RU2811698C1 - Microlife biological drug - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: composition for canning alfalfa for the production of feed for farm animals is characterized by the following: it consists of strains with the following concentrations of CFU/ml: rod-shaped Lactobacillus plantarum VKPM V-5337 — up to 3.5*10¹⁰ CFU/ml and coccal form of Lactococcus lactis B-4989 — up to 3.5*10¹⁰ CFU/ml, Enterococcus faecium B-4489 — up to 1*10¹⁰ CFU/ml, propionic acid bacteria Propionibacterium freudenreichii B-9653 — up to 1*10¹⁰ CFU/ml, as well as Bacillus subtillis B-4506 — up to 1*10¹⁰ CFU/ml, taken in equal volume fractions.

EFFECT: invention makes it possible to expand the arsenal of preparations for preserving plant materials, ensuring high safety and quality of the finished alfalfa feed.

1 cl, 6 tbl, 2 ex

Description

The invention relates to the field of production of feed for farm animals, namely to bacterial starters for preserving plant feed raw materials using certain types and strains of microorganisms, and can be used in technological processes for the production of haylage, silage and silage from dried grasses.

The biotechnological processes for the production of haylage, silage and silage are identical to each other and differ only in the degree of wilting, i.e. dry matter content (DM). Thus, the moisture content of dried grass haylage according to GOST 55452-2021 is 40-55%, The moisture content of the silo according to GOST 55986-2022 is more than 70%. Silage is dried silage or haylage with high humidity. According to GOST 55986-2014, silage is silage with a moisture content of 60-70%.

The main succulent feeds for cattle with traditional feeding technology during the winter-stall period, and even more so with the modern year-round uniform housing and feeding system, are haylage, cured grass silage and corn silage, during the procurement of which significant losses of nutrients are allowed, the formation of harmful and toxic compounds that cause a decrease in their quality [Theory and practice of production and use of bulk feeds / Sh.K. Shakirov, O.L. Shaitanov, E.O. Krupin, R.P. Idiatullina and [etc.] // 2nd edition, revised and expanded. - Kazan: “FEN” AS RT. - 2021. - 291 p.]. This leads to a significant shortage of livestock products, so the development of new effective technologies for preserving green grasses in order to increase the safety and quality of the feed obtained from them still remains relevant [Feed preparation technology / S.Ya. Zafren. - M.: Kolos. - 1977. - 240 p.; Preparation of silage and haylage using domestic biological preparations / V.A. Bondarev, V.M. Kosolapov, V.P. Klimenko, A.N. Krichevsky. - M.: FGBNU All-Russian Research Institute of Feeds named after. V.R. Williams. - 2016. - 212 p.].

Currently, the main raw material for making haylage and silage from dried plants is alfalfa. Alfalfa is difficult to ensile using the traditional method because it contains a lot of water, protein, potassium, calcium and other alkalizing substances and little sugar. As grass stands age, its nutritional value quickly decreases. When ensiling green alfalfa mass without additives, the activity of butyric acid and putrefactive bacteria continues in the silage even after the existing sugars have been consumed. Undesirable protein breakdown also continues. At the same time, the content of amine nitrogen increases, which leads to spoilage of silage during storage. Together with the released juice, valuable carbohydrates and part of the already formed lactic acid are lost. Alfalfa haylage deserves special attention because it exhibits the most favorable fermentation [Microbiological and biochemical processes during silage of alfalfa with different levels of dry matter / I.I. Filatov, T.T. Kuznetsova, L.G. Safronova // Siberian Bulletin of Agricultural Science. - 1978. - No. 5. - P. 44-47], accompanied by low losses of nutrients, quite comparable to the losses that occur during artificial drying [Haylage or silage? Features of canning legumes and cereals / Yu.A. Pobednov // Problems of biology of productive animals. - 2016. - No. 2. - P. 42-54]. However, the success of ensiling the wilted mass depends, first of all, on an uncontrollable factor - the high number of epiphytic lactic acid bacteria, since their number on plant covers largely determines their qualitative composition. Available data show [Fehrmann E. Jaresverlauf des epiphytischen Mikrobenbesatzes auf einen Graslandstandort / E. Fehrmann, Th. Müller // Das Wirtschaftseigene Futter. - 1990. - Bd. 36. - H. 1. - P.66-78], that if when the number of epiphytic lactic acid bacteria is ≤ 10 ³ CFU/g of green mass, they are usually represented by 3-4 low-active coccal forms, then when the number is ≥ 10 ⁵ CFU/g - only a single highly active bacillus Lactobacillus plantarum . According to E.I. Kvasnikova and I.V. Shchelokova [New in the microbiology of feed silage / E.I. Kvasnikov, I.F. Shchelokov // Izv. USSR Academy of Sciences, biological series. - 1968. - No. 4. - P. 543-556] in silage with a high dry matter content, Lactobacillus plantarum tends to outstrip other lactic acid bacteria in growth, which indicates its improved adaptability to fermentation conditions on dried mass.

It is known that alfalfa, even when dried to a dry matter content of 40-45% or more, differs from other feed crops in its increased content of freely bound water, high buffer capacity and sugar deficiency, in which maximum acidification of the feed is achieved only after 15 days. During this period, clostridia still show their activity [Theoretical aspects of ensiling dried grasses / Yu.A. Pobednov // Feed production. - 1998. - No. 8. P. 21-25]. This creates the prerequisites for butyric acid fermentation, while other types of undesirable microflora do not receive noticeable development [Theoretical background and methods of canning corn and herbs based on the regulation of microbiological processes: method. instructions / Yu.A. Pobednov. - St. Petersburg: Biotrof LLC. - 2017. - 52 p.]. Based on this, the principle of alfalfa canning is based on the biological characteristics of butyric acid bacteria. The fact is that, unlike lactic acid bacteria, butyric acid bacteria are present on plants in the form of spores, the germination of which requires water. Consequently, the less moisture the plant contains available to microbes, the slower the spores of butyric acid bacteria will germinate.

To obtain a wilted mass of alfalfa that is free from the accumulation of butyric acid during haylage and ensiling, it should be quickly dried in the field to a dry matter content of ≥ 40%, it should be carefully sealed in storage, and measures should be taken to accelerate the process of lowering the pH to a value of - 4, 6-4.7 - critical for butyric acid bacteria. At the same time, eliminating butyric acid fermentation is by no means an essential task that should be solved when preparing silage from wilted grasses. It is equally important to eliminate the vital activity of other microorganisms undesirable during ensiling, the main of which are enterobacteria and yeast. It is precisely because of the slowness of their spontaneous acidification that the number of enterobacteria can reach a significant value, practically not inferior to the number of lactic acid bacteria at the beginning of ensiling [Role of microflora in forage conservation / G. Pahlow // Landbauforschung-Völkenrode. - 1991. - Sonderheft 123. - R. 26-36].

Thus, with spontaneous acidification of dried herbs, as with the slow fermentation of freshly cut mass, it is often not possible to eliminate unwanted fermentation products. However, when accelerating the acidification of such a mass, for example, with the help of bacterial preparations, this ensures enzymatic processes in almost any case [Theoretical aspects of ensiling dried grasses / Yu.A. Pobednov // Feed production. - 1998. - No. 8. - P. 21-25].

The development of biological preservatives that accelerate fermentation processes based on lactic acid bacteria when preserving green herbs has been going on for more than 100 years. The theoretical prerequisites for their use were based on the idea of a more complete and rapid fermentation of sugar contained in the silage mass into lactic acid with the help of introduced lactic acid bacteria. The resulting lactic acid in an anaerobic environment helps eliminate the development of unwanted microorganisms, primarily enterobacteria and butyric acid bacteria. Currently, this provision is the basis for the development of new bacterial preparations [Preparation of silage and haylage using domestic biological preparations / V.A. Bondarev, V.M. Kosolapov, V.P. Klimenko, A.N. Krichevsky. - M.: FGBNU All-Russian Research Institute of Feeds named after. V.R. Williams. - 2016. - 212 p.].

