General Biotechnology: Lecture Course. Part II - Blinov, V. A. 2004

Production of single-cell protein. Large-scale microbiological production of enzyme preparations

♦ Non-traditional protein sources.

♦ Raw material base for Single-Cell Protein synthesis.

♦ Process flow diagram for fodder biomass production.

♦ Prospects for biomass utilization.

♦ Production of enzyme preparations for various Applications.

Food is a complex of mixed products that differ in chemical composition, nutritional properties, and biological effects. Some food products serve as sources of structural and growth substances, others as Energy Sources, while still others ensure the supply of essential, vital, biologically active components to the body.

The Main sources of structural compounds are foods of animal origin. Their most important constituent part, protein, contains Essential Amino acids that are favorably balanced for the Synthesis of the body's own protein compounds.

In Human Nutrition, meat and meat products are not only the primary source of complete protein, but also of fat, a complex of Vitamins, and minerals. Protein digestibility is at least 96%. Proteins from plant-based foods also play an important role, although their digestibility is lower, ranging within 70-85%. However, a rational ratio of animal and plant food products ensures optimal protein nutrition through the mutual supplementation of their amino acid profiles.

Energy sources in human nutrition are mainly foods rich in CARBOHYDRATES and fats. Finally, all niche foods are to some extent sources of biologically active, regulatory substances.

In nutrition, It is important not only to supply the body with energy and structural Materials. Food must also regulate physiological Functions, biochemical and behavioral reactions, reduce the risk of diseases, accelerate recovery from various illnesses, and promote longevity. To this end, modern diets incorporate bifidobacteria, bifidogenic factors of various origins, dietary fiber, amino acids, Peptides, vitamins, Fatty acids, Choline, Glycosides, specific minerals, etc. The range of such BIOLOGICALLY ACTIVE SUBSTANCES is steadily expanding.

Protein deficiency in the diet of animals and humans leads to profound negative changes. These manifest as a negative nitrogen balance, hypoproteinemia, and disturbances in colloidal-osmotic and Water-Salt METABOLISM. The consequences of prolonged protein shortage include Various Forms of anemia, dysfunctions of the nervous, endocrine, and cardiovascular systems, profound metabolic shifts, growth arrest, emaciation, etc. Particularly severe disorders develop at a young age. It must be emphasized that not only complete absence of protein in food is dangerous, but also its insufficient intake or the consumption of low-quality protein. Dietary protein must contain all amino acids, especially essential ones, be close in composition to The amino acid profile of the body's proteins, and be easily digestible in the gastrointestinal tract.

Non-traditional protein sources. Currently, the global dietary protein deficit exceeds 15 million tons per year. This shortage of an essential food component is difficult to replenish because it is associated with the reduction of arable land, soil depletion, salinization and erosion, urbanization, crop losses due to pests, crop diseases, biosphere pollution, as well as a decrease in livestock population and productivity caused by an inadequate feed base, etc. The shortage of feed protein on a global scale, according to FAO UN data, is estimated at approximately 30 million tons per year.

Fundamentally changing this situation is possible only through biotechnological means. Moreover, the producers of food and, particularly, feed protein can be Bacteria, Yeasts, microalgae, micro- and macromycetes. Microbial biomass has a very high growth rate and contains up to 70-80% protein, which is rich in essential Amino Acids and other important biologically active substances.

Raw material sources for microbial Protein Synthesis are quite substantial and readily available. These include petroleum paraffins, various alcohols including methanol and ethanol, natural gas, and various industrial wastes, especially from the meat and dairy industries, as well as agriculture. Plant biomass serves as an accessible and cheap nutritional source for bacteria.

From a technological standpoint, yeasts are the best among them. They are easy to cultivate under industrial conditions. Yeasts grow and multiply rapidly on virtually any substrates, are resistant to contaminating microflora, contain more protein than cereal grains, and are only slightly inferior in Amino Acid Composition to milk protein and fish meal. Yeasts are very rich in many vitamins, namely: thiamine, riboflavin, pantothenic and nicotinic acids, pyridoxine, Folic acid, as well as choline, Inositol, etc. Yeast Cells contain Trace Elements and a significant amount of fat, dominated by Unsaturated fatty acids. One kg of fodder yeast contains 1.03-1.16 feed units. The disadvantages of yeasts include a thick Cell wall and a high nucleic acid content.

