtain fodder biomass were plant waste hydrolysates, prehydralizates and sulphite liquor - waste products from the pulp and paper industry.
Interest in carbohydrate raw materials as the main renewable source of carbon has also increased significantly from an environmental point of view, since it can serve as the basis for creating a waste-free technology for processing plant products.
Due to the fact that hydrolysates are a complex substrate consisting of a mixture of hexoses and pentoses, the types of yeasts C. utilis, C. scottii and C.tropicalis, which, along with hexoses, are capable of assimilating pentoses, as well as transferring the presence of furfural in the medium.
The composition of the nutrient medium, in the case of cultivation on a hydrocarbon feedstock, significantly differs from that used for growing microorganisms on a hydrocarbon substrate. In hydrolysates and sulphite lye there are a small amount of almost all the microelements necessary for yeast growth. The missing amounts of nitrogen, phosphorus and potassium are introduced in the form of a general solution of ammophos salts, potassium chloride and ammonium sulfate.
Fermentation is carried out in air-lift devices designed by Lefrancois-Marillet with a volume of 320 and 600 m3. The yeast cultivation process is carried out in a continuous mode at a pH of 4.2-4.6. The optimum temperature is from 30 to 40 ° C.
Fodder yeast obtained by cultivation on hydrolysates of plant raw materials and sulfite lye has the following composition (in%): protein - 43-58; lipids - 2.3-3.0; carbohydrates - 11-23; ash - up to 11; humidity - no more than 10.
One of the promising substrates in the production of fodder biomass are peat hydrolysates, which contain a large amount of easily digestible monosaccharides and organic acids. Additionally, only small amounts of superphosphate and potassium chloride are added to the nutrient medium. The source of nitrogen is ammonia water.
In terms of quality, the fodder biomass obtained from peat hydrolyzates surpasses yeast grown on plant waste.
L.V. Timoshchenko, M.V. Chubik
Requirements for nutrient substrates,
used in biotechnological processes. Natural
raw materials of plant origin. Waste
various industries as raw materials for biotechnological processes.
Chemical and petrochemical substrates used as
raw materials for biotechnology.
Industrial biotechnology Microorganism protein production
Substrates for the cultivation of microorganisms in order to obtain protein
Microorganisms use a wide variety of substrates as sources of matter and energy - normal paraffins and oil distillates, natural gas, alcohols, vegetable hydrolysates and industrial waste.
For growing microorganisms for protein, it would be nice to have a carbon-rich, but cheap substrate. This requirement is fully met by normal (unbranched) oil paraffins. The yield of biomass can reach when using them up to 100% of the mass of the substrate. The quality of the product depends on the purity of the paraffins. When using paraffins of a sufficient degree of purification, the obtained yeast mass can be successfully used as an additional source of protein in animal diets. The world's first large feed yeast plant with a capacity of 70,000 tons per year. was launched in 1973 in the USSR. As a raw material, n-alkanes isolated from oil and several types of yeast capable of rapid growth on hydrocarbons were used: Candida maltosa, Candida guilliermondii, and Candida lipolytica. In the future, it was waste from oil refining that served as the main raw material for the production of yeast protein, which grew rapidly by the mid-1980s. exceeded 1 million tons per year, and in the USSR fodder protein received twice as much as in all other countries of the world combined. However, in the future, the scale of production of yeast protein from oil hydrocarbons sharply decreased. This happened both as a result of the economic crisis of the 90s, and because of a number of specific problems associated with this production. One of them is the need to clean the finished feed product from oil residues that have carcinogenic properties.
There are few areas in our country suitable for growing soybeans, which are the main source of protein supplements. Therefore establishedlarge-scale production of feed yeast on n-paraffins... There are several factories with a capacity from 70 to 240 thousand tons per year.Liquid refined paraffins are used as raw materials.