For the first time, a preparation with lactic acid bacteria for preserving green feed was proposed by A.A. Garder back in 1932 [Scientific principles of canning green feed / A.A. Zubrilin. - M.: Ogiz-selkhozgiz. - 1947. - 391 p.]. Subsequently, over 50 different bacterial preparations, intended mainly for ensiling corn and freshly cut grass, were developed and offered for production. In the early 80s of the last century, scientists at the All-Russian Research Institute of Biotechnology based on lactic acid bacteria proposed “Biosil” and “Litosil”. The first preparation consisted of two cultures of rod-shaped and two streptococcal forms of lactic acid bacteria. The second drug “Litosil” is made from two cultures of rod-shaped and one streptococcal form of lactic acid bacteria. The main disadvantage of the proposed bacterial preparations is the weak resistance of epiphytic microflora, as well as the lack of sugar in most types of perennial grasses to form lactic acid in the amount necessary to acidify the mass to pH 4.2-4.3. Therefore, when using the proposed drugs, the results of grass silage, in most cases, were negative.

The prerequisite for the creation of a new generation of bacterial preparations, consisting mainly of osmotolerant lactic acid bacteria, capable of actively functioning on mass with a high dry matter content, was the data that a significant part of epiphytic lactic acid bacteria exhibit their activity only in conditions of high humidity of silage raw materials and, as a result This is not very suitable for development in dried grasses [New in the use of lactic acid bacteria in grass silage / Yu.A. Pobednov, F. Weisbach, G. Palov // Feed production. - 1997. - No. 8. - P. 24-28]. The effectiveness of these drugs is manifested when ensiling grass with a dry matter content of at least 25%. Therefore, the effect of using bacteriological preparations containing such strains of lactic acid bacteria is many times greater.

In this regard, research to improve biological preparations based on bacterial cultures is aimed at selecting lactic acid bacteria with increased osmotolerance, which use not only sugars, but also oligosaccharides for their nutrition. In this regard, the drug “Biotrof” is known [RU 2168909, 06/20/2001], consisting of a single species of rod-shaped lactic acid bacteria Lactobacillus plantarum VNIISKHM 78 D. The drug turned out to be reliable only when preserving easily and difficult to silage herbs, dried to a moisture content of 60-70%.

It is also known to use biological preparations for grass silage, such as the rod-shaped Lactobacillus plantarum 8P-A3 [RU 2011138042 A, 03/20/2013], Lactobacillus plantarum VKPM 5337 [SU 1784641, 12/30/1992] and the coccal form Streptococcus faecium No. 500 [SU 1514766, 10/15/1989], Enterococcus

durans VKPM B 10093 with an aqueous extract of the stevia plant [RU 2555596 C2, 07/10/2015], and in combination with other strains of rod-shaped bacteria Lactobacillus plantarum - 38 VKPMA-4174, Lactobacillus casei M 76 VKPM V-34389, Lactobacillus delbrueckii subsp. bulgaricus - 298 VKPM B-2455, Lactococcus lactis “t” VKPM B-5284 [RU2268299 C2, 01/20/2006]. However, the results of studies conducted using such preparations on freshly cut hard-to-silage grasses turned out to be significantly lower than expected due to the intensive development in the first 2-3 days of unwanted aerobic bacteria, which also use sugars for their nutrition. They are completely ineffective for ensiling freshly cut and dried high-protein legumes, which are non-silageable plants due to lack of sugar [Preparation of silage and haylage using domestic biological preparations / Bondarev V.A., Kosolapov V.M., Klimenko V.P., Krichevsky A.N. M.: FGBNU All-Russian Research Institute of Feeds named after. V.R. Williams. - 2016. - 212 p.]. The preparations turned out to be reliable only when preserving easily and difficult to silage herbs, dried to a moisture content of 60-70%. In addition, in wilted grasses at the beginning of their ensiling, due to the content of a large residual air, lactic acid fermentation mainly occurs according to the heterofermentative type, causing an increase in nutrient losses from gas formation and a decrease in the quality of feed for fermentation products [Role of microflora in forage conservation / G Pahlow // Landbauforschung-Volkenrode. - 1991. - Sonderheft 123. - P. 26-36].

There are known preparations for preserving green feed using complex strains of rod-shaped lactic acid bacteria and coca forms; representatives of such preparations are “Biosib” and “Bioamide-3”, which respectively contain Lactococcus lactis, Lactobacillus plantarum and Propionibacterium freudenreichii [htts://ru .calameo.com/read/ 0055096144690fblde67c] and Lactococcus lactis subsp. lactis VKPM B-3123, Lactobacillus plantarum VKPM - 10965, Propionobacterium sp. VKPM B-6085 [Instructions for the use of the feed additive “Bioamide-3” for silage and haylage of feed. Approved by the deputy head of the head of Rosselkhoznadzor on April 19, 2016].

A bacterial starter has also been developed [SU 1091549 A1, 06/10/1997] for feed silage, consisting of lactic acid bacteria of the rod-shaped form Lactobacterium pentoaceticum , the coccal form of PMB Streptococcus lactis diastaticus AMK and propionic acid bacteria Propionobacterium shermanii PKB.

The purpose of rod-shaped lactic acid and propionic acid bacteria is to accelerate lactic acid fermentation; coccal bacteria should limit the activity of yeast and thereby stimulate lactic acid fermentation. In addition, it is known that rod-shaped homofermentative bacteria, being the most powerful producers of lactic acid, cannot develop at pH 5.5-7.0. Therefore, the composition of the preservative includes coke forms of bacteria, especially Enterococcus faecium, which in this process play a special “starting” role; such bacteria multiply quickly and contribute to the onset of active fermentation [Hughes, AD (1969). The non-protein nitrogen composition of grass silages. I. The estimation of the basic amino acids and non-volatile amines by chromium atography on a weak cation exchange resin. J.agric. Sci., Camb. 72, 459 rub.]. The pH value decreases to 5.0. Next, fermentation is continued by lactic acid bacteria Lactobacillus plantarum and others, reducing the pH value to 4.0, and this position is maintained throughout the entire canning period. Such complementarity in fermentation processes is used in the creation of both domestic and foreign biological products [Silostan-Russia, FaedtechTM Silage F-10 - Czech Republic, Biostabil plus - Austria, etc.].

Bacteria with amylolytic and cellulolytic activity are also used for ensiling difficult-to-silage crops. Some strains, such as lactobacilli and streptococci, are capable of hydrolyzing starch [Biology of lactic acid bacteria / E. Kvasnikov // Acad. Sciences of the UzSSR. Institute of Botany. - Tashkent: Academy of Sciences of the UzSSR. - 1960. - 351 p.; Microbiology / E.N. Mishustin, V.T. Yemtsev. - M.: Agropromizdat. - 1985. - 270 p.]. These strains of Lactobacillus plantarum and Streptococcus lactis subsp. diastaticus are found in clover haylage and are used in alfalfa ensiling. The number of amylolytic bacteria depends both on the type of plant material used in the silage and on the stage of its maturation. In freshly cut plants they are represented mainly by coccal forms, the number of which is 5-10% of the total number of lactic acid bacteria (LAB). The peak reproduction of lactic acid bacteria occurs 1-3 days after laying silage, and the largest number of amylolytic microorganisms was found in the period from 3 to 15 days. The content of amylolytic lactic acid bacteria (LAB) in corn silage does not exceed 5×10 ⁵ CFU/g, and their activity gradually decreases. At the same time, in rapeseed and alfalfa silos the number of amylotic lactic acid bacteria reaches 5 × 10 ⁶ CFU/g [Microflora of amylolytic and lactic acid bacteria silage / T.N. Golovich, N.K. Kovalenko // Microbiological Journal. - 1994. - T.56 (2). - P. 3-7]. It is obvious that when ensiling any feed, strains of lactic acid bacteria that digest simple carbohydrates rather than hydrolyze starch gain an advantage. AMKB, producing extracellular amylyses, provides simple carbohydrates not only to themselves, but also to other strains, which is the basis for the relationship between LAB and AMKB in the community of microorganisms.