Bacteria are also protein producers. They are characterized by a high growth rate. The protein content in their biomass is 70-80%. With a significant amount of Methionine, bacteria are easily amenable to Selection, making it possible to obtain highly productive strains. Their disadvantages include difficult sedimentation due to small cell sizes, significant sensitivity to infections, especially phage infections, and a high nucleic acid content in the biomass.

Recently, interest in Algae as protein sources has increased significantly. Microalgae, as phototrophs, use only atmospheric carbon dioxide to form their biomass. It is known that 0.1 hectares of pond surface can yield as much protein as 14 hectares of bean crops. Some peoples have consumed marine and oceanic algae since ancient times. For example, over 300 recipes incorporating algae are known in Japan.

The cultivation of algae as aquaculture is a thriving branch of biotechnology. Special attention is currently given to the blue-green alga spirulina (Spirulina platensis and Spirulina maxima). Its biomass meets the highest standards of dietary protein and is rich in vitamins A, D, and B-complex. Algal protein is suitable for both feed and food purposes.

Another example: more than 300 species of edible plants grow in Siberia, excluding mushrooms and algae. Among them, water chestnut, snake knotweed, lyme grass, nettle, oleaster, mannagrass, burdock, arrowhead, sorrel, etc., can serve as additional sources of dietary protein. Furthermore, many of these plant species surpass cultivated plants in nutritional and flavor qualities or have no analogues at all.

Finally, mushrooms, both lower and higher, can be an important source of high-quality protein. The high Nutritional Value of the fruiting bodies of higher mushrooms has been known for a long time. However, their gross harvesting in natural conditions naturally cannot meet the ever-increasing demand for protein. Therefore, attempts were made to cultivate macromycete mycelium under industrial conditions. This was based on the assumption that the mycelium would match the flavor and nutritional qualities of mushroom fruiting bodies. This turned out not to be the case. The vegetative body of higher mushrooms (mycelium) lacked the taste, aroma, and nutritional value of the fruiting bodies, while the medium for cultivating the mycelium proved to be very complex and expensive. At the same time, micromycete mycelium has been used for a long time and enjoys well-deserved popularity. For instance, in Indonesia, peanut cakes are overgrown with mold Fungi of the genus Rhizopus. In Japan, the mold fungus Aspergillus oryzae is cultivated on soybeans. In China, a peculiar delicacy is obtained by growing fungi of the genus Mucor on soybeans. There are many such Examples. Importantly, thanks to micromycetes, starch-containing food is enriched with protein and becomes similar to meat products. It is likely that micromycetes will be one of the promising, non-traditional sources of dietary protein in the near future.

Admittedly, compared to reference protein, fungal proteins are limited in the sum of Sulfur-Containing Amino Acids (Cysteine and methionine). At the same time, they are rich in Lysine, the main amino acid lacking in cereal protein. This makes it possible to formulate balanced food and feed mixtures based on grain and fungal biomass.

Raw material base for single-cell protein synthesis. Obtaining microbial protein requires a carbon-rich yet cheap substrate. Normal (straight-chain) petroleum paraffins (n-paraffins) meet this requirement. A high biomass yield (up to 100% of the substrate weight) is ensured by the high carbon content, while product quality depends on the degree of paraffin purity. If the paraffins are insufficiently purified, the yeast biomass contains non-metabolizable components: benzene derivatives, D-amino acids, Lipids with an odd number of carbon atoms in fatty acids, and protein-based toxins. Therefore, petroleum paraffins must be thoroughly purified. Yeasts grown on n-paraffins are used in amounts of 8-15% of the total protein in the diet for fattening cattle, pigs, sheep, and broilers.

Methanol is another promising carbon source for cultivating protein producers. It is obtained by microbial synthesis from wood, straw, and municipal waste. The difficulty of using methanol lies in the fact that its molecule contains only one carbon atom, whereas the synthesis of most Organic compounds proceeds via two-carbon molecules. Compounds with an odd number of carbon atoms are generally not indifferent to the Organism. Bacteria of the genus Methylomonas are used as producers. Methanol is assimilated by bacteria, yeasts, fungi, and actinomycetes. Producing protein from methanol is more economical than using n-paraffins. For example, the product "Pruteen" (UK) contains 72% crude protein and is used as a high-protein additive to compound feeds in the diets of pigs, poultry, fur-bearing animals, and as a milk replacer for calves. Along with methanol, methane and carbon monoxide serve as Energy Sources for A number of microorganisms.

