Method of producing a probiotic feed supplement

Abstract

The invention relates to the field of biotechnology, and more particularly to producing protein feed products by microbiological synthesis. A method of producing a feed supplement is proposed which comprises producing galactose-sucrose products by hydrolysing whey and plant waste with succinic acid and native yeast enzymes for subsequently growing yeast cultures. For this purpose, the whey and plant waste hydrolysate is mixed with yeast in the following ratio of ingredients: 90% whey and plant waste hydrolysate; 0.03-0.05% succinic acid; 10% Saccharomyces cerevisiae yeast. Producing a feed supplement having the claimed composition will make it possible to significantly enhance the biological value of feeds and also increase feed efficiency, while at the same time reducing production costs. Adding the proposed supplement to a compound feed will assist in stabilizing the nutritional balance of the body and enhancing livestock productivity. In addition, using whey for growing yeast will be beneficial to the environment.

Description

[0001] METHOD FOR PRODUCING A PROBIOTIC FEED ADDITIVE

[0002] The invention relates to the field of biotechnology, in particular, to the production of feed protein products by microbiological synthesis.

[0003] The current rapid development of agriculture is largely driven by the use of modern, high-tech technologies. One such area is biotechnological production. Biotechnological methods can help solve the problem of balanced animal nutrition.

[0004] The high efficiency of using yeast protein in animal husbandry has necessitated the development of various methods for yeast production.

[0005] For example, a method for producing feed protein is known that involves processing waste plant materials by hydrolysis and cultivating yeast on the resulting hydrolysate. Pre-treatment of the substrate is carried out by acid hydrolysis at a pH of 0.8-1.3 and a temperature of 118-123°C for 30 minutes. Coffee grounds are used as waste plant materials, and the yeast biomass is the biomass of Saccharomyces cerevisiae [Patent RU 2393719, published 10.07.2010].

[0006] The disadvantages of hydrolysis with mineral acids include insufficient formation of simple carbohydrates for the subsequent fermentation process, due to the fact that during acid hydrolysis not only starch is hydrolyzed to glucose, but also the destruction of glucose itself and the destruction of pentatomic sugars to furfural and hydroxymethylfurfural occur.

[0007] A method for producing feed microbiological protein is known, which involves grinding grain production waste, diluting the raw material with water (W) and heating it to a temperature of 65-58°C at a heating rate of 1.5°C / min in the presence of alkali or alkaline earth metal hypochlorite (0.2-0.8% of W) and ammonium sulfate (0.1% of W). The mass is maintained at this temperature for 1 hour, after which it is treated with amylosubtilin at 50°C for 1.5-2.5 hours. The resulting substrate is used to grow protein-producing yeast. The method makes it possible to obtain a feed product with an increased protein content [Patent RU 2091492, published 09.27.1997]. The disadvantages of known methods that include hydrolysis or enzymolysis stages are the complexity of the technological process, duration, labor intensity, as well as the high cost of proteolytic or amylolytic enzymes, which leads to increased costs and decreased competitiveness of the finished product.A method is known for producing a protein feed product based on an enzymatic lysate of grain raw materials, the method involves preparing the grain raw materials by grinding under pressure with shear, preparing an aqueous suspension with a dry matter concentration of 15-16%, carrying out enzymatic lysis with the formation of glucose of 10-13%, introducing nitrogen and phosphorus sources to obtain a nutrient medium, introducing Saccharomyces cerevisiae yeast, growing, thickening and drying [Patent RU 2562146, published 10.09.2014].

[0008] The disadvantages of this method are the use of valuable raw materials - grain and flour, the labor-intensive preparation of raw materials, grinding, mixing with water in a ratio of 1:5, the consumption of a large amount of water for the preparation of the nutrient medium, the use of stirring devices, the introduction of additional sources of minerals, which increases the cost of the final product.

[0009] The closest analogue (prototype) is a method for producing feed microbiological protein, which includes processing milk whey by enzymolysis, which is carried out using native enzymes secreted by yeast cultures, while the yeast cultures are fed in a ratio of 0.5-1.0% to the dry substances of the milk whey, with the formation of a suspension. This suspension contains up to 4 wt. % of the glucose-galactose component. An autolysate of yeast cultures is also obtained. Next, the autolysate of yeast cultures - 70-80% - is mixed with the glucose-galactose component - 20-30%. The invention makes it possible to obtain a feed additive in which the main components of autolysates, namely essential amino acids and vitamins, are preserved, with a significant reduction in the cost of the production technology [Patent RU 2295869, published. 03.27.2006].