The use of preparations containing amylolytic lactic acid bacteria also has a number of disadvantages: the preparations do not have sufficient antagonistic activity against molds and pathogenic bacteria, are little or completely ineffective when ensiling freshly cut grass, as well as corn and other silage crops with excess sugar content, do not produce the desired result when ensiling dried grasses belonging to the group of non-silageable plants [Mamaev A.A. The effectiveness of canning herbs with Bacillus subtilis and the use of the resulting feed in cattle diets: Dis. Ph.D. agricultural Sciences: 06.02.02 / A.A. Mamaev. - Moscow. - 2005. - 133 p.]. In addition, strains of lactic acid bacteria with amylolytic activity are rarely observed in nature. Therefore, researchers are trying to obtain such strains using genetic engineering methods [Cloning and expression of cellulase and xylanase genes in Lactobacillus plantarum / T. Scheirlinck, J. De Meutter, G. Arnaut, H. Joos, M. Claeyssens and F. Michiels // Applied Microbiology and Biotechnology. - 1990. - No. 33. - R. 534-541].

Weather conditions during the period of harvesting succulent feed have a significant impact on the quality of the silage mass and, consequently, on the quality of the finished feed and its productive effect. So, according to G.I. Domracheva et al. [The influence of propionic acid bacteria on the quality

silage, growth and development of young animals / G.I. Domracheva, Yu.V. Kononov, A.E. Maidanovich // Scientific. tr. Sib. Scientific research Institute of Agricultural Sciences animals. Omsk. 1970. No. 15. P. 173-177] in conditions of high humidity, it is advisable to use starters based on propionic acid bacteria, which can reduce the acidification of the final product, and, consequently, reduce the loss of nutrients in it. In dry years, on the contrary, the use of lactic acid bacteria is preferable to propionic acid bacteria.

This problem is solved by using a starter [RU 2265655 C2, 12/10/2005] for feed silage, consisting of 8 cultures, which involves growing lactic acid and propionic acid bacteria, mixing the resulting cultures in various ratios and drying, where an association of strains is used as lactic acid bacteria Lactobacillus salivarius - LT1, LT2, LT3, LT5, Enterococcus durans - LT4, LT6, Lactobacillus plantarum - LT7 and propionic acid bacteria strain Propionibacterium freudenreichii - LT8. The general disadvantages of such drugs are technological difficulties in production, their high cost and lack of guarantee of high efficiency due to strong intrapopulation competition of microorganisms.

To increase the efficiency of ensiling of difficult and non-silageable crops, it has been proposed to use both individual polyenzyme preparations and in combination with bacterial cultures for the deep hydrolysis of indigestible carbohydrates (cellulose, hemicellulose, pentosans and pectin substances) in order to provide the silage mass with the necessary amount of sugar to optimize fermentation processes , eliminating the vital activity of unwanted microorganisms, as well as increasing the energy value of feed.

The need to jointly use different types of enzymes when ensiling perennial grasses leads D.R. Seal [Bacteria and enzymes as product to improve silage preservation / D.R. Seal // Developments in Silage. 1987. P. 47-61]. The author notes that it is most advisable to create a complex of enzymes from pectinases, amylases, hemicellulases and cellulases in order to provide the silage mass with the necessary amount of sugar during the hydrolysis of complex carbohydrates in the first two days of ensiling.

It is known to use the domestic enzyme preparation “Ferkon” for ensiling dried perennial grasses, created on the basis of a complex of hydrolytic and lyase enzymes: cellulase and xylanase. The hydrolysis of complex carbohydrates produces a sufficient amount of sugar, which creates favorable conditions for the development of lactic acid bacteria [Efficiency of alfalfa silage technology with the new biological preparation “Ferkon” / A.A. Anisimov // Your rural consultant. - 2006. - No. 4. - P. 28-30]. The disadvantage is that the preparation, containing only enzymes for the narrowly targeted hydrolysis of complex carbohydrates, is designed for the presence in the ensiled plant mass of a sufficient amount of natural epiphytic lactic acid bacteria capable of producing the optimal amount of lactic acid to eliminate butyric acid and putrefactive fermentation. In addition, the drug "Ferkon" is quite expensive.

A well-known biological preservation preparation is Sil-All (USA), containing a set of enzymes (cellulase, hemicellulase, pentonase, amylase) in combination with rod-shaped and coccal forms of lactic acid bacteria Lactococcus faecium, Lactobacillus plantarum, Pediococcus acidilactici, Lactobacillus salivarius. The use of Sil-All biopreservative when making silage leads to an increase in the preservation of dry matter, crude protein, nitrogen-free extractive substances (NFES) and energy value in general. At the same time, the content of crude fiber decreases by 16.5% [Efficiency of using the biological additive Seal-All when making silage / N.V. Pristach, A.A. Tsoi // News of the St. Petersburg State Agrarian University. - 2007. - No. 6. - P. 73-78]. However, silage prepared using the biological product Sil-All has a high cost of finished feed.

A well-known biological preservation product is Feedtech™ Silage F18 (Czech Republic) for the preservation of legumes and cereals, consisting of two types of lactic acid bacteria Pediococcus acidilactici, Lactobacillus plantarum, and the enzyme Cellulose-xylanase. When this drug is introduced into a cereal-legume mixture of herbs, an increase in the safety of crude protein, the amount of sugars and metabolic energy is observed compared to silage with spontaneous acidification [The influence of biopreservatives on the safety of nutrients in silage / V.V. Mikhalev, I.D. Arnautovsky, V.N. Kondratyev // Feeding farm animals and feed production. - 2012. - No. 4. - P. 3-8]. The use of a biopreservative in the green mass of corn is inferior in terms of the level of preservation of crude protein to the biopreservatives “BioSil” and “Sila-Prime” [Efficiency of using biological preservatives when ensiling green mass of corn / E.G. Artemuk, V.A. Satishur //Bulletin of the Brestskaga University. Gray 5 Chemistry. Biology. Knowledge of the earth. - 2016. - No. 1. - P. 13-16]. The disadvantage of the drug is its high market cost, which affects the cost of finished feed and livestock products.

In this direction, active research is being carried out on the development of complex drugs by Lallemand under the general brand “Biotal”. The biological preparation Biotal Axcool Gold is also used in the preparation of silage and haylage from dried perennial grasses, which consists of three enzymes - amylase, β-glucanase and xylanase, and two types of bacteria Lactobacillus buchneri and Pediococcus pentosacius. However, according to V.A. Bondareva et al., [Preparation of silage and haylage using domestic biological preparations / V.A. Bondarev, V.M. Kosolapov V.P. Klimenko, A.N. Krichevsky - M.: FGBNU All-Russian Research Institute of Feeds named after. V.R. Williams. - 2016. - 212 pp.] when ensiling alfalfa using Biotal Axcool Gold, the silage is slightly acidified, as a result, traces of butyric acid are found in it and quite a lot of ammonia (0.31%) in the dry matter. The intensity of hydrolysis of complex carbohydrates was insufficient and had no effect on improving the quality of feed.