The Use of ethanol as a substrate for microbial protein synthesis eliminates Structure/149.html">The problem of purifying biomass from anomalous metabolic products with an odd number of carbon atoms. The cost of ethanol production is somewhat higher than that of methanol. Such technology is used in the Czech Republic, Spain, Germany, Japan, and the USA. In these and other countries, technological processes have been developed for producing protein from natural gas using methane-assimilating bacteria Methylomonas, methanol-utilizing Hypomicrobium and Pseudomonas, etc.

Special attention of researchers is drawn to plant biomass, which contains a large amount of sugars. These include Cellulose, consisting of glucose molecule residues, and hemicelluloses, consisting of residues of arabinose, galactose, mannose, fructose, and xylose. Yeasts are grown on the liquid sugar-containing fraction of the hydrolysate. In addition to plant hydrolysates, fodder yeast production utilizes post-alcohol distillery stillage, sunflower husks, cottonseed hulls, bast fiber production waste (flax and hemp shives), beet pulp, potato-starch production waste, as well as waste from the brewing, fruit and vegetable, and canning industries, etc. Cereal straw, which is usually proteinized (e.g., by yeast Fermentation) or whose digestibility is increased through the action of enzyme preparations (pectofoetidin G3x in combination with celluloviridin G3x or glucavamorin Px), is used as raw material for the Hydrolysis industry. Methods for the direct bioconversion of Photosynthesis products and their derivatives into single-cell protein using higher and lower fungi are widely employed. For this purpose, cellulose-degrading fungi such as Chaetomium cellulolyticum, as well as Aspergillus niger, Trichoderma, etc., are utilized.

Significant prospects are opened up by the PRODUCTION OF MICROBIAL protein from whey. Around 200 million tons of whey are generated globally every year. Each ton of whey contains 50 kg of milk sugar, up to 10 kg of high-value protein, 1.5 kg of fat, as well as vitamins, microelements, and other components. Using whey directly is inefficient both for feed production and animal feeding. This is because the degree of whey utilization decreases as its proportion in the diet increases. Moreover, this leads to digestive disorders, and The conversion of whey protein into animal body protein is extremely low. This also applies to dried whey, as the animal organism assimilates only 20% of it due to an unfavorable ratio of carbohydrates, proteins, and mineral salts.

In this regard, The production of milk-protein concentrates is more rational. Here, whey proteins are used to make skimmed milk powder substitutes, food additives, and baby food components. These proteins are incorporated into milk chocolate, candies, egg and pasta products, and find applications in the meat, baking, and confectionery industries. Milk sugar is utilized in the medical and food industries, in the production of various beverages, and so on. However, the volume of industrial whey Processing still does not exceed 50–60% of the total amount produced.

Lactose can serve as an energy source for many species of microorganisms, as well as a raw material for the microbial Production of organic acids, Enzymes, alcohols, vitamins, and, of course, protein biomass. Yeasts are most commonly used to obtain such biomass. It has been established that the conversion rate of whey protein into microbial protein by yeast is 20 times higher than the degree of its conversion into animal protein. In addition, most yeast species enrich the whey with vitamins. Various strains of the genera Saccharomyces, Kluyveromyces fragilis, Candida, Trichosporon, and Torulopsis are used as producers.

The USE OF MICROORGANISMS for food purposes involves addressing a number of issues, both technological—such as finding producer strains, selecting raw materials, and establishing suitable cultivation conditions—and biomedical, ensuring the safety of the resulting products. Highly purified microbial protein isolates should be used for food purposes rather than whole-cell or partially refined biomass. Protein isolates need to be endowed with the taste, color, odor, and texture of familiar foods. The most challenging aspect is food texture, which is an engineering task. Already today, Microbial and Plant protein texturates are produced in a number of countries. For instance, soy proteins are used to manufacture ham, bacon, sausages, and other products.

Thus, humanity has already embarked on restructuring the food production system, transitioning to the use of unlimited renewable resources and their transformation into food, feed, and technical-grade products.