[0010] The disadvantages of the analogue are the short shelf life of the liquid feed additive and the lack of a wide range of microelements.

[0011] Below are the beneficial properties of the substances used in the proposed feed additive.

[0012] Whey is a promising raw material. The theoretical yield of whey is approximately 90% of the processed milk mass, and it contains approximately 50% of milk solids. The significant volumes of whey and its high nutritional value necessitate its complete collection and rational use [Khramtsov, A.G. The Phenomenon of Whey: Monograph. St. Petersburg: Profession, 2011, 802 p.].

[0013] An analysis of the structure of secondary dairy raw material processing shows that 25-27 million tons of secondary dairy raw materials are generated annually in the agricultural sectors of the Republic of Kazakhstan, a significant portion of which is not used for food purposes. Therefore, expanding the range of new types of dairy products and increasing their output is only possible with the rational use and waste-free processing of all components of dairy raw materials. The problem of waste-free production at the present level is only possible through the organization of scientific programs to develop a technology for processing secondary raw materials and the production of new generation dairy products with increased biological value for functional nutrition [Astakhova N. Use of secondary raw materials in the dairy industry / / Science and Life. - 2012. - No. 2]. At a number of milk processing plants, whey is simply discharged into the sewer, which contributes to the deterioration of the environmental situation, since 1 m3 water contaminated with whey is equivalent to 400 m 3 industrial waters. Considering that during the production of cheese, cottage cheese and casein, whey is formed in colossal quantities (approximately 90% of the milk used), this problem is of particular importance. Draining whey creates an environmental problem, since its polluting potential exceeds that of domestic wastewater by 500-1000 times. In most countries, its discharge is prohibited and punishable by law. The cost of treating wastewater contaminated by a dairy plant processing 100 tons of milk per day is equivalent to the cost of treating wastewater in a city with a population of 80,000 people [Tsydenzhapov A., Budaeva A. V. Ecological project "Whey: benefit or harm? - 2017. URL: https: / / infourok.ru>proekt-po-ekologii ... sivorotka-polza... ].

[0014] The reasons for the restraint of whey processing are insignificant investments, lack of funds for the implementation of modern technologies and the purchase of equipment, and the liberalism of the environmental service regarding its discharge into wastewater. At the same time, the organization of industrial processing of whey allows to increase the profit from product sales by 28-30% [Sokolenko G.G. et al. Biotechnology of yeast-whey product / / Bulletin of OrelSAU, - 2016. - No. 5. - P. 79-83]. Whey is a rich source of whey proteins for food, biological and functional applications, which pass into whey after the primary processing of milk. The introduction of products obtained from whey, including whey proteins, has not achieved widespread distribution due to the complexity of its processing (low dry content, high energy costs for processing) [Pais Chanfrau, J.M., Nunez Perez, J., et al., Milk whey - from a problematic byproduct to a source of valuable products, Prensa Med.[Argent., 2017, vol. 103, no. 4, p. 1]. Unprofitable methods of whey processing often include loss-making treatments associated with discharge into wastewater or the simple use of low-value powders, which are restricted by environmental regulations. Certainly, it is advisable to use the valuable components contained in whey dry matter based on the development of new technologies [Kassem Jihan. Future challenges of whey proteins, Int. J. Dairy Sci., 2015, vol. 10, no. 4, p. 139].

[0015] The lack of energy-saving industrial technologies for the waste-free use of whey, both its entire dry matter and its individual protein fractions, is one of the most important challenges in dairy processing. These shortcomings are being addressed by developing new methods for whey processing, isolating protein fractions, and creating products with specific functional and nutritional properties [Photis, P., Paschalia, K., Technological utilization of whey toward sustainable exploitation, J. Adv. Dairy Res., 2019, vol. 7, no. 231, p. 1].