The use of preparations based on lactic acid bacteria, which use complex carbohydrates for their nutrition, has not yet resulted in positive results, since not a single strain of the known forms of these microorganisms ferments starch, a very accessible compound for other bacteria. In this regard, the attention of researchers is switched to the selection of bacteria of other genera that can participate in the fermentation of complex carbohydrates [Haylage or silage? Features of canning legumes and cereals / Yu.A. Pobednov // Problems of biology of productive animals. - 2016. - No. 2. - P. 42-54]. In addition, to guarantee an increase in the safety and quality of silage from almost any plant raw material, it is necessary to use a biological preparation that, along with a bactericidal and well-defined fungicidal effect. This goal can be achieved by creating drugs based on cultures of the genus Bacillus subtilis [Mamaev A.A. The effectiveness of canning herbs with Bacillus subtilis and the use of the resulting feed in cattle diets: Dis. ...cand. agricultural Sciences: 06.02.02 / A.A. Mamaev. - Moscow. - 2005. - 133 p.].

It is known that, despite the fact that the bacteria Bacillus subtilis belong to the group of aerobes, they always show a tendency to anaerobiosis [Bacteria of the group of hay and potato bacilli / V.A. Mirzoeva. - M.: USSR Academy of Sciences. - 1959. - 175 p.]. At the same time, their belonging to the group of aerobes allows them to quickly consume the oxygen contained in the mass, creating anaerobic conditions in it, and the tendency to anaerobiosis contributes to the long-term functioning of bacteria in anaerobic conditions, leading to the fermentation of carbohydrates according to the homofermentative lactic acid type, without destroying the protein of the feed to its final products , that is, to ammonia [Fermentation of carbohydrates and related compounds / W. Wood // Metabolism of bacteria. - M.: Foreign publishing house. lit. - 1963. - P. 9-62]. In addition, unlike lactic acid bacteria, microorganisms of the species Bacillus subtilis form the exoenzyme amylase, which breaks down starch into glucose, maltose and oligoglucosides, followed by their fermentation into organic acids [World of Microbes. In three volumes / R. Steinier, E. Edelberg, J. Ingram. - M.: Mir. - 1979. - Volume 2. - 334 p.], which makes it possible to successfully use this crop when making silage from difficult and even non-silageable grasses. Bacillus subtilis synthesizes a number of antibiotics that are active against both bacteria, including butyric acid bacteria, and fungi, including molds and yeasts, and also synthesizes amino acids and vitamins [Gegen Pilze wirksame antibiotika der Bacillus subtilis Gruppe / W. Loeffler, W. Katzer, S Kremer et al. // Forum Microbiol. - 1990. - V. 13. - P. 156-163], which ensures high safety and quality of feed regardless of the degree of acidification.

It is known to use a two-component preparation [RU 2204911 C2, 05/27/2003] for ensiling and preserving dried herbs, created on the basis of osmotolerant strains of lactic acid bacteria Lactobacillus plantarum mixed with bacteria of the species Bacillus subtilis , which helps to preserve nutrient losses, improve the quality and energy nutritional value of silage . Meanwhile, bacteria of the species Bacillus subtilis , even without combination with osmotolerant strains of lactic acid bacteria, are capable of providing a high preservative effect and, in comparison with the following, have a number of noticeable advantages [RU 2012126499 A, 12/20/2013]. These bacteria are very well adapted for preserving green mass. They are undemanding to the moisture content of raw materials, they can preserve not only dried (up to 30-40% DM) hard-to-silage plants, but also other plants, regardless of their sugar-buffer ratio, as well as which group the freshly cut mass belongs to - to group of hard-to-silage and easy-to-silage crops [Efficiency of preserving dried herbs with the drug “Biotrof” and using the resulting feed in cattle rations / Yu.A. Pobednov, A.A. Mamaev // Current problems of technology for preparing feed and feeding farm animals. - Dubrovitsy. - 2006. - P. 22-24]. During the conservation process, they release special substances - bacterocins, which directly act on harmful microflora [Laptev G.Yu. Development of biological preparations to increase the nutritional value and efficiency of feed use: dissertation. ...doc. biol. Sciences: 03.00.23, 06.02.02 / G.Yu. Laptev. - Saint Petersburg. - 2009. - 352 p.].

A known drug containing the bacterial strain Bacillus subtilis VKPM B-11353 [RU 2539762 C1, 01/27/2015] for preserving green herbs. Multicomponent liquid biological preparations are also known, for example, “Silostan”, consisting of strains of bacteria Bacillus subtilis in combination with two rod-shaped lactic acid bacteria Lactobacillus plantarum and Lactobacillus casei [Efficiency of the microbiological starter culture “Silostan” with a description of the mechanism of action of selected bacteria during feed ensiling // Efficient livestock farming. - 2015. - No. 5 (114). - P. 48-49], “Lactis” in combination with two rod-shaped lactic acid bacteria Lactobacillus plantarum , Lactobacillus buchneri , four coccus-shaped Pediococcus pentosaceus , Pediococcus acidilactici , Enterococcus faecium , Streptococcus lactis and two propionic acid bacteria Propionibacterium freudenreichii , Propionibacterium acidipropionici [ The influence of biopreservatives “Laktifit” and “Lactis” on the safety of corn silage / A.D. Prudnikov, A.N. Nikitin, A.A. Puzik, L.A. Demyanova // International scientific research journal. - 2017. - No. 12-3 (66). - P. 130-133].

The multicomponent dry biopreservative “Sila-Prime”, jointly produced in Israel and the USA, is known, which consists of seven microorganisms: complementary rod-shaped lactic acid bacteria Lactobacillus plantarum, Pediococcus pentosaceus, Pediococcus acidilactici, Enterococcus faecium, Lactobacillus casei, Streptococcus lactis and Bacillus subtillis [Comparison of the effectiveness of liquid and dry biopreservatives when making corn silage / A.Yu. Marchenko, N.V. Bychenko, N.N. Zabashta, E.N. Golovko // Collection of scientific studies of the Krasnodar Scientific Center for Animal Science and Veterinary Medicine. - 2020. - T. 9. No. 2. - P. 105-108].

The general disadvantages of the preparations described above are that if, with the combined use of two or three-component preparations, for example, lactic acid and propionic acid bacteria with the bacteria Bacillus subtillis to optimize fermentation processes, there is a decrease in nutrient losses, which leads to the biodiversity of a complementary consortium, then with multicomponent options with 7 and 8 bacteria, due to the strong intrapopulation competition of microorganisms, the effectiveness of their use in canning herbs, even corn, is reduced compared to other preservatives. In addition, such drugs significantly increase the cost of finished feed.

In connection with the above, it is urgent to develop an effective and affordable liquid biological preparation for any difficult and non-silage grasses with broader functional properties associated with the microorganisms Bacillus subtilis , providing guaranteed good nutritional value and safety of the finished feed.

The advantage of a liquid biological preparation compared to dry preparations is the rapid introduction of microorganisms into the process

fermentation, which speeds up the onset of the process, the disadvantage is the short shelf life of the drug.

Thus, the technical problem to be solved by the invention is the expansion of the arsenal of preparations for preserving plant raw materials, ensuring high safety and quality of the finished feed from difficult-to-silage crops (alfalfa), reducing the readiness period and improving its preservation.

The technical result of the claimed invention consists in a specially selected multicomponent composition of strains of rod-shaped and coccal forms of bacteria (consortium), which is a well-coordinated complementary mechanism covering all phases of the fermentation processes of raw materials. The consortium effectively and quickly reduces pH levels, preventing unwanted nutrient losses and guaranteeing the aerobic stability of the finished feed.

The consortium of strains has a high energy of acid formation due to the accumulation of lactic acid, is capable of rapid breakdown of mono- and disaccharides and polysaccharides, and has an antagonistic effect against pathogenic and opportunistic bacteria and fungi, which determines the positive effect of the biological preparation (preservative, starter culture) on microbiological processes when ensiling (preserving) difficult-to-silage plants, and to optimize the microflora of the digestive system of cattle.