Technological flowchart for obtaining feed biomass. Let us emphasize once again the advantages of biomass production via microbial synthesis over other protein sources. First, it features a high rate of biomass accumulation, which is 500–5,000 times higher than that of plants or animals. Second, microbial cells accumulate large amounts of protein (up to 60% in yeast and up to 75% in bacteria by dry weight). Third, the production of microbial protein lacks multiple stages: the Biosynthesis process proceeds under mild conditions at a Temperature of 30–45 °C, a pH of 3–6, and a pressure of 0.1 MPa. It is less labor-intensive compared to agricultural production and the organic synthesis of proteins.

The production of microbial protein is the highest-tonnage sector in biotechnology. The fundamental technological flowchart for cultivating feed biomass is as follows:

♦ Isolation of the pure culture. Producer strains from test tubes are transferred into conical shaker flasks containing a sterile nutrient medium. The flasks are placed on a shaker, and cultivation is carried out under optimal temperature and pH conditions while monitoring The Development of microorganisms;

♦ the pure culture in the log phase is transferred into a small seed bioreactor (500 L) with a nutrient medium, the pH of which is adjusted to 5.5–5.8 using aqueous ammonia or milk of lime. First, about 40 L of the medium is fed into the apparatus, diluted 4–4.5 times with sterile water, and, under intensive aeration, the remaining amount of the nutrient medium (80–100 L) is added, bringing the medium pH to 4.5–5.5. A microbial suspension with a volume of 1.5–2 L (approximately 0.4%) is introduced from the shaker flasks, and cultivation is carried out until the medium accumulates 3.5–4.0 g of cells/L in terms of absolute dry matter (ADM). This typically takes 15–18 hours;

♦ the suspension from the small seed bioreactor is fed into a 4–5 m³ apparatus previously filled with nutrient medium (~200 L) and sterile water (1.2–1.5 m³). Aeration is turned on, and cultivation is carried out for 10–12 hours with a continuous feed of 70–75 L/h of nutrient medium and The addition of aqueous ammonia to maintain the target pH;

♦ next, the seed culture is grown in a 15–20 m³ fermenter. The apparatus is filled to 10% (by volume) with sterile or boiled water, about 0.5 m³ of nutrient medium is introduced, and the entire Contents of the previous apparatus (2.5–2.7 m³) are pumped in completely. The growth of the inoculum without suspension withdrawal continues for 8–9 hours under intensive aeration and with a constant feed of nutrient medium (170–200 L/h) until biomass accumulates in the fermenter to an amount of 4–5 g ADM/L. After this, the seed culture begins to be drawn off to the main production line at a rate of 1.3–1.7 m³/h while simultaneously feeding in fresh nutrient medium.

The fermentation process lasts from 5 to 10 days, after which the inoculum preparation cycle restarts. The preparatory stages of production include the preparation of nutrient salt and trace element solutions necessary for the normal development of microorganisms. This section has its own technological flowchart. Typically, the set of mineral components is grouped into two solutions that are fed in parallel to the main fermenter: a solution of all Macronutrients (N, P, K), the required amount of which is 5–70 g/L; and a solution of micronutrients (Mg, Mn, Fe, Zn, etc.), the concentrations of which do not exceed 5–10 mg/L.

Process streams from all preparatory departments (air compression, raw material storage and preparation, seed culture production, preparation of nutrient salt and trace element solutions, process water, aqueous ammonia, sterile culture broth) proceed to the main production stage—the fermentation stage. The main apparatus in this department is the fermenter, a continuous stirred-tank Reactor for the liquid phase that ensures:

the GROWTH AND DEVELOPMENT of microorganism populations within the liquid phase volume;

The transport of nutrients to the microbial cells;

the removal of Metabolic waste products from the microbial cells;

the removal of heat generated by cellular activity from the medium.