Methyl alcohol is considered one of the promising carbon sources for the cultivation of high-quality protein producers. It can be obtained by microbial synthesis on substrates such as wood, straw, municipal waste. The use of methanol as a substrate is difficult due to its chemical structure: the methanol molecule contains one carbon atom, while the synthesis of most organic compounds is carried out through two-carbon molecules. About 25 yeast species, including Pichia polymorpha, Pichia anomala, Yarrowia lipolytica, can grow on methanol as the only source of carbon and energy. Bacteria are considered the best producers on this substrate, because they can grow on methanol with the addition of mineral salts. Methanol-based protein production processes are quite economical. According to the ICI concern (Great Britain), the cost of a product produced on methanol is 10-15% lower than in a similar production based on highly purified n-paraffins. High-protein products from methanol are obtained by companies from a number of developed countries of the world: Great Britain, Sweden, Germany, USA, Italy. Protein producers are bacteria of the genus Methylomonas. Growing methylotrophic bacteria such as Methylophilus methylotrophus on methanol is beneficial because they use one-carbon compounds more efficiently. When growing on methanol, bacteria produce more biomass than yeast. The first oxidation reaction of methanol in yeast is catalyzed by oxidase, and in methylotrophic prokaryotes, by dehydrogenase. Genetic engineering work is underway to transfer the methanol dehydrogenase gene from bacteria to yeast. This will combine the technological advantages of yeast with the efficiency of bacterial growth.
The use of ethanol as a substrate eliminates the problem of purifying biomass from abnormal metabolic products with an odd number of carbon atoms. The cost of such production is somewhat higher. Ethanol-based biomass is produced in Czechoslovakia, Spain, Germany, Japan, and the USA.
In the USA, Japan, Canada, Germany, Great Britain, technological processes for obtaining protein using natural gas have been developed. The biomass yield in this case can be 66% of the substrate weight. The process developed in the UK uses a mixed culture of Methylomonas bacteria that metabolize methane, Hypomicrobium and Pseudomonas that metabolize methanol, and two types of non-methylotrophic bacteria. The culture is characterized by a high growth rate and productivity. The main advantages of methane (by the way, the main component of natural gas) are availability, relatively low cost, high conversion efficiency into biomass by methane-oxidizing microorganisms, a significant content of protein in biomass, balanced in amino acid composition. Bacteria growing on methane tolerate acidic environments and high temperatures well, and therefore are resistant to infections.
Mineral carbon - carbon dioxide can also be a substrate for microbial synthesis. Oxidized carbon in this case is successfully reduced by microalgae using solar energy and hydrogen-oxidizing bacteria using hydrogen. A suspension of algae is used for livestock feed. For the operation of installations for the cultivation of algae, stable climatic conditions are required - constant air temperatures and the intensity of sunlight.
The most promising is the production of protein using hydrogen-oxidizing bacteria, which develop due to the oxidation of hydrogen by atmospheric oxygen. The energy released in this process is used for the assimilation of carbon dioxide. As a rule, bacteria of the genus Hydrogenomonas are used to obtain biomass. Initially, interest in them arose during the development of closed life support systems, and then they began to be studied from the point of view of their use as producers of high-quality protein.At the Institute of Microbiology of the University of Göttingen (Germany), a method for the cultivation of hydrogen-oxidizing bacteria has been developed, in which it is possible to obtain 20 g of dry matter per 1 liter of cell suspension. Perhaps in the future, these bacteria will become the main source of dietary microbial proteins.
Plant biomass is an extremely accessible and fairly cheap source of carbohydrates for the production of microbial protein. Any plant contains a variety of sugars. Cellulose is a polysaccharide made up of glucose molecules. Hemicellulose consists of the remains of arabinose, galactose, mannose, fructose. The problem is that wood polysaccharides are linked by rigid oxyphenylpropane units of lignin, a polymer that is almost indestructible. Therefore, wood hydrolysis occurs only in the presence of a catalyst - mineral acid and at high temperatures. In this case, monosaccharides are formed - hexoses and pentoses. Yeast is grown on a liquid containing sugar fraction of the hydrolyzate. During acid hydrolysis of wood, a number of by-products (furfural, melanins) are formed, and due to high temperatures, caramelization of sugars can occur. These substances interfere with the normal growth of yeast, they are separated from the hydrolyzate and used whenever possible. Strains of Candida scotti and C. tropicalis are used as producers.