The technical problem is solved, and the specified technical result is achieved by the claimed composition for canning alfalfa for the production of feed for farm animals, characterized by the fact that it consists of strains with the following concentrations of CFU/ml: rod-shaped Lactobacillus plantarum VKPM V-5337 - up to 3.5* 10 ¹⁰ CFU/ml and coccal form of Lactococcus lactis B-4989 - up to 3.5*10 ¹⁰ CFU/ml, Enterococcus faecium B-4489 - up to 1*10 ¹⁰ CFU/ml, propionic acid bacteria Propionibacterium freudenreichii B-9653 - up to 1 *10 ¹⁰ CFU/ml, as well as Bacillus subtillis B-4506 - up to 1*10 ¹⁰ CFU/ml, taken in equal volume fractions.

The biological preparation "MicroLife" is a specially selected multicomponent consortium of strains of rod-shaped and coccal forms of various microorganisms, it is a well-coordinated complementary mechanism covering all phases of the fermentation processes of raw materials, which optimizes their direction, effectively and quickly reduces the pH level, preventing unwanted losses of nutrients, and guarantees the stability of the finished feed.

The biological preparation "MicroLife" consists of strains of lactic acid bacteria at the following concentrations (CFU/ml):

rod-shaped Lactobacillus plantarum VKPM V-5337 - up to 3.5*10 ¹⁰ CFU/ml and coccal form Lactococcus lactis B-4989 - up to 3.5*10 ¹⁰ CFU/ml, Enterococcus faecium B-4489 - up to 1*10 ¹⁰ CFU/ml, deposited in the collection of the Federal State Budgetary Institution "All-Russian Research Institute of Phytopathology" (FGBNU "VNIIF"),

propionic acid bacteria Propionibacterium freudenreichii B-9653 - up to 1*10 ¹⁰ CFU/ml, as well as Bacillus subtillis B-4506 - up to 1*10 ¹⁰ CFU/ml, deposited in the collection of the Federal State Budgetary Institution "Federal Center for Toxicological, Radiation and Biological Safety" (FGBNU "FCTRB-VNIVI").

To obtain the biological preparation "MicroLife", each strain is first grown.

Strains of Propionibacterium freudenreichii VKPM V-9653 and Enterococcus faecium VKPM V-4489 are grown for 48 hours at a temperature of +37°C in MRS broth to a strain concentration of over 1.0 × 10 ¹⁰ CFU/ml, MRS broth contains (g/ l):

glucose - 20.0;

peptone from casein - 10.0;

meat extract - 8.0;

yeast extract - 4.0;

sodium acetate - 5.0;

disubstituted potassium phosphate - 2.0;

disubstituted ammonium citrate - 2.0;

twin-80 - 1.0;

magnesium sulfate - 0.2 and

manganese sulfate - 0.04.

Separation of bacterial cells from the culture medium is carried out by centrifugation at 8000 rpm for 5 minutes at 12°C under sterile conditions. The cell sediment is washed twice with physiological solution (with the addition of di- and monosubstituted sodium phosphates, which are mixed in quantities necessary to create a pH of 7.2) under the same conditions, they are resuspended and a bacterial suspension is prepared with a titer of each strain of 1.0 × 10 ¹⁰ CFU/ml.

Strains of Lactobacillus plantarum VKPM V-5337 and Lactococcus lactis VKPM V-4989 are grown for 24-48 hours at a temperature of +37°C in MRS broth of the above composition to a concentration of over 3.5×10 ¹⁰ CFU/ml. The separation of bacterial cells of the strain is carried out similarly to the above method. The titer of each strain is 3.5×10 ¹⁰ CFU/ml.

Bacillus subtilis strain VKPM B-4506 with a concentration of 1.0×10 ¹¹ CFU/ml is grown for 24 hours at a temperature of +37°C in Luria-Bertoni broth of the following composition: (g/l):

tryptone - 10.0;

yeast extract - 5.0;

sodium chloride - 5.0.

The separation of bacterial cells of the strain is carried out similarly to the above method. The strain concentration is 1.0×10 ¹¹ CFU/ml. To obtain the liquid biological preparation "MicroLife", the resulting bacterial suspensions are mixed together, including strains Enterococcus faecium VKPM V-4489, Lactobacillus plantarum VKPM V-5337, Lactococcus lactis VKPM V-4989, Propionibacterium freudenreichii VKPM V-9653 and Bacillus subtilis VKPM V- 4506, in equal volumetric ratios and added to a medium containing beet molasses, soy hydrolysate, sodium chloride, nipagin and water, mix intensively and incubate. The total concentration of all bacteria in the biological preparation "MicroLife" must be at least 1*10 ¹¹ CFU/ml, and is achieved within 3 days at a temperature of +37°C (the concentration is controlled in accordance with the requirements for working with pathogens 3-4 pathogenicity group according to SP 1.3.2322-08). The concentration of isolated cultures of microorganisms is monitored by a visual microscopic method in accordance with accreditation (accreditation certificate RA.RU.21PU48). Counting of microorganism cells is carried out under a microscope in a Goryaev chamber (MiniMed, Russia) according to A.I. Netrusova [Netrusov A.I. Workshop on microbiology / A.I. Netrusov. - M.: Academy, 2005. - 608 p.]. If the MicroLife product does not meet the declared quality criteria, it is rejected and not allowed for use.

The appearance of the consortium is a transparent straw-yellow liquid with opalescence and a characteristic odor. There may be a whitish sediment that breaks up when shaken. The finished product is stored in a clean, dry room protected from light at a temperature of +4 to 20°C for 3 months from the date of production.

Application of the biological preparation "MicroLife" carry out in an amount of at least 2.5 ml per 1 ton of phytomass (biomass). In practice, the dose of the drug is calculated based on the concentration of bacteria (titer) per 1 g of preserved biomass. Titer 250 thousand CFU/g of green biomass - this corresponds to 2.5 ml/t of biomass. The concentration of 250 thousand CFU/g of biomass is chosen as the arithmetic mean of the amount of known biopreservatives added per 1 g of preserved biomass - based on an analysis of the titer of existing domestic and foreign biopreservatives (more than 50 items) used in the Russian Federation when preparing feed [Molodkin V. Feed preservatives. Market trends // 04/03/2016. Electronic resource: https://www.tsenovik.ru/articles/obzory-i-prognozy/kormovye-konservanty-tendentsii-rynka/, date of access: 07/07/2022], many drugs have a titer mainly in the range of 200- 300 thousand CFU/g of green mass, but not less than 100 thousand CFU/g [V. Molodkin, 2016].

The biological preparation "MicroLife" is added to the biomass by spraying. To distribute the drug evenly throughout the biomass, the drug is preferably diluted with water in an amount that allows the solution to be evenly sprayed over the biomass, but does not increase the moisture content of the dried biomass. The optimal amount of water is 3 liters per 1 ton of biomass.

Minor changes in temperature during canning (below 8°C and above 18°C) do not lead to a significant disruption of the chemical composition and nutritional value of alfalfa haylage.

It is known that the main (main) fermentation phase when preparing canned feed ends after 21 days [Mitrik T. Silage. Illustrated practical guide / T. Mitrik. 2012. - 81 p.], and this state of stability (preservation) continues until the opening of the trench. For laboratory studies, the preservation period is maintained within 30-45 days, and for semi-production [Methodological recommendations for studying in laboratory conditions the preservative properties of chemicals used in forage ensiling / M.T. Taranov, V.L. Vladimirov, P.A. Naumenko and others - VIZH, Dubrovitsy. - 1983. - 25 p.] it takes up to 45-60 days.

To assess the effectiveness of the proposed drug for canning alfalfa, compare the performance of haylage obtained using the claimed biological preparation "MicroLife" - a prototype, with the performance of haylage obtained by canning alfalfa without the use of starters (preservatives) - a control sample, and with the performance of haylage obtained canning alfalfa with the domestic drug “Bioamide-3” (BIOAMID, Saratov) known on the market - a comparison sample.