[0016] Currently, the majority of whey is used to feed livestock in its natural form. At the same time, livestock farming is experiencing a shortage of complete protein and domestically produced feed additives containing it. One solution to this problem is to establish whey processing facilities to produce feed additives containing complete animal protein. The most efficient way to utilize whey is to bioconvert its components into protein by culturing single-celled organisms that can utilize lactose as an energy source and convert mineral nitrogen into complete protein. Through microbial synthesis, the whey acquires new qualitative properties, enriching it with vitamins, enzymes, organic acids, and other biologically active compounds.Yeast, lactic acid bacteria, microscopic fungi, and mixed cultures of various microorganisms are used for the bioconversion of milk whey. Of all known microorganisms, yeast has the highest coefficient of conversion of whey into microbial protein. They are distinguished by a high growth rate and are widely used in biotechnological production to obtain protein, ethanol, and as a source of enzymes [Yarovoy S. A. et al. Complex effect of inulin and milk whey on the development of yeast of the genus Saccharomyces / / Milk processing. - 2010. - No. 7. - P. 52-55]. Yeast biomass obtained by bioconversion of milk whey is a physiologically complete high-protein product that can be used for food and feed purposes. Despite the low cost of milk whey as a raw material and its high biological and nutritional value, it currently finds virtually no use in our country.Whey contains immune-boosting components such as lactoferrin, immunoglobulin, a full range of B vitamins, as well as vitamin C, niacin, choline, vitamin A, vitamin E, and biotin, as well as micro- and macroelements such as calcium, potassium, potassium, potassium, iron, and zinc. Whey contains all essential amino acids. Whey carbohydrates are primarily lactose (90%). Curd whey contains 0.7-1.6% glucose and 0.05-0.45% fat. Almost all the salts and microelements in milk are transferred to whey. The absolute content (%) of the main ash elements in whey is as follows: potassium - 0.09-0.19, magnesium - 0.009-0.02, calcium - 0.04-0.11, sodium - 0.03-0.05, phosphorus - 0.04-0.10, chlorine - 0.08-0.11. The energy value of whey is 36% of the energy value of cow's milk, and their biological value is approximately the same.

[0017] The presence of milk sugar (lactose) in whey makes it invaluable because it is completely absorbed by the body. It is the body's most desirable carbohydrate, preventing fat formation in cells and having a beneficial effect on the gastrointestinal tract. The proteins found in whey include essential amino acids, which the body cannot produce and must obtain through food. These proteins also participate in the formation of red blood cells and in protein synthesis in the liver. Whey proteins are extremely valuable compared to other natural proteins.

[0018] 100 g of whey contains: proteins - 0.846 g; fats - 0.36 g; carbohydrates - 5.14 g. The energy value is 27 kcal - 112 kJ [Khramtsov A.G. The phenomenon of whey: monograph, St. Petersburg: Profession, 2011, 802 p.].

[0019] Succinic acid is a universal intermediate metabolite formed during the interconversion of carbohydrates, proteins, and fats in plant and animal cells. Succinic acid stimulates energy production in cells, promoting ATP production. This means that the conversion of succinic acid in the body is associated with the production of energy necessary for life. As the load on any system increases, its functioning is maintained primarily through the oxidation of succinic acid. This is why succinic acid has a non-specific therapeutic effect on a number of pathological conditions of various etiologies. Furthermore, succinic acid has actoprotective and antiviral effects.

[0020] By participating in the tricarboxylic acid cycle, it enhances anabolic processes in the body, helps normalize metabolism, improve immunobiological reactivity, and promote resistance to a number of infectious diseases. Succinic acid exhibits an antihypoxic effect, enhancing cellular respiration and facilitating oxygen uptake by cells. It has a powerful antioxidant effect, neutralizing free radicals, supporting calcium transport, eliminating metabolic acidosis, and stimulating steroidogenesis. By enhancing the production of inhibitory mediators in the cerebral cortex, it has an anti-stress effect. Succinic acid reduces the effects of toxic substances, bacterial toxins, and mycotoxins on the body, activates a number of enzymatic systems, improves growth, development, productivity, and survival in animals, and exhibits general strengthening and restorative properties [Patent RU 2591993, published July 20, 2016].

[0021] Yeast contains 48-50% protein, which is similar in amino acid composition to animal proteins and therefore has significant nutritional value. The digestibility of yeast proteins reaches 85%. Baker's yeast contains, on average, the following percentages: water - 68-75, protein - 13-14, glycogen - 7-8, fiber - 1.8, fat - 0.9-2.0, ash - 1.77-2.5. Yeast contains many vitamins and growth substances. It contains vitamins: D, B1, B2, PP, pantothenic acid, phosphoric acid and biotin - a powerful growth stimulant. Yeast is rich in various enzymes and hormones, including those that stimulate sexual activity and fertility in animals. Yeast is a single-celled organism that can survive in the rumen of a cow and "work" in the natural microbial population. In animal husbandry, the ability of yeast to absorb oxygen helps create anaerobic conditions in the digestive tract, which are necessary for the growth of anaerobic bacteria.