The largest producers of raw materials for the hydrolysis industry are woodworking enterprises, whose waste reaches tens of millions of tons annually. Unfortunately, waste from the production of bast fibers (from flax and hemp), potato starch production, brewing, fruit and vegetable, canning industries, beet pulp are not used rationally or not at all.
Methods for direct bioconversion of photosynthetic products and their derivatives into protein using fungi deserve special attention. These organisms, due to the presence of powerful enzyme systems, are able to utilize complex plant substrates without pretreatment. Studies of the conditions for the bioconversion of plant substrates into microbial protein are being actively carried out in the USA, Canada, India, Finland, Sweden, Great Britain, in our country and other countries of the world. However, there are few data in the literature on the large-scale production of proteins of microbial origin. The best known and most advanced to the stage of industrial implementation is the "Waterloo" process, developed at the University of Waterloo in Canada. This process, based on the cultivation of the cellulose-destroying fungi Chaetomium cellulolyticum, can be carried out both in submerged culture and by the surface method. The protein content of the final product (dried mushroom mycelium) is 45%. The Finnish company "Tampella" has developed the technology and organized the production of the protein feed product "Pekilo" on the waste of the pulp and paper industry. The product contains up to 60% protein with a good amino acid profile and a significant amount of B vitamins.
In most milk producing countries, the traditional way of utilizing whey is feeding it to animals. The degree of conversion of whey protein to animal protein is very low (1700 kg of whey is needed to produce 1 kg of animal protein). In the last 10-15 years, high-quality proteins have been isolated from whey by ultrafiltration, on the basis of which substitutes for skimmed milk powder and other products are made. Concentrates can be used as food additives and components of baby food. Whey is also used to produce milk sugar - lactose, which is used in the food and medical industries. With all this, the volume of industrial processing of whey is 50-60% of its total production. Consequently, there is a large loss of the most valuable milk protein and lactose. Moreover, there is a problem of waste disposal, since the process of natural decomposition of whey is extremely slow.Whey lactose can serve as an energy source for many types of microorganisms, as a raw material for the production of microbial synthesis products (organic acids, enzymes, alcohols, vitamins) and protein biomass. Of all known microorganisms, yeast has the highest conversion rate of whey protein to microbial protein. The ability to assimilate lactose is found in about 20% of all known yeast species. Yeast fermenting lactose is much less common. Active catabolism of lactose is especially characteristic of yeast from the genus Kluyveromyces. This yeast can be used to obtain feed protein, ethanol, β-glucosidase preparations on whey.
For the first time, whey-based yeast was grown in Germany. Various strains of saccharomycetes were used as producers. Methods for obtaining microbial products based on the use of lactose as a monoculture and a mixture of yeast and bacteria have been developed. Currently, yeasts of the genera Candida, Trichosporon, Torulopsis are used as producers. In terms of biological value, whey with yeast grown in it significantly exceeds the original raw material and it can be used as a milk substitute. The above list of microorganisms and processes for obtaining protein in unicellular organisms is not exhaustive. However, the potential of this new industry is far from being fully exploited. In addition, we do not yet know all the possibilities of the activity of microorganisms as protein producers, but as our knowledge deepens, they will be expanded.