Canning is carried out in laboratory and semi-production conditions. The indicators of haylage obtained using the compared preparations (preservatives) in laboratory and semi-production conditions are determined in accordance with [Methodological recommendations for studying in laboratory conditions the preservative properties of chemical preparations used in forage ensiling / M.T. Taranov, V.L. Vladimirov, P.A. Naumenko and others - VIZH, Dubrovitsy. - 1983. - 25 p.].

Determination of dry matter (humidity) in laboratory and semi-production conditions is carried out in a microwave oven according to the method described in the book [L. Durst and M. Wittmann. Feeding farm animals. Per. from German edited by and with a preface. Ibatullina I.I., Provatopova G.V. - Vinnitsa, Nova Kniga, 2003. - 384 p.].

Zootechnical analysis is carried out according to the methods described [Zootechnical analysis of feed: 2nd ed., add. reworked / E.A. Petukhova. - M.: Agropromizdat. - 1989. - 239 p.] using an automated set of instruments from VELP Scientific (Italy).

Microbiological analysis is carried out according to the methods described by E.Z. Tepper [Workshop on microbiology / E.Z. Tepper, W.K. Shilnikova, G.I. Pereverzeva. - M.: Kolos. - 1993. - 175 p.].

Organoleptic evaluation is carried out in accordance with the requirements of GOST [GOSTR 55452-2021. Hay and haylage. General technical conditions. - M.: Standardinform. - 2021. - 12 p.].

Example 1

Research in laboratory conditions is carried out at the Tatar Scientific Research Institute of Agriculture - a separate structural unit of the Federal Research Center of the Kazan Scientific Center of the Russian Academy of Sciences (hereinafter TatNIISKH - OSP FRC KazSC RAS) in accordance with [Methodological guidelines for chemical conservation of feed and testing them on animals / K.M. Solntsev and others - M.: Kolos. - 1980. - 24 p.].

The raw material (green mass) is mown, dried in accordance with the requirements of GOST 55452-2021 to contain 45-55% of the dry matter of the haylage mass of alfalfa of the Guzel variety of the second year of use, grown in the conditions of the suburban farm of the Agricultural Production Complex "Plemennoi Zavod im. Lenin" Atninsky district of the Republic of Tatarstan.

The green mass is mowed in dry weather using a self-propelled mower-conditioner Class disco 9300 (Germany) and wilted until reaching 45-55% dry matter (humidity 55-45%). Determine the dry matter content in biomass % according to the method [L. Durst and M. Wittmann. Feeding farm animals. Per. from German edited by and with a preface. Ibatullina I.I., Provatopova G.V. - Vinnitsa, Nova Kniga, 2003. - 384 p.]. The dried mass is selected using a self-propelled forage harvester "Jaguar 850" (Germany) with a grinding of 2-3 cm. Then the biomass is divided into three equal parts of 12 kg on the basis that three repetitions are carried out for each compared experiment in dynamics of 3, 7, 15th and 30th days of aging. In the conditions of the veterinary laboratory, 12 kg of dried alfalfa biomass are weighed on mechanical scales VT 8908-50N (IVZ, Russia) and spread in a thin layer on the table.

In laboratory conditions, to ensure uniform distribution of drugs throughout the biomass, the required dose of the drug is mixed with the calculated amount of distilled water in a measuring cup. The solution is thoroughly mixed and sprayed evenly using a medical syringe. 36 ml of distilled water is sprayed onto 12 kg of wilted alfalfa biomass at the rate of 3 liters of water per 1 ton of biomass.

The MicroLife drug is added to one part of the biomass, for which 36 ml of distilled water is poured into a glass and the required dose of the drug is added dropwise at the rate of 2.5 ml per 1 ton of biomass - 30 μl. The solution is then mixed, filled into a syringe and sprayed onto the biomass.

The second part of the biomass is sprayed with an aqueous solution of the drug “Bioamide-3” in an amount according to the instructions for use [https://bioamid.com/netcat_f1les/l8/54/h_b94e3alf27410d4add3169b4e0213129, access date 01/18/2023], for which in 36 ml of distilled 18 mg of the drug is dissolved in water at the rate of 1.5 g per 1 ton of biomass.

The third part of the biomass acts as a control; distilled water is added to it in an amount of 36 ml per 12 kg of biomass.

All biomass is thoroughly manually mixed, divided into 1 kg portions and packaged using a Redmond rvs-m 020 vacuum packer (China) into sealed vacuum bags (Redmond, China) supplied by the manufacturer (Redmond, China). A total of 36 packages - 12 packages each for haylage obtained with the use of the compared drugs and without the drug. The packaged packages are kept in special darkened unheated semi-underground concrete vat pits at a temperature of +8-18°C. The quality indicators of the compared haylages in dynamics are determined on the 3rd, 7th, 15th and 30th days of aging; for this purpose, the packages are opened and the compared haylages are analyzed. For the mathematical reliability of the results, the indicators of the haylage samples obtained using the compared drugs are determined as the arithmetic mean of the indicators from three packages (three times) for each compared drug on the 3, 7, 15 and 30 days of aging. The average quality indicators of compared alfalfa haylage obtained in laboratory conditions are presented in Tables 1 and 2 - acidity (pH), total amount of organic acids, as well as the amount of lactic, acetic and butyric acids - in Table 1, chemical composition and nutritional value (metabolizable energy and amount of sugars) per 1 kg of haylage - in table 2.

An organoleptic assessment of the compared haylage samples demonstrates that all samples have a pronounced smell of pickled fruits and vegetables without signs of mustiness, are yellow-green in color, and without mold. The structure of the stems and leaves is preserved, the consistency is not smearable. It has been established that a significant difference in acidity is achieved (pH) from the 7th to the 30th day of fermentation in samples using biological preparations and is respectively 0.18-0.20 (on the 7th day), 0.17-0.12 (on the 15th day) and 0.26-0.28 (on the 30th day) units compared to control values. The amount of organic acids already on the 3rd day of aging reaches its maximum concentration and is 7.97 abs.% for the sample using the Bioamide-3 preparation, 7.95 abs.% for the sample using the MicroLife preparation, versus 2. 50 abs.% in the control sample. During the aging process, the concentration of organic acids tends to decrease, and on the 30th day of fermentation it is: for a sample using the Bioamide-3 preparation - 6.73 abs.%, for a sample using the MicroLife preparation - 6, 20 abs.%, and exceeds the value for the control sample by 1.01 and 0.48 abs.%, respectively. A similar pattern is observed in the accumulation of lactic acid in haylage samples.

It should be noted that the concentration of acetic acid in all samples using the MicroLife drug from the 3rd to the 30th day of aging is higher by 0.23-0.42 abs.% compared to control samples and by 0.08 -0.38 abs.% compared to samples using the Bioamide-3 preparation, which indicates a high guarantee of “aerobic stability” of haylage during its removal.

The chemical composition and nutritional value of experimental alfalfa haylage over time, depending on the canning preparation used, are presented in Table 2. The content of dry substances in the samples obtained with the MicroLife preparation exceeds the same control values on the 3rd day of aging by 1.71% ( 50.40), on the 7th day by 2.48% (50.48), on the 15th day by 2.34% (50.24) and on the 30th day by 2.74% (50.40). 24). And in relation to the samples obtained using the drug “Bioamide-3”, the excess of the dry matter values in the indicators is 0.63-1.36%, with the exception of the 7th day, where the dry matter content is 5.85 % (50.81).

During the fermentation process, the biological preparation "MicroLife" helps to increase the preservation of crude protein in haylage. The level of crude protein on the 7th day for the sample obtained using the MicroLife drug exceeds the value of the control sample by 0.62%. With longer aging, the level of crude protein in samples using the MicroLife drug exceeds that in both the control sample and in samples using the Bioamide-3 drug by 0.30-0.31% and 0.04-0. 09% respectively. The level of crude fiber for the entire period of aging in the test haylage samples is higher than the control values - for samples using the Bioamide-3 preparation by 0.18-0.55%, for samples using the MicroLife preparation - by 0.06- 0.85%.