This is followed by the other Stages of the feed biomass production flowchart:

♦ thickening of the microbial suspension. Here, the biomass concentration is increased to 12–16% ADM by mechanically separating most of the intercellular moisture in the minimum possible time. Separators, as well as flocculation, coagulation, flotation, or decantation, are used for this purpose;

♦ heat Treatment of the suspension. Heating the microorganisms to a temperature of 75–85 °C for 10–40 minutes results in the death of the producer strain and virtually all accompanying microflora;

♦ concentration of the suspension in the evaporation unit to a concentration of 23–25% ADM. A 3-effect vacuum evaporation plant is used for this: the temperature is 90 °C in the first effect, 75 °C In the second, and 60 °C in the third;

♦ drying. This unit produces the finished product with a moisture content of ~10% (by weight). Convective dryers (spray, fluidized bed, belt, and drum dryers) are most commonly used for this;

♦ granulation and drying. Usually, dry biomass containing 8–10% (by weight) moisture represents the finished product and is sent to the warehouse for the consumer after packaging. If the product needs to be obtained in pellet form, dry and wet (post-evaporation) biomass in a 1:1 ratio is fed into the granulator. Here, the wet biomass adheres to the dry particles, and the entire mass with a moisture content of 45–50% moves through the apparatus, forming granules that are then fed into a fluidized bed dryer. The granules are dried to a residual moisture content of 8–10% (by weight) using hot air or flue gases at a temperature of 260–300 °C;

♦ packaging and wrapping of the finished product. The dry biomass enters a receiving hopper and is packaged in 25–30 kg valve paper bags. These bags are placed on special pallets, which are then transported to the warehouse or shipped to the consumer.

A mandatory stage in obtaining protein substances is the purification of gas-air emissions (GAEs). These represent large volumes of air containing living microbial cells, protein dust, and other products of microbial synthesis. GAEs are fed by a fan into a Venturi scrubber, where they mix with water injected under pressure. The atomized water forms tiny droplets upon which impurities are adsorbed and partially coagulated. Then, the gas is partially separated from the liquid in an inertial separator, passes through a cyclone droplet separator, and the purified air is directed to a chimney stack or released into the atmosphere. The contaminated water enters a settling tank, where most of the impurities are separated. Cleaner water is sent back for the recirculation spray of the Venturi scrubber, while the contaminated portion (15–25%) goes to wastewater treatment.

Prospects for Biomass Utilization. Large-scale cultivation of industrial MICROORGANISMS AND THE utilization of their biomass represent one of the primary sources of protein for humans and animals. The raw material base for microorganisms is practically inexhaustible, biomass growth is rapid and intensive, and the protein composition of unicellular organisms is highly consistent, typically well-balanced in its amino acid profile.

The biomass of industrial microorganisms can be used to produce a wide range of products for various applications, playing a vital role in human economic activity.

Bioproducts

Cysteine, methionine, lysine — enhancing the nutritional value of proteins:

The biomass of industrial microorganisms is already widely used to produce microbial preparations (soil fertilizers, plant growth stimulants and regulators) and microbial polymers (enzyme proteins, immunobiological preparations, interferons, Antibiotics, Hormones, etc.).

Production of Enzyme Preparations for Various Applications. The biotechnological industry is conventionally divided into large-scale production and fine biosynthesis products. The production of ethanol, fodder and baker's yeasts, organic acids and amino acids, organic Solvents, Polysaccharides, and enzymes belongs to the large-scale category. Fine synthesis encompasses the production of antibiotics, hormones, medical (purified) enzymes, Pharmaceuticals, and various biochemical Reagents.

Enzymes used in industrial or semi-Industrial processes, with the exception of papain (which is extracted from papaya fruit and serves as a meat tenderizer), are of microbial origin. Four enzymes are produced in the largest quantities. In 1980, 530 tons of proteases, 350 tons of glucoamylase, 320 tons of a-amylase, and 70 tons of glucose isomerase were produced. The industrial application areas of microorganisms and the uses of major microbial enzymes are as follows:

♦ production of fermented alcoholic beverages and food products (soy sauce, vinegar, pickled vegetables, cheeses, yogurt, lactic acid beverages, beer, cider, wine, sake, etc.);

♦ production of physiologically active substances (Vaccines and microbial bioinsecticides, baker's and fodder yeasts, amino acids, mononucleotides, vitamins, Steroids, carotenoids, Gibberellins, and other plant growth hormones);

♦ production of solvents and organic acids (ethanol, butanol, acetone, acetic, citric, fumaric, and lactic acids);

♦ production of polysaccharides (dextrans, levans, Mannans, xanthan Gums);

♦ production of antibiotics (Penicillins, streptomycins, kanamycins, neomycins, Tetracyclines, bacitracin, etc.);

♦ pollution reduction (treatment of industrial effluents, liquid wastes, disposal of waste and garbage, etc.);

♦ microbial leaching of ores and disposal of mining waste;

♦ application in cheesemaking (pressed curd, soft, semi-soft, hard, and very hard cheeses, etc.).