Raw materials and composition of culture media for biotechnological production The nutrient medium provides vital activity, growth, development of a biological object, effective synthesis of the target product. An integral part of the nutrient medium is water, nutrients that form true solutions (mineral salts, amino acids, carboxylic acids, alcohols, aldehydes, etc.) and colloidal solutions (proteins, lipids, inorganic compounds - iron hydroxide). Individual components can be in a solid state of aggregation, can float, be evenly distributed throughout the volume as a suspension, or form a bottom layer.Raw materials for culture media in biotechnological production
The raw materials used to obtain the target product should be non-scarce, inexpensive, and as readily available as possible: molasses - a by-product of sugar production, oil and natural gas components, agricultural waste, woodworking and paper industries, etc. Most often, food waste is used as components of nutrient media. Beet molasses - a waste product from sugar production from beets, is rich in organic and mineral substances necessary for the development of microorganisms. It contains 45-60% sucrose, 0.25-2.0% invert sugar, 0.2-3.0% raffinose. In addition, molasses contains amino acids, organic acids and their salts, betaine, minerals, and some vitamins. Used for industrial production of citric acid, ethanol and other products. Molasses stillage is a waste of molasses-alcohol production. The chemical composition of vinasse depends on the composition of the original molasses and varies widely. According to its chemical composition, molasses stillage is a full-fledged raw material for the production of feed yeast, which does not require the addition of growth substances, since it contains a sufficient amount of vitamins. The content of dry matter in natural stillage is 8-12%, in evaporated stillage - 53%. Grain and potato stillage is a waste of alcohol production. The content of soluble dry substances is usually 2.5-3.0%, including 0.2-0.5% of reducing substances, there are sources of nitrogen and trace elements. It is used to obtain microbial protein. Brewing waste (brewer's grains and malt sprouts) and unmalted barley waste are a suitable, but small, source of digestible carbohydrate for microbial protein production. For the production of feed yeast, this raw material is appropriately hydrolyzed and introduced into the nutrient medium in a ratio of 8: 0.2: 0.05 (pellet: sprouts: barley waste). Wheat bran is a waste of milling production, it is used for the preparation of nutrient media in the solid-phase cultivation method. They have a rich chemical composition and can be used as the only component of the nutrient medium. Since wheat bran is an expensive product, it is mixed with cheaper components: sawdust, malt sprouts, fruit pomace, etc. Whey is a waste from the production of cheese, cottage cheese and casein. In this regard, a distinction is made between cheese, cottage cheese and casein whey. In terms of chemical composition and energy value, this product is considered "half milk". Milk whey is very rich in various biologically active compounds, its dry residue contains on average 70-80% lactose, 7-15% protein substances, 2-8% fat, 8-10% mineral salts. In addition, whey contains a significant amount of hormones, organic acids, vitamins and microelements. The presence of carbon sources easily assimilated by many types of microorganisms in milk whey, as well as various growth factors, makes it one of the most valuable nutrient media for obtaining products of microbial synthesis, for example, for the production of protein preparations on an industrial scale. Of great importance is the fact that the use of milk whey does not require special complex preparation, and the culture liquid after the growth of microorganisms can be used for food and feed purposes without processing.
Microbial biomass production is the largest microbiological production. Microbial biomass can be a good protein supplement for pets, birds and fish. Microbial biomass production is especially important for countries that do not cultivate soy on a large scale (soy flour is used as a traditional protein feed additive).
When choosing a microorganism, the specific growth rate and biomass yield on a given substrate, stability during continuous cultivation, and cell size are taken into account. Yeast cells are larger than bacteria and are easier to separate from liquids by centrifugation. Large cell polyploid yeast mutants can be grown. Currently, only two groups of microorganisms are known that possess the properties necessary for large-scale industrial production: these are the yeast of the genus Candida on n-alkanes (normal hydrocarbons) and the bacteria Methylophillus methylotrophus on methanol.
Microorganisms can be grown on other nutrient media: gases, oil, waste coal, chemical, food, wine and vodka, woodworking industries. The economic benefits of using them are obvious. So, a kilogram of oil processed by microorganisms gives a kilogram of protein, and, say, a kilogram of sugar - only 500 grams of protein. The amino acid composition of yeast protein practically does not differ from that obtained from microorganisms grown on conventional carbohydrate media. Biological tests of preparations made from yeast grown on hydrocarbons, which were carried out both in our country and abroad, showed that they did not have any harmful effect on the organism of the tested animals. The experiments have been carried out on many generations of tens of thousands of laboratory and farm animals. Unprocessed yeast contains nonspecific lipids and amino acids, biogenic amines, polysaccharides and nucleic acids, and their effect on the body is still poorly understood. Therefore, it is proposed to isolate protein from yeast in a chemically pure form.Freeing it from nucleic acids has also become easy.
In modern biotechnological processes based on the use of microorganisms, yeast, other fungi, bacteria and microscopic algae serve as protein producers.
From a technological point of view, yeast is the best of them. Their advantage lies primarily in manufacturability: yeast is easy to grow under production conditions. They are characterized by a high growth rate, resistance to extraneous microflora, are able to assimilate any food sources, are easily separated, and do not pollute the air with spores. Yeast cells contain up to 25% dry matter. The most valuable component of yeast biomass is protein, which, in terms of amino acid composition, surpasses the protein of cereal grain and is only slightly inferior to proteins of milk and fish meal. The biological value of yeast protein is determined by the presence of a significant amount of essential amino acids. In terms of the content of vitamins, yeast surpasses all protein feeds, including fish meal. In addition, yeast cells contain trace elements and a significant amount of fat, which is dominated by unsaturated fatty acids. When feeding fodder yeast to cows, milk yield and fat content in milk increase, and the quality of fur improves in fur animals. Of interest are yeasts possessing hydrolytic enzymes and capable of growing on polysaccharides without their preliminary hydrolysis. The use of such yeast will avoid the expensive stage of hydrolysis of polysaccharide-containing waste. More than 100 yeast species are known to thrive on starch as the sole carbon source. Among them, two species are especially distinguished, which form both glucoamylases and β-amylases, grow on starch with a high economic coefficient and can not only assimilate, but also ferment starch: Schwanniomyces occidentalis and Saccharomycopsis fibuliger. Both species are promising producers of protein and amylolytic enzymes on starch-containing waste. The search is underway for such yeasts that could break down the native cellulose. Cellulases have been found in several species, for example, in Trichosporon pullulans, but the activity of these enzymes is low and there is no need to talk about the industrial use of such yeasts. Yeast from the genus Kluyveromyces grows well on inulin, the main storage substance in Jerusalem artichoke tubers, an important forage crop that can also be used to obtain yeast protein.
Recently, bacteria have begun to be used as protein producers, which have a high growth rate and contain up to 80% protein in the biomass. Bacteria lend themselves well to selection, which makes it possible to obtain highly productive strains. Their disadvantages are difficult sedimentation due to small cell sizes, significant sensitivity to phage infections, and a high content of nucleic acids in the biomass. The latter circumstance is unfavorable only if the food use of the product is envisaged. There is no need to reduce the content of nucleic acids in the biomass used for animal feed, since uric acid and its salts formed during the destruction of nitrogenous bases are converted in the animal body into allantoin, which is easily excreted in the urine. In humans, an excess of uric acid salts can contribute to the development of a number of diseases.
The next group of protein producers are mushrooms. They attract the attention of researchers due to their ability to utilize the most diverse organic raw materials: molasses, milk whey, plant and root crop juice, lignin - and cellulose-containing solid waste from the food, woodworking, and hydrolysis industries. Mushroom mycelium is rich in protein substances, which are closest to soy proteins in terms of the content of essential amino acids. At the same time, the protein of mushrooms is rich in lysine, the main amino acid that is missing in the protein of cereals.This makes it possible to formulate balanced food and feed mixtures on the basis of grain and mushroom biomass. Mushroom proteins have a fairly high biological value and are well absorbed by the body.
The fibrous structure of the grown culture is also a positive factor. This allows you to imitate the texture of the meat, and with the help of various additives, its color and smell. The mushroom mycelium is usually stored frozen.
Fungi use glucose and other nutrients as a substrate, and ammonia and ammonium salts are a common source of nitrogen. After the completion of the fermentation stage, the culture is subjected to heat treatment to reduce the content of ribonucleic acid, and then the mycelium is separated by vacuum filtration.
Algae can also serve as sources of protein substances. With a phototrophic method of feeding and biomass formation, they use atmospheric carbon dioxide. Algae are grown, as a rule, in the surface layer of ponds, where as much protein can be obtained from an area of 0.1 hectares as from 14 hectares of beans. Algae protein is suitable not only for feed, but also for food purposes.
Finally, good protein producers are duckweed, which accumulate protein up to 45% of the dry weight, as well as up to 45% of carbohydrates. However, despite their small size, they do not belong to the aforementioned protein producers (microorganisms), since they are not only multicellular organisms, but also belong to higher plants.
MINISTRY OF EDUCATION OF THE RUSSIAN FEDERATION SYKTYVKAR STATE UNIVERSITY Department of Botany Abstract on the topic: PROTEIN PRODUCTION
Performer: student 243 gr.
Aniskina Maria
Lecturer: Ph.D., Associate Professor,
Shergina N.N.
Syktyvkar 2000
CONTENTS _______ 2
INTRODUCTION __________ 3
1.Protein of unicellular organisms 4
1.1. Obtaining microbial protein on lower alcohols__ 4
1.2. Obtaining protein substances from carbohydrate raw materials ____ 7
2.Mushroom protein (mycoprotein) ________ 8
REFERENCES _______ 10
INTRODUCTION
Microorganisms began to be used in the production of protein products long before the emergence of microbiology. Suffice it to mention all kinds of cheese, as well as products obtained by fermentation of soybeans. Both in the first and in the second case, the nutritional basis is protein. When these products are developed with the participation of microbes, a profound change in the properties of protein-containing raw materials occurs. The result is food products that can be stored longer (cheese) or more convenient to consume (bean curd). Microbes play a role in the production of some meat products for storage. So, in the manufacture of some varieties of sausages, acidic fermentation is used, usually with the participation of a complex of lactic acid bacteria. The resulting acid contributes to the preservation of the product and contributes to the formation of its special taste.
This, perhaps, limits the use of microorganisms in the processing of proteins. The possibilities of modern biotechnology in these industries are small, with the exception of cheese making. The cultivation and collection of microbial mass processed into food is another matter: here biotechnology can manifest itself in its entirety.
For many important indicators, the biomass of microorganisms can have a very high nutritional value. To a large extent, this value is determined by proteins: in most species, they make up a significant proportion of the dry mass of cells. For decades, the prospects for increasing the share of microbial protein in the total balance of protein produced worldwide have been actively discussed and investigated.
The production of such protein involves the large-scale cultivation of certain microorganisms that are collected and processed into food. To realize the fullest possible transformation of the substrate into microbial biomass, a multifaceted approach is required. Growing microbes for food is of interest for two reasons.First, they grow much faster than plants and animals: the time for doubling their numbers is measured in hours. This shortens the time it takes to produce a certain amount of food. Secondly, depending on the grown microorganisms, various types of raw materials can be used as substrates. As for substrates, here you can go in two main directions: process low-quality waste products or focus on readily available carbohydrates and obtain microbial biomass containing high-quality protein from them.
1.1. Obtaining microbial protein on lower alcohols
Cultivation in methanol. The main advantage of this substrate is its high purity and absence of carcinogenic impurities, good solubility in water, high volatility, which makes it easy to remove its residues from the finished product. The biomass obtained on methanol does not contain undesirable impurities, which makes it possible to exclude the purification stage from the technological scheme.
However, it is necessary to take into account during the process such features of methanol as flammability and the possibility of the formation of explosive mixtures with air.
Both yeast and bacterial strains have been studied as producers using methanol in constructive metabolism. In yeast, Candida boidinii, Hansenula polymorpha and Piehia pastoris were recommended for production, the optimal conditions for which (t = 34-37 ° C, pH = 4.2-4.6) allow the process to be carried out with an economic coefficient of substrate assimilation up to 0.40 at a flow rate in the range of 0.12-0.16 h-1. Among bacterial cultures, Methylomonas clara, Pseudomonas rosea and others are used, capable of developing at t = 32-34 ° C, pH = 6.0-6.4 with an economic coefficient of substrate assimilation up to 0.55 at a flow rate of up to 0.5 h; one.
Features of the cultivation process are largely due to the used producer strain (yeast or bacteria) and the conditions of asepsis. A number of foreign companies offer to use yeast strains and carry out cultivation in the absence of strict asepsis. In this case, the technological process takes place in an ejection-type fermenter with a capacity of 75 tons of ACW per day, and the specific consumption of methanol is 2.5 t / t ACW.
When cultivating yeast under aseptic conditions, columnar or earlift type devices with a capacity of 75-100 t ACV / day are recommended at a methanol consumption of up to 2.63 t / t ACV. In both cases, the cultivation process is carried out in one stage, without the stage of "ripening" with a low concentration of the substrate (8-10 g / l).
In a number of countries, bacterial strains are used as producers; the process is carried out under aseptic conditions in airliphite or jet type fermenters with a capacity of 100-300 t / day and a methanol consumption of up to 2.3 t / t ASB. Fermentation is carried out in one step at low alcohol concentrations (up to 12 g / l) with a high degree of methanol utilization.
The most promising in terms of its design is the jet fermenter of the Institute of Technical Chemistry of the Academy of Sciences of the German Democratic Republic. The fermenter with a volume of 1000m3 consists of sections located one above the other and interconnected by shaft overflows. The fermentation medium from the lower section of the fermenter through a pressure pipeline is supplied by centrifugal circulation pumps to the upper shaft overflows, through which it passes to the lower section, while sucking air from the gas pipeline. Thus, the medium flows from section to section, constantly sucking in new portions of air. Falling jets in mine overflows provide intensive aeration of the medium.
The nutrient medium is continuously supplied to the area of the upper shaft overflow, and the microbial suspension is removed from the remote circuits. At the stage of isolation for all types of producers, the separation of granulation is provided in order to obtain a finished product in granules.
Fodder yeast obtained on methanol has the following percentage composition: crude protein 56-62; lipids 5-6; ash 7-11; moisture 8-10; nucleic acids 5-6.Bacterial biomass is characterized by the following composition: crude protein 70-74; lipids 7-9; ash 8-10; nucleic acids 10-13; humidity 8-10.
In addition to methanol, ethanol is used as a high-quality raw material, which has low toxicity, good solubility in water, and a small amount of impurities.
Yeast (Candida utilis, Sacharomyces lambica, Hansenula anomala, Acinetobacter calcoaceticus) can be used as microorganisms that produce protein on ethyl alcohol as the only carbon source. The cultivation process is carried out in one stage in fermenters with high mass transfer characteristics at an ethanol concentration of no more than 15 g / l.
Ethanol-grown yeast contains (%): crude protein 60-62; lipids 2-4; ash 8-10; moisture up to 10.
1.2. Obtaining protein substances from carbohydrate raw materials
Historically, one of the first substrates used to obtain fodder biomass was plant waste hydrolysates, prehydralizates and sulphite liquor - waste products from the pulp and paper industry. Interest in carbohydrate raw materials as the main renewable source of carbon has also increased significantly from an environmental point of view, since it can serve as the basis for creating a waste-free technology for processing plant products.