[0022] The benefits of using yeast in animal husbandry have been proven by numerous studies over the past decades. Yeast prefers an acidic environment and grows well at a pH of 4.5-6.5. It survives well in the rumen at a pH of 6.0-6.5, producing more vitamins, amino acids, and nucleotides. Active yeast cells have adsorption and buffering properties, making them a reservoir of nutrients and a pH buffer. Yeast has become widely used in dairy cow nutrition due to its ability to ferment. Yeast uses rumen oxygen for growth, thereby improving conditions for the growth of cellulolytic bacteria (anaerobes). Furthermore, probiotic yeast produces enzymes that break down feed nutrients, including fiber.Consistent and rapid fermentation of crude fiber increases bacterial protein production, enhances the formation of free fatty acids—an energy source for the body—and reduces ammonia levels in the rumen, as it is used to produce bacterial protein. Probiotic yeast activity in the rumen of ruminants reduces lactic acid formation, which helps control rumen acidity. Ultimately, the influence of yeast on rumen fermentation has a beneficial effect on cow health, contributing to increased milk production and milk quality.

[0023] Yeast culture preparations contain specialized dried yeast cells and some essential components of their growth medium. The cells are dried to preserve their viability. Therefore, the yeast must be robust enough to withstand the drying and pelleting processes. For maximum effectiveness, it must be metabolically active in the animal's body. Yeast cells do not need to divide (reproduce), but they must be alive to consume oxygen, secrete specific peptides, and utilize sugars in the rumen.

[0024] Yeasting is an effective way to increase protein in animal diets. This enriches the grain with protein (increasing its content by 1.5 to 2 times). This protein has high biological value, contains all essential amino acids, and is 90 to 95% digestible.

[0025] Using feed sugar, yeast cells rapidly multiply and accumulate significant amounts of protein and vitamins, with yeast protein having a high biological value. This property, along with the presence of vitamins (B complex), enzymes (amylase, maltase, zymase, etc.), and plant insulin (glucokinin), explains the beneficial effects of yeast-infused feed on animal appetite, health, reproduction, and feed utilization. Yeast fermentation allows for savings of up to 25% in concentrated feed. Feeding yeast concentrates has a beneficial effect on animal health and increases productivity by 15-20%. Yeast, a special type of bioregulator, is believed to be effective in the rumen and less effective in the post-rumen digestive tract. The use of yeast results in:

[0026] 1) Modifying rumen pH. One of the benefits of adding a yeast-based supplement to animal feed is that it optimizes rumen conditions during carbohydrate digestion, leading to a decrease in rumen pH. Including a yeast-based supplement in feed reduces the drop in rumen pH. Since yeast cannot directly assimilate these acids, it stimulates certain microorganisms in the rumen to utilize these acids, thereby raising rumen pH.

[0027] 2) Improved feed utilization. Higher amounts of propionate byproducts were found in the rumen of dairy cattle fed yeast cultures. This indicates that a higher percentage of the hydrogen involved in metabolism is involved in propionate production than in methane production, resulting in improved feed utilization. 3) Increased metal digestibility. Yeast helps absorb chelated forms of trace elements in feed, including magnesium, zinc, copper, manganese, selenium, cobalt, and potassium, making them bioavailable. These trace elements largely determine the development and resistance of animals to disease.

[0028] 4) Stimulation of the proliferation and activity of anaerobic bacteria in the rumen. Yeast has a naturally pleasant aroma that improves the palatability of feed, thereby increasing its intake. Furthermore, yeast contains a complex of B vitamins, many different amino acids, and some unknown growth factors that stimulate the growth of certain anaerobic bacteria found only in ruminants. These components are released in the rumen following yeast autolysis. The number of certain anaerobic bacteria in the rumen, including cellulosic and hemicellulosic bacteria, can increase tenfold if dairy cattle consume yeast cultures, resulting in increased digestibility of cellulosic biomass. Thus, the symbiotic relationship between yeast and rumen bacteria (which digest the bulk of fiber) is beneficial for both groups of microorganisms.

[0029] For yeasting, barley and corn meal, flour, grain waste, corn cobs, straw, and succulent feed are used, but before this, all feed must be thoroughly ground (the higher the degree of grinding, the more successful and better their saccharification and yeasting will be).