The Role of enzymes in medical Diagnostics is increasingly growing. For instance, they are used to determine Cholesterol, glucose, uric acid, and other substances in Blood serum. Prenatal Diagnosis of Sickle cell anemia can be performed by treating Amniotic Fluid germ cell DNA with restriction enzymes. This eliminates The Need for fetal blood sampling.

Technologically, it is significant that certain microorganisms secrete enzymes from their cells into the surrounding environment, which facilitates Isolation and Purification. Excreted enzymes include Hydrolases, amylases, proteases, cellulases, etc. The mechanisms governing The excretion of such large molecules as enzymes are not yet fully understood. It is hypothesized that enzymes are released into the environment via exocytosis.

The isolation of enzymes from biomass is a complex process. Several methods exist for converting dissolved enzymes into insoluble particulate form: salting out, precipitation via temperature and pH adjustment, solvent precipitation, application of high-molecular-weight polymers, Metal Ions, and complexes. For instance, to precipitate a-amylase, substantial amounts of ethanol are added to the bioreactor while stirring the medium. Following decantation, the precipitate is compacted in a filter press and dried in a vacuum dryer. As a result, 73.5 kg of high-activity preparation is obtained from 5,000 L of medium. It is then standardized with gypsum to achieve the required activity level.

Technological processes for producing enzyme preparations can be divided into two groups. In the first case, fermentation is carried out using the submerged method in a liquid nutrient medium; in the second case, surface culture is employed, growing on a specially prepared loose and moistened nutrient medium.

The MAIN STAGES OF the submerged cultivation method for enzyme producers:

♦ obtaining inoculum According to the scheme: initial producer culture → stock culture grown in flasks on a shaker → seed culture grown in an inoculator → seed culture grown in a seed tank. The volume of the seed tank typically constitutes up to 10% of the industrial fermenter volume;

♦ preparation of nutrient media;

♦ Sterilization of Nutrient media using membranes or high temperatures;

♦ air purification before and after aeration;

♦ Production-scale cultivation. At this stage, it is necessary to monitor biomass growth, enzyme accumulation, changes in nutrient medium composition, acidity, aeration, etc.

Submerged cultivation is more advanced than surface culture, as it readily lends itself to mechanization and automation, and facilitates a smoother, simpler scale-up to industrial volumes. This process must be carried out under strictly aseptic conditions. However, the Enzyme Concentration in submerged cultures is generally much lower than in aqueous extracts from surface cultures.

In the surface method, the culture grows On the surface of a solid, moistened nutrient medium. The disadvantages of this method include: the need for a large surface area for air contact with the loose substrate, which often results in a slow, non-intensive process; the labor-intensive handling required for washing, sterilizing, and transporting shallow trays, as well as filling and emptying them; and the fact that cultivation takes place under non-aseptic conditions.

Advantages of the surface method: higher final enzyme concentration per unit mass of the medium, ease of cultivation and formulation into commercial products, lower electricity requirements, etc.

Microbial cultures grown by the surface method, as well as culture broths from submerged cultivation, contain a high concentration of ballast substances: producer biomass, unconsumed medium components, and metabolic byproducts. The actual enzyme content accounts for only about 1% in surface cultures and no more than 0.1% in submerged cultures.

The isolation and purification of enzymes is a labor-intensive and costly process. Therefore, crude enzyme preparations are widely used in the leather and alcohol industries, as well as in animal feed additives. In contrast, highly purified enzymes are mandatory for the food industry, textile manufacturing, microbiological synthesis, and medicine. Purification leads to a significant increase in the specific activity of the preparation. This is a critical objective, achieved through a diverse array of preparative techniques applied in non-standard sequences and various combinations. Fundamentally, the Technology for Obtaining purified enzymes involves the following stages:

extraction from surface culture;

separation of the solid phase;

concentration of enzyme solutions; ultrafiltration;

precipitation with organic solvents;

salting-out;

sorption purification;

drying;

Standardization of enzyme preparations.