Due to the fact that hydrolysates are a complex substrate consisting of a mixture of hexoses and pentoses, the species of yeast C.utilis, C.scottii and C.tropicalis have become widespread among industrial producer strains, which, along with hexoses, are capable of assimilating pentoses, as well as transferring the presence of furfural in the environment.
The composition of the nutrient medium in the case of cultivation on a hydrocarbon feedstock significantly differs from that used for growing microorganisms on a hydrocarbon substrate. In hydrolysates and sulphite lye there are a small amount of almost all the trace elements necessary for yeast growth. The missing amounts of nitrogen, phosphorus and potassium are introduced in the form of a general solution of ammophos salts, potassium chloride and ammonium sulfate.
Fermentation is carried out in air-lift devices designed by Lefrancois-Marillet with a volume of 320 and 600 m3. The yeast cultivation process is carried out in a continuous mode at a pH of 4.2-4.6. The optimum temperature is from 30 to 40 ° C.
Fodder yeast obtained by cultivation on hydrolysates of plant raw materials and sulfite liquors has the following composition (%): protein 43-58; lipids 2.3-3.0; carbohydrates 11-23; ash - up to 11; humidity - no more than 10.
One of the promising substrates in the production of fodder biomass are peat hydrolysates, which contain a large amount of easily digestible monosaccharides and organic acids. Additionally, only small amounts of superphosphate and potassium chloride are added to the nutrient medium. The source of nitrogen is ammonia water. In terms of quality, the fodder biomass obtained from peat hydrolyzates surpasses yeast grown on plant waste.
Mycoprotein is a food product consisting mainly of the mycelium of the fungus. In its production, the Fusarium graminearum strain isolated from the soil is used. The mycoprotein is produced today in a pilot plant by the continuous growing method. Glucose and other nutrients are used as a substrate, and ammonia and ammonium salts are sources of nitrogen. After the completion of the fermentation stage, the culture is subjected to heat treatment to reduce the content of ribonucleic acid, and then the mycelium is separated by vacuum filtration.
If we compare the production of mycoprotein with the synthesis of animal proteins, then a number of its advantages will be revealed. In addition to the fact that the growth rate is higher here, the transformation of the substrate into protein is incomparably more efficient than when the food is assimilated by domestic animals. This is reflected in table 1.
It is worth recalling that animal feed should contain a certain amount of protein, up to 15-20%, depending on the type of animals and the way they are kept.The fibrous structure of the grown culture is also a positive factor; the texture of the mycelium mass is close to that of natural products, therefore, the texture of the meat can be imitated in the product, and, due to additives, its taste and color. The density of the product depends on the hyphal length of the grown mushroom, which is determined by the growth rate.
Table 1. Conversion efficiency for protein formation for various animals and Fusarium graminearum.
| Original product | Products and services | ||
| Protein, g | Total, g | ||
| Cow | 1 kg feed | 14 | 68 beef |
| Pig | 1 kg feed | 41 | 200 pork |
| Chicken | 1 kg feed | 49 | 240 meat |
| Fusarium graminearum | 1 kg carbohydrates + inorganic nitrogen | 136 | 1080 cell mass |
Following extensive research into the nutritional value and safety of the mycoprotein, the USDA has approved its marketing in England. Its nutrient content is shown in Table 2.
Table 2. Average composition of mycoprotein and comparison with that of beef.
| Components | Composition,% (dry weight) | |
| mycoprotein | steak | |
| Protein | 47 | 68 |
| Fats | 14 | 30 |
| Alimentary fiber | 25 | Traces |
| Carbohydrates | 10 | 0 |
| Ash | 3 | 2 |
| RNA | 1 | Traces |
1. Biotechnology: Principles and Applications. Ed. I. Higgens and others. Moscow: "Mir", 1988
2. Biotechnology. Production of protein substances. V.A.Bykov, M.N. Manakov and others. Moscow "Higher School", 1987
3. Vorobieva A.I. Industrial microbiology. Ed. Moscow University, 1989