As for the fat content in the obtained haylage samples, this indicator on the 3rd day of aging in all compared samples was almost the same. The level of crude fat in control samples on the 7th, 15th and 30th days was 1.62-1.63%. On the 30th day of storage, the advantage in this indicator was with the experimental sample (2.39%) and the comparison sample (2.09%), the difference being 0.47 and 0.77%, respectively, compared to the control.

The content of nitrogen-free extractive substances (NFES) in the compared samples of haylage prepared with biological preparations is higher than the values of control samples. For samples obtained using the MicroLife drug, this difference in dynamics was: on the 3rd day - 0.91%, on the 7th day - 0.44%, on the 15th day - 0.58% and on 30 day - 3.28%, for samples obtained using the drug "Bioamide-3" - 0.94; 0.03; 0.41 and 2.55% respectively.

The level of total sugars in the studied haylage samples, which is characterized as the main energy source for the life of microorganisms, tends to decrease depending on the duration of aging and the preservative used. In general, the maximum content of the amount of sugars is detected on the 3rd and 7th days of aging in experimental variants, which is 28.71-25.44 mg/kg and 29.23-25.24 mg/kg, respectively, against 26.78 mg/kg and 24.00 mg/kg in the control, or higher than the control value by 7.21-6.00% and 9.52-5.17%.

Changes in the chemical composition of haylage during its maturation also affected the amount of metabolic energy. Thus, if in the experimental samples with the MicroLife drug during the period from 3 to 30 days of aging the metabolic energy indicators fluctuated in the range of 4.30-4.65 MJ/kg, then in the comparison samples the indicators turned out to be slightly lower than 4.25-4. 56 MJ/kg, versus 4.16-4.26 MJ/kg in the control. When opening the haylage on the 30th day of aging, the difference in this indicator compared to the control (4.16 MJ/kg) turned out to be maximum - in samples with MicroLife preparations it was higher by 11.78% (4.65 MJ/kg), in samples with the drug “Bioamide-3” it is higher by only 9.62% (4.56 MJ/kg).

The nutritional value of the feed is calculated based on the metabolic energy indicator; the higher the indicator, the higher the quality of the resulting feed. From the 15th to the 30th day of aging, the indicators of metabolic energy in the test samples - 4.36 and 4.65 MJ/kg exceed the indicators of the comparison samples - 4.61-5.54% and control samples - 4.19-4, 19%, which makes the claimed drug more effective compared to the drug “Bioamide-3” for canning alfalfa without the use of starter cultures.

The quality indicators of the compared haylages in dynamics, obtained when using the compared drugs on the 3rd, 7th, 15th and 30th days, demonstrate that the process of fermentation of haylage with the use of the claimed drug "MicroLife" proceeds a little faster than with the use comparison drug, and significantly faster compared to the control. In addition, the fermentation time of haylage with the MicroLife preparation is significantly less than that indicated in the recommendations currently used. Thus, the data in tables 1 and 2 demonstrate that the characteristics of haylage obtained after 7 days of aging do not differ much from the characteristics of silage obtained after 15 and 30 days of aging. The acidity indicator (pH) on the 30th day compared to the indicator on the 7th day changed slightly - it decreased by 0.27 units. The content of lactic and acetic acids on the 30th day decreased by only 0.4% and 0.08%, respectively, compared to the indicators on the 7th day. The content of crude protein on the 30th day decreased by 0.39%, crude fiber - by 0.32%, crude fat - by 0.06%, BEV - by 2.6%, metabolic energy - by 0.35% compared with the indicators on the 7th day. The only indicator that shows a significant change is the amount of sugars - 25.24 g/kg on the 7th day, 19.62 g/kg on the 15th day and 18.00 g/kg on the 30th day.

The fermentation process of haylage with the MicroLife preparation proceeds more intensively, where after 7 days of aging the acidity level is 4.79 units. with a sufficient concentration of organic acids for further preservation, which is confirmed by the results on the 3rd, 7th, 15th and 30th days of fermentation. Despite the fact that from the data presented it can be concluded that the use of the proposed drug "MicroLife" for canning alfalfa after a week of fermentation makes it possible to obtain haylage with good potential characteristics, but the silage must be kept for at least 30 days after laying in accordance with GOST 55452- 2021.

Example 2

To assess the effectiveness of the proposed drug for canning alfalfa in semi-production conditions, compare the indicators of haylage obtained by the claimed biological preparation "MicroLife" - a prototype, with the indicators of haylage obtained by canning alfalfa without the use of starters (preservatives) - a control sample, and with the indicators of haylage, obtained by canning alfalfa with domestic and foreign preparations known on the market such as: “Bonsilage Alpha” (Schaumann Agri, Brunn am Gebirge, Austria), “Bioamide-3” (BIOAMID, Saratov), and “Lactis” ( Institute of Crimea Agrotechnology, Simferopol) - comparison samples.

In semi-production conditions, comparisons of preparations for canning alfalfa are carried out on the basis of LLC named after. Timiryazev, Baltasinsky district of the Republic of Tatarstan.

The green mass of alfalfa of the Guzel variety of the second year of use is mowed in the beginning of budding phase using a self-propelled mower-conditioner "Polesie KPR-9" (Belarus), the mass is selected, dried according to GOST 55452-2021 to a haylage moisture content of 45-55%, crushed to a length of 2- 3 cm with a Jaguar 830 forage harvester (Germany). Biomass humidity is controlled using the express method [L. Durst and M. Wittmann. Feeding farm animals. Per. from German edited by and with a preface. Ibatullina I.I., Provatopova G.V. - Vinnitsa, Nova Kniga, 2003. - 384 p.]. The resulting haylage mass is distributed into five equal parts weighing 75 kg each, weighed on a mechanical scale VT 8908-50N (IVZ, Russia) and distributed in a thin layer on a concrete area covered with film (for example, polyethylene).

In semi-production conditions, to ensure uniform distribution of the compared drugs throughout the biomass, the dried biomass is sprayed with drug solutions using a universal trigger for spraying liquid SONAX ProfiLine 0.5 l (Germany). For this purpose, distilled water is mixed in the trigger container with the appropriate preparation in the required ratio, taking into account that for 1 ton of dried alfalfa biomass, optimally 3 liters of water (for 75 kg - 225 ml of water) are thoroughly mixed and the solutions of preparations are evenly sprayed over the biomass.

A solution of the MicroLife drug is added to one part of the biomass, for which 225 ml of distilled water is poured into the trigger container and the dose required at the rate of 2.5 ml of the MicroLife drug per 1 ton of biomass is added dropwise - 187.5 μl. The container is closed, the solution is mixed and the first part of the haylage biomass is sprayed.

A solution of the drug “Bonsilage Alpha” is added to the second part of the haylage biomass, obtained by dissolving 150 mg of the drug in 225 ml of water. The amount of the applied drug is 2 g of the drug per 1 ton of biomass according to the instructions for use [https://schaumann.ru/product/bonsilazh-alfa, access date 01/18/2023].

The third part of the haylage biomass is treated with a solution of the drug “Bioamide-3” (112.5 mg of the drug in 225 ml of water). The amount of the drug applied according to the instructions for use [https://bioamid.com/netcat_files/ 18/54/h_b94e3a1f27410d4add3169b4e0213129, access date 01/18/2023] is 1.5 g of the drug per 1 ton of biomass.

A quarter of the biomass is sprayed with a solution of the drug “Lactis” - 150 μl of the drug, 225 ml of water. The amount of the drug is 2 ml per 1 ton of biomass in accordance with the instructions for use [https://www.lactis-agro.ru/index.php/2017-01-26-16-11-11/about-lactis/use, date of access: 01/18/2023].

A fifth of the dried biomass acts as a control - 225 ml of water without a preservative is added to it.

To ensure uniform distribution of drug/water solutions, each part of the biomass is thoroughly mixed and divided into 3 more equal parts. For the mathematical reliability of the results, the indicators of the haylage samples obtained using the compared drugs are determined as the arithmetic mean of the indicators from three packages (three times repeated) for each compared drug. All biomass is placed in hermetically sealed, heavy-duty, two-layer plastic bags with ties from Liberhaus praktischc producte (Biosphere Polymer LLC, Russia) with a volume of 60 liters (15 bags). After displacing the air, the filled bags are laid in a row in a haylage trench with a volume of 1000 tons to a depth of 1.5 m in order to create fermentation conditions similar to production conditions at the average long-term positive ambient temperature. After opening the trench after 45 days of aging, haylage samples are subjected to zootechnical and microbiological studies.

The average quality indicators of compared alfalfa haylage obtained under semi-production conditions are presented in Tables 3 and 4 - acidity (pH), total amount of organic acids, as well as the amount of lactic, acetic and butyric acids - in Table 3, chemical composition and nutritional value (metabolizable energy and the amount of sugars) per 1 kg of haylage - in table 4. The data indicate that the acidity (pH) of the haylage samples for the test samples and comparison samples do not differ significantly from each other, and the pH values are 4.73-4.78 units . compared to 4.92 units. in the control sample.

The maximum accumulation of organic acids as a result of enzymatic processes occurs in haylage samples obtained using the Bioamide-3 preparation and the MicroLife preparation; these figures are 6.06 and 5.70 abs.%, respectively, against 5.03 abs. .% in the control sample. In the haylage sample obtained using the Bonsilage Alpha preparation, the content of organic acids is 4.49 abs.%, which is 0.54 abs.% lower than the value of the control sample. Such differences were also reflected in the concentration of lactic acid in the compared samples. So, if in samples of haylage using the drug "Bioamide-3" and using the drug "MicroLife" the concentration of lactic acid is 5.57 and 5.06 abs.%, respectively, then in samples of haylage using the drug "Bonsilage Alpha" and using the drug "Lactis" - 3.91 and 4.74 abs.%, respectively. However, samples of haylage using the drug "Bonsilage Alpha" and using the drug "MicroLife" are distinguished by an increased content of acetic acid - respectively 0.57 and 0.64 abs.% versus 0.40 abs.% in the control sample, with a difference of 0 .17 and 0.24 abs.%, respectively. This indicator indicates the high “aerobic stability” of the sample during its aging and removal.

Canning alfalfa with different biological preparations has different effects on the chemical composition and nutritional value of the resulting haylage samples. The indicators for the chemical composition and nutritional value presented in Table 4 demonstrate that the most dry matter when preserving biomass is contained in samples of haylage obtained using the drug "Bonsilage Alpha" and using the drug "MicroLife", the values are 50.95 and 50 .92% versus 48.92% in the control sample with a difference of 2.03 and 2.00%, respectively.

A similar picture is revealed by the content of crude protein in haylage samples obtained using the drug "Bonsilage Alpha" (9.68%) and using the drug "MicroLife" (9.62%), where the protein level is higher by 0.40 and 0 .34%, respectively, compared to the control sample (9.28%). As for the crude fiber content, no significant differences were found among the compared samples.

The level of crude fat for a given period of silage aging was higher in haylage samples obtained using the Bioamide-3 preparation (1.40%) by 0.09%, and with the use of the MicroLife preparation (1.58%) - by 0 .27% compared to the control sample (1.31%).

In samples of haylage obtained using the drug "Bonsilage Alpha" and using the drug "MicroLife", the maximum content of BEV was revealed, which was 17.35 and 17.08%, which is higher than the control value by 1.17 and 0.90%, respectively. .

Calculations of the energy value of haylage, based on the results of the actual nutrient content, show that the maximum values of metabolizable energy in 1 kg of natural feed are found in samples of haylage obtained using the MicroLife drug and using the Bonsilage Alpha drug, which is 4.47 and 4.44 MJ, which is higher than the control sample by 5.2% and 4.5%, respectively. In the haylage samples obtained using the Bioamide-3 preparation and the Lactis preparation, the difference in these indicators was only 2.6 and 2.1%, respectively.

There was no significant difference in the amount of sugars during canning of the dried alfalfa mass in the test sample and in the comparison samples (10.73-12.64), while these indicators were lower by 17.6-38.6% compared to the control sample (14.87). The amount of sugars gradually decreases. This is a natural process, since with the additional introduction of highly active artificially selected lactic acid and other microorganisms into the haylage mass, in order to achieve the optimal pH concentration of the environment, they intensively use feed sugars for their life as an energy source of nutrition, thereby leading to a decrease in its level in the finished products. haylons.

To calculate the concentration of bacteria of various physiological groups, use [Workshop on microbiology / E.Z. Tepper, W.K. Shilnikova, G.I. Pereverzeva. - M.: Kolos. - 1993. - 175 p.]. The concentrations of various physiological groups of microorganisms in alfalfa haylage are presented in Table 5. The maximum values for the total microbial number (TMC) - 9.20*10 ⁶ CFU/g and 10.03*10 ⁶ CFU/g were detected in the control sample and in the sample with using the drug "Lactis". In other samples, these indicators are lower than for the control sample by 22.89-28.08%. According to the concentration of lactic acid bacteria - the most necessary for the fermentation processes of alfalfa haylage biomass, it is noted that the concentration of lactic acid bacteria is significantly higher in the test sample (3.16 times), in the comparison samples (2.14-2.97 times) compared to the control sample. The highest numbers of lactic acid bacteria were observed in haylage using the MicroLife preparation and using the Bonsilage Alpha preparation, which is 3.16 and 2.97 times higher, respectively, compared to the control sample (17.66 * 10 ⁶ CFU/g) .

A similar picture is observed with regard to the number of propionic acid bacteria. So, if in samples obtained using the drug "MicroLife" and using the drug "Bonsilage Alpha", their number is 6.00 * 10 ⁶ CFU/g and 5.20 * 10 ⁶ CFU/g, then using the drug "Bioamide-3" and using the drug "Lactis" - 4.33*10 ⁶ CFU/g and 4.67*10 ⁶ CFU/g, which is 2.03 higher than the control sample; 1.76; 1.46 and 1.58 times respectively.

All compared haylage samples contain undesirable facultative aerobic microorganisms - yeast. The lowest values for this indicator are samples of haylage using the drug "MicroLife" and using the drug "Bonsilage Alpha" - 0.76 * 10 ⁴ CFU / g and 0.97 * 10 ⁴ CFU / g, which is 80 lower than the control sample. 51% and 75.13% respectively. For haylage samples using the Bioamide-3 preparation and using the Lactis preparation, this difference is 63.33% and 49.49%, respectively, compared to the control sample (3.90*10 ⁴ CFU/g).

An assessment of the economic effectiveness of compared preparations for canning alfalfa is carried out by comparing the cost of haylage presented in Table 6, as well as the cost of feed quality indicators, such as metabolic energy, crude protein and the amount of sugars.

The data in Table 6 indicates that the most expensive is the sample of haylage obtained using the drug “Bonsilage Alpha”, the cost of 1 ton of which is 1208 rubles, which is 14.2% higher than the cost of the control sample (1058 rubles).

The cost per unit of metabolic energy and crude protein is also higher compared to the control sample for haylage samples obtained using the Bonsilage Alpha preparation, by 9.2%, and by 0.4%, respectively, when using the Lactis preparation. At the same time, the indicated indicator of the prototype is 3.6% lower than that of the control sample.

A similar trend is observed in relation to the cost of 1 g of preserved sugars. So, if in samples of haylage obtained using the drug "Bonsilage Alpha" and using the drug "Lactis", these indicators are 0.113 and 0.093 rubles, which is higