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

Agricultural Biotechnology I

♦ Industrial production of feed additives.

♦ Biotechnological modification of plant-based feeds.

♦ Bacterial starters. Premixes and Probiotics in animal husbandry.

Industrial production of feed additives. Farm animal feeding must ensure maximum genetically predetermined productivity while maintaining animal health and reproductive function. Currently, more than 500 Different types of feeds and feed additives are used in animal Nutrition. These include waste products from the oil extraction and food industries, microbial synthesis products, macro- and microelement salts, vitamin, enzyme, amino acid, and antibiotic supplements, as well as tranquilizers, sorbents, antioxidants, flavoring agents, etc.

Based on their origin, all feeds are classified into the following categories:

♦ plant-based feeds;

♦ animal-based feeds;

♦ mineral feeds;

♦ products of microbiological origin;

♦ food industry by-products;

♦ products of chemical synthesis.

An essential component of feeds is feed additives—any dietary supplements that regulate the quantity and ratio of nutrients and BIOLOGICALLY ACTIVE SUBSTANCES while ensuring animal health and peak productivity. The Role of biotechnology in producing feeds of various origins is immense. In the modern world, overcoming the persistent deficit of feed for farm animals and poultry is achievable only through this science. Several plant-based feed products have been obtained biotechnologically: hydrolyzed molasses, micro-milled and technologically processed wood, RUK-1, and RUK-2.

Feed Hydrolysis molasses contains up to 30% dry matter, including 83–96% Monosaccharides. Depending on the type of woody raw material, glucose can account for 38–80% of the total monosaccharides in the dry matter. In hardwood and softwood hydrolyzates, xylose accounts for 25% and 13%, respectively. Furthermore, the developed technology allows the Separation of harmful volatile impurities (methanol, formaldehyde, acetone, volatile phenols, furfural, lignohumic substances), as well as excess gypsum and ammonium sulfate, from the molasses.

Micro-milled and technologically processed wood is produced through the combined thermomechanical and chemical Treatment of wood. This disrupts the ordered Structure OF THE wood and induces partial polymer hydrolysis. The resulting feed additive contains up to 20% readily digestible sugars.

Further research into improving wood Processing technology for feed production led to The Development of a selective acid hydrolysis method. First, incomplete hydrolysis of wood polymers is carried out. This depolymerizes only the hemicellulose, which makes up 19–20% of aspen sawdust. The resulting feed product, consisting of mono- and Oligosaccharides, was designated as RUK-1. It is produced using a zero-waste technology. Meanwhile, the poorly hydrolyzable Cellulose fraction, together with Lignin, forms a lignocellulose complex. This complex undergoes further hydrolysis and can subsequently be used as a second feed product, RUK-2. The liquid fraction obtained after the initial hydrolysis is utilized for growing fodder Yeast. It has been shown that when such a hydrolyzate is fed into the bioreactor at a rate of 26–30 m3/h, the yeast concentration reaches 29–33 g/L, with a yeast yield of 53.7% relative to the reducing substances used. From one ton of absolutely dry wood, it is possible to obtain 700 kg of RUK-2 and up to 120 kg of yeast.

A variation of selective hydrolysis is the cultivation of yeast on solid particles of lignocellulose, i.e., heterophasic cultivation. Following Fermentation, the biomass is separated along with the solid phase via filtration. The dried product contains up to 20% protein.

Biotechnological modification of plant-based feeds. Harvesting alfalfa, clover, and grass mixtures can yield over 1 ton of protein per hectare per season. The cost of protein from green mass is 2.5–5 times lower than that of grain protein. Currently, several Methods for obtaining protein concentrates have been proposed: juice expression, protein coagulation followed by centrifugation, and drying. These methods are quite complex, expensive, and energy-intensive. Of greater appeal is the technology of anaerobic fermentation of plant juice, chemical-biological protein coagulation, and pulp ensiling. In this spontaneous fermentation process, total acidity and the acid ratio are controlled. Once a specific pH is reached, protein coagulation occurs; sometimes flocculants or chemical preservatives are added to enhance the process.

During anaerobic fermentation, the nutritional properties of plant protein are improved due to the inactivation of Trypsin inhibitors and Alkaloids, as well as the transformation of phenols and Unsaturated Fatty acids. In addition, bacterial biomass with a high Methionine content binds to the plant protein. As a result, the quality of the feed product is significantly enhanced, and its chemical composition approaches that of skim milk. Furthermore, the shelf life of such a product is substantially extended. The quality of ensiled pulp is improved by inoculating it with lactic acid Bacteria.

The juice fermentation process can be implemented using batch or continuous (semi-continuous) technologies. In the first approach, the fermentation-coagulation vessel is gradually filled with freshly expressed juice. When the pH drops to 4.2–4.5, the juice is ready to be fed to animals (pigs, young cattle) either without separating the coagulate or by isolating a portion of the protein with the coagulate. In the continuous or semi-continuous process (the second method), fresh juice is fed continuously or in portions into the central part of the fermentation-coagulation vessel, displacing the fermented juice from the apparatus. The coagulate is periodically discharged from the bottom. With this technology, and due to the complete Filling of the fermentation vessel with liquid, anaerobic conditions are maintained, preventing mold growth. The apparatus can operate for weeks without stoppage or cleaning. Before starting fermentation, about 10% actively fermenting juice or an acid-forming bacterial starter suspension must be introduced into the clean apparatus.

The process of obtaining fractionated green mass (brown, protein-free juice and pulp) is technically straightforward and low in energy consumption. The resulting juice is virtually free of cellulose and contains 1–3% protein.

Silaging and haylage making. Bioconversion is an effective way to extend the shelf life of feeds. Immediately after the plant Organism's defense system is compromised, Yeasts and acid-forming bacteria become active. In the process, CARBOHYDRATES are converted into alcohol and organic acids. This results in the natural preservation of plant products, essentially a pickling process. The main objective of advanced feed conservation technologies (chemical preservation, artificial dehydration, preservation with inert gases, etc.) is to minimize dry matter losses while maintaining high product quality.

Silaging is a complex microbiological and biochemical process used to preserve various perishable, succulent plant Materials. Silaging is based on Lactic acid fermentation. Moreover, The amount of lactic acid in silage should be 2–3 times greater than that of acetic acid, and the silage pH should range between 4.2 and 4.4. At this pH level, putrefactive bacteria cannot thrive. However, if aerobic conditions arise, lactic acid is destroyed by aerobic microflora, leading to silage spoilage due to putrefactive processes, accumulation of butyric acid, ammonia, trimethylamine (herring-like odor), botulinum bacilli, and Molds.

Haylage making is a type of feed conservation derived from perennial and annual grasses wilted to a moisture content of 40–55%. Feed preservation is ensured not by high acidity, but by the physiological dryness of the initial raw material stored under anaerobic conditions; the haylage pH is 4.4–5.6.

Feed silaging is always accompanied by dry matter losses of up to 15–20%, which is associated with the METABOLIC ACTIVITY OF the microflora. Chemical preservation helps retain nutrients in the feed and suppresses the growth of putrefactive and butyric acid bacteria. Currently, over 1,000 feed preservatives are known. According to their mode of action, they are classified into:

♦ inorganic acids: sulfuric, hydrochloric, phosphoric, and their salts;

♦ antibacterial acids: formic, propionic, benzoic, and mixtures thereof;

♦ antibacterial salts: sodium nitrite, sodium benzoate;

♦ Antibiotics: streptomycin, bacitracin.

Examples of feed preservatives used in Russia include VIK-1, consisting of formic acid (27%), acetic acid (27%), propionic acid (26%), and Water (20%), as well as VIK-2, which consists of 80% formic acid, 9% acetic acid, and 11% propionic acid. Sulfite liquors and other agents are used for preserving corn, etc.

A modern biotechnological approach to feed stabilization and bioconversion involves The Use of enzyme preparations of microbial or fungal origin. Currently, numerous Enzymes are in use, such as Pectafoetidin P10x and Amylosubtilin G3x. The dosage of purified enzyme preparations typically ranges from 0.02 to 0.005%, and for unpurified ones, from 0.5 to 1% of the raw material mass. The letter X indicates that the enzyme preparations have not undergone preliminary purification. The numbers characterize the activity level relative to the native culture, while P stands for surface cultivation and G for deep (submerged) cultivation.

During chemical preservation of feeds, carbohydrates are consumed to a lesser extent in The formation of organic acids. The use of preservatives is justified if:

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where R_K represents the costs associated with the production and application of the preservative for cultivating a unit of feed; Z_K and Z_C are the costs of nutrient losses in the preserved and ensiled feeds, respectively; and R_C denotes the costs associated with the ensiling of the given feed.

It is known that the conservation of 1 m³ of green mass juice leads to the formation of 15 kg of organic acids from carbohydrates, which is accompanied by a loss of approximately 5% of dry matter and 70% of nutrients. For stabilizing juice intended for short-term feeding to animals (1–3 days), anaerobic fermentation is more economically advantageous than the application of chemical preservatives. In this case, dry matter losses are negligible, and the resulting acetate directly undergoes oxidation. However, a certain adaptation period—typically 7–20 days—is required for pigs, calves, and chicks to adjust to such acidified feed.

Losses of feed dry matter occur not only during The conversion of carbohydrates into acids driven by the development of acid-forming bacteria, but also during the storage period. In all cases where Microbial growth is not suppressed (optimal pH, Temperature of 15–20 °C), substrate is consumed to maintain bacterial viability. From a biotechnology perspective, these expenditures are unproductive. However, if METABOLISM is suppressed—for instance, by the action of inhibitors (such as acids)—the unproductive energy costs of microorganisms are mainly associated with maintaining required gradients, resynthesizing macromolecules, repair, reactivation, and various defense mechanisms. Constantly occurring proteolysis is not a negative process, as Amino Acids are readily utilized. Nevertheless, if the protein-free filtrate is discarded rather than used, it represents a loss of feed protein.

To minimize the loss of medium components, especially during the summer period, the microflora should be targeted with additional agents that suppress their metabolic activity:

♦ increasing the osmotic pressure. This is achieved either by raising the concentration of substances to induce dehydration or by adding sodium chloride to a concentration of 1–1.5%;

♦ adding extra chemical inhibitors to the medium: acids, acid mixtures, formalin, sodium pyrosulfite, etc. For instance, The addition of 0.1% formalin to an acid coagulant with a pH of 4.2 almost completely suppresses the metabolic activity of microorganisms.

The anaerobic biotransformation of carbohydrates into acids is justified not only as a method for preserving the liquid fraction of plant green mass, but also for pulp. However, doing so is technically more complex. The pulp must be densely packed in trenches or silos to completely exclude air and prevent the proliferation of aerobic microbes. It should be borne in mind that the moisture content of pulp (73–75%) is lower than that of grass; therefore, Homofermentative lactic acid bacteria predominantly develop in it while the growth of other forms is suppressed. It has been shown that when ensiling a pulp-grass mixture with chemically treated wheat straw and barley meal, the moisture content of the ensiled mass is 50%. In this mass, acid-forming bacteria multiply maximally by the sixth day at a temperature of 20 °C. Such feed contains about 10% protein, 31% fiber, 1.3% fat, 9.5% ash, and up to 40% nitrogen-free extractives. Butyric acid is absent in such a product, and the Nutritional Value of the feed is increased by 21%.

It should be emphasized that the processing of plant biomass yields Three types of feed: protein coagulant, from which protein-vitamin paste is produced; fermented juice formed after the separation of the protein coagulant; and the residual plant material remaining after juice expression in the form of pulp.

Protein coagulant contains 15–22% protein on a dry matter basis and is typically fed to animals during the winter period. The protein content in protein-vitamin paste can reach up to 50%. Leaves of alfalfa, clover, and sugar beet are used to produce such paste. Fermented brown juice contains 7–12% dry matter, 1–3% protein, 1–1.5% organic acids, 4–5% nitrogen-free extractives (total easily digestible carbohydrates), 1–2% ash, and 40–50 mg/dl carotene. The juice is used as a feed additive for animals, particularly pigs (1.5 liters per HEAD per day), or processed into feed yeast.

Bacterial starters. The ensiling process can be managed by artificially enriching the green mass with specific cultures of lactic acid bacteria capable of actively multiplying in it and directing the ripening of silage along the desired pathway. To this end, biomass is grown and subsequently transitioned into an anabiotic state.

Starters for feed ensiling are typically prepared on The basis of Lactobacillus plantarum bacteria. Cultures of homofermentative lactic acid bacteria such as L. acidophilus, L. faecalis, and Streptococcus lactis, or heterofermentative ones like L. brevis, are also employed. It is desirable to use cultures that produce L-lactic acid, which undergoes metabolism and serves as an energy source much like carbohydrates.

Along with monocultures, culture mixtures are used in the preparation of ensiling starters. Bacteria with amylolytic and cellulase activity should be incorporated into the starters. The combined Application of Enzymes and starters is also frequently practiced. The use of mixed cultures allows for the more efficient ensiling of substrates with varying compositions.

Lactic acid bacterial starters are prepared by deep (submerged) fermentation followed by the separation and drying of The Cell mass. A suitable nutrient medium is sterile skimmed milk with an elevated dry matter content (up to 16%). To achieve this, milk powder and a 0.1% sodium citrate solution are added to the starters. The inoculum accounts for 1% of the medium volume. Bacterial multiplication is carried out without aeration. Lactic streptococci are propagated at 30 °C for 12–16 h, and lactic rods at 40 °C for 6 h. Afterward, the culture liquid is neutralized with a 20% sodium hydroxide solution back to the initial acidity of the sterile milk. The liquid starter is dried in a spray dryer with an incoming air temperature of 130–140 °C. In the spraying zone, the temperature must not exceed 48–50 °C. The residual moisture of the dry starter is 5–7%. The survival rate of streptococci upon drying under these conditions is 18–33%, and that of acidophilic rods is 7–8%.

Using bacterial starters, a concentrate with a paste-like consistency is prepared. One gram of such a concentrate contains 52–100 billion viable lactic acid rods. Its residual moisture is 70–72%, and the optimum pH is 4.5–4.7. The concentrate is stored at 4–6 °C with the addition of 0.003% potassium bromide. For long-term storage, the paste-like concentrate is dried, frozen, or the biomass is lyophilized using special protective media.

Two approaches are used to increase the protein content in plant feeds:

♦ cultivating microorganisms on starch-containing raw materials. This increases the protein content and enriches the product with Vitamins. For instance, all B-group vitamins and various other growth- and metabolism-stimulating substances are present in yeast;

♦ introducing hydrolytic enzymes. It has been demonstrated that the addition of such enzymes to feeds increases the live weight gain of animals and poultry by an average of 10–15% and reduces feed costs per 1 kg of gain by 5–7%.

The technological process for obtaining a protein-enzyme preparation consists of two stages: preparation of the inoculum and main fermentation. For example, when cultivating the yeast-like culture Endomycopsis fibuligera R-574, the nutrient medium should contain (in %): molasses, 5.0, or various feed meals, 10; (NH₄)₂HPO₄, 0.3; CaCl₂, 0.04. The initial pH of the medium is 6.8–7.2, and the cultivation temperature is 30–32 °C. The inoculum multiplies on the molasses medium within the fermenter. It is introduced into the culture liquid at a rate of 1%. The cultivation of the seed material lasts 13–16 h.

Yeast culture in the main fermenter is grown at 30-32 °C with constant agitation and aeration. The main fermentation process lasts for 12 hours. It is important to note that living Cells are difficult for animals to digest, meaning a portion of the yeast protein remains unabsorbed. To overcome this, the culture liquid is heated to 90 °C for half an hour. The finished product cannot be stored for long periods, so once fermentation is complete, it is sent directly to farms to be fed to livestock mixed with other feeds.

Protein enrichment of starch- and lignocellulose-containing raw materials is also carried out via solid-state fermentation. For instance, when cultivating Aspergillus niger, the protein content in the biomass can reach 17-20 %, while the acids produced by the culture protect the medium from contamination. A major drawback of solid-state fermentation is the difficulty of heat dissipation. Therefore, the process must be carried out in a thin layer, which increases the required equipment size.

Premixes and probiotics in animal husbandry. A premix is a homogeneous blend of finely ground micro-additives and a carrier, used to enrich compound feeds as well as protein-vitamin supplements.

In addition to vitamins, Trace Elements, and amino acids, premixes contain compounds that exert a stimulating effect on the animal organism, as well as substances that protect feed and prevent quality degradation. Premixes help improve feed palatability and utilization efficiency (antioxidants, emulsifiers, enzymes, flavoring agents, etc.). Therapeutic and prophylactic compounds (such as furazolidone and sulfadimezin), sedatives (tranquilizers), and Surfactants (detergents) are also incorporated into premixes.

Common carriers include wheat bran, finely ground wheat grain, feed yeast, and soybean meal. Premixes are added to respective compound feeds for various species and groups of animals at a rate of 1% (10 kg per 1 t). Below is an example (Table 1).

Table 1

Premix formulation for high-yielding cows, per 1 t (All-Russian Institute of Animal Husbandry)

As is well known, about half of all harvested grain is used as feed on livestock farms. Fodder grain can be used much more efficiently by increasing its content of protein and other metabolic-enhancing feed additives. Our country produces over one million tons of microbial protein supplements annually, which enhances the nutritional value of nearly 20 million tons of grain feed. These are primarily yeasts grown on various low-cost nutrient media. Adding a ton of yeast to poultry grain rations yields an extra 1-1.5 t of meat or 25-30 thousand eggs, and in pig farming - 0.4-0.6 t of meat, while saving about 5-7 t of fodder grain.

Thus, biotechnology provides agriculture with highly nutritious feed additives derived from yeasts, Fungi, and microorganisms. Agricultural waste is used to produce various economically significant substances, including Essential Amino Acids, alcohols (ethyl, methyl, butyl, etc.), diverse organic acids (citric, lactic, gluconic, acetic, propionic, fumaric, etc.), Solvents (acetone, butanol), carbohydrates and their derivatives (dextran, levan, xanthan, alginic acid, pullulan, scleroglucan, etc.), and microbial enzymes.

Recently, probiotics have been drawing increasing attention. This term was introduced into scientific literature in 1985. According to the modern definition, probiotics are preparations based on live, specially selected strains of microorganisms or specific substances of microbial, plant, or animal origin. When administered to an organism, probiotics positively alter the endogenous microflora, ultimately exerting a beneficial effect on the physiological Functions and biochemical Reactions of the host. Other dietary supplements that selectively stimulate the GROWTH AND REPRODUCTION of bacteria natural to humans and animals are referred to as prebiotics. Combined products containing both probiotics and prebiotics are called synbiotics. The term "eubiotics" is synonymous with "probiotics".

Probiotics are comparable in efficacy to certain ANTIBIOTICS AND CHEMOTHERAPEUTIC agents. They do not adversely affect the gastrointestinal microflora, do not disrupt ecology, and promote better Digestion and assimilation of food and feed. For instance, cellobacterin is a probiotic developed from more than 100 microorganism associations isolated from the rumen of domestic and wild animals. This preparation improves the digestibility of plant fiber and significantly increases productivity and total egg weight in laying hens. Recently, effective microorganism preparations have garnered widespread attention; as a new generation of probiotics, they exert significant positive effects on crops, animals, and humans.

According to B.V. Tarakanov (2001), the probiotic lactoamilovorin "when fed to piglets, calves, and broiler chickens consistently provides the following effects: inhibition of Escherichia, Salmonella, and hemolytic bacteria in the intestine; stimulation of microorganisms that hydrolyze complex Polysaccharides; increased intake of concentrated feeds; enhanced enzymatic activity in the Small Intestine; stimulation of non-specific animal resistance; prophylactic and therapeutic effects against diarrheal diseases; improved animal survival and live weight gain; and pronounced anticholesterolemic effect. Streptophagin inhibits amylolytic streptococci and their activity in the forestomachs; increases cellulolytic activity and the count of hemicellulose-hydrolyzing bacteria; shifts fermentation pathways toward greater propionate and butyrate production in the rumen; and raises milk fat levels by 0.2-0.3 % during indoor housing and by 0.15-0.47 % during the transition to pasture."

Over a dozen probiotic preparations for veterinary and human medicine have been produced recombinantly using spore-forming microorganisms of the genus Bacillus. These preparations (Table 2) exhibit a highly diverse range of positive activities (Bakulina L.F. et al., 2001).

Table 2

Spectrum of activity of probiotics based on spore-forming microorganisms of the genus Bacillus

Microbial preparations are increasingly utilized to improve feeding efficiency. Some contain live symbiotic microorganisms of the gastrointestinal tract (propiovit, propyocide, azoacid). For instance, dry acidophilus and propiovit are used to prevent gastrointestinal disorders and enhance the productivity of young stock. A complex of microorganism strains is employed to combat dysbiosis and balance gastrointestinal biocenoses, with propiovit and azoacid falling into this category. Bifidobacteria show great promise in pig farming; this preparation exerts therapeutic and prophylactic effects against acute gastrointestinal disorders, normalizes intestinal microflora composition, and stimulates the GROWTH AND DEVELOPMENT of piglets. Spore-forming aerobic bacteria are also effective in treating gastrointestinal diseases in farm animals. Alongside fodder yeast, fungal-origin preparations—including those produced via biotechnological recipes—are finding growing application in animal feeding.

Other microbial products are obtained using microorganisms that are not part of the normal digestive tract microflora of animals. These preparations consist of killed bacteria and serve as protein supplements.

Thus, the application of biotechnology in agriculture opens up highly promising Prospects. For animal husbandry, biotechnologists should primarily focus on producing adequate quantities of feed protein from bacteria, fungi, yeasts, and Algae, as well as through the bioconversion of renewable plant biomass. Another vital task is the development of advanced Prevention, diagnostic, and treatment methods for animal infectious diseases utilizing restriction fragment length polymorphism of viral genomes, DNA or RNA diagnostic probes, Monoclonal Antibodies, genetically engineered and antigen Vaccines, various probiotics, etc.