[0030] When yeast is added to feed, rapid yeast growth occurs, accompanied by alcoholic fermentation. Simultaneously, lactic acid bacteria proliferate, producing lactic, acetic, formic, and other volatile acids. As a result, the feed's pH increases to 3.8–4.2, inhibiting the growth of putrefactive bacteria. Yeast is capable of synthesizing protein from simple nitrogen compounds, increasing the overall protein content of the feed.

[0031] The essence of the yeast fermentation process is that during reproduction, yeast uses non-protein nitrogenous compounds (amides) from the grain to synthesize proteins in its own cells. This feed increases the content of complete proteins, enzymes, B vitamins, and estrogens [Khudenko S. Feed Fermentation. URL: https: / / vk.com / topic-111928158 _39746566].

[0032] The technical problem addressed by the proposed invention is the production of a high-quality feed additive for the preparation of feeds balanced in protein, amino acids and vitamins.

[0033] The technical problem is solved by a method for producing a feed additive that involves obtaining galactose-sucrose products by hydrolyzing whey and plant waste with succinic acid for subsequent cultivation of yeast cultures. This involves mixing the hydrolyzed whey and plant waste with yeast in the following ratio: 90% whey and plant waste hydrolyzed, 0.03-0.05% succinic acid, and 10% Saccharomyces cerevisiae yeast.

[0034] Galactose-sucrose products from the processing of dairy and plant waste are obtained by hydrolysis with succinic acid and fermentation with native enzymes secreted by yeast cultures.

[0035] The technical result is the production of a feed additive with the stated composition, which allows for a significant increase in the biological value of feed and feeding efficiency, while significantly reducing the cost of production technology.

[0036] The proposed method for obtaining the feed additive is carried out as follows.

[0037] 1. First, hydrolyze plant waste and whey with succinic acid. Add 0.5 g of succinic acid to 200 ml of whey and heat with constant stirring until the acid dissolves. Then, add the whey with succinic acid to 800 ml of native whey. This yields 1,000 ml of a 0.05% succinic acid solution in whey. To perform hydrolysis, grind the plant waste to a particle size of 10-100 µm. Add 600 ml of a 0.05% succinic acid solution in whey to 1 kg of crushed raw material until the moisture content reaches 45-50%. The mixture is placed in a fermenter and hydrolyzed at 70°C for 120 minutes. 2. Fermentation and drying of the mixture. The most suitable producer of biological synthesis of nitrogenous substances in milk whey is yeast, which has the ability to use lactose for nutrition.For this purpose, a thick suspension is prepared from pressed baker's yeast, which is added at a rate of 50 ml per liter of lactic-vegetable hydrolysate. The yeast is cultivated in a fermenter at a temperature of 28-30°C for 10-12 hours with an air flow of 2-4 l / min, a stirrer speed of 1000-1200 rpm and a pH of 5.0-5.5. Bran is then added to reduce the moisture content to 20-25%. The resulting product is then dried in drying equipment at a temperature not exceeding 50°C. After these procedures, the probiotic feed supplement is ready for use. The results of the laboratory study of the developed supplement are presented in Table 1.

[0038] Table 1 Physicochemical parameters of the probiotic feed additive

[0039] As evidenced by the data presented in Table 1, in the feed additive obtained using the technology we developed, the amount of crude fiber and crude fat increased by 1%, and crude protein by 0.88%.

[0040] The issue of rational use of whey remains a pressing issue in many countries. Insufficient use of whey in industrial processing leads to significant emissions and negative environmental impacts. In this context, using whey as a feed additive offers significant potential. It enriches feed rations with valuable nutrients such as protein, vitamins, enzymes, and microelements, which can positively impact animal productivity. Therefore, the developed biotechnology and formulation of a probiotic feed additive based on whey, grain waste, yeast, and succinic acid represent a promising solution for improving livestock farming efficiency and addressing the issue of whey disposal.

Claims

Invention formula A method for producing a feed additive, characterized by the fact that it includes hydrolysis of milk whey and plant waste, which is carried out using succinic acid and native enzymes secreted by yeast cultures (Saccharomyces cerevisiae), at a temperature of 70° C for 120 minutes, yeast cultures are added in an amount of 10% by weight to the hydrolysate of milk whey and plant waste, cultivated at a temperature of 28-30° C for 10-12 hours and an air supply of 2-4 l / min at a stirrer rotation speed of 1000-1200 rpm, followed by the addition of bran to 20-25% moisture and drying of the resulting product on drying equipment at a temperature of no more than 50° C.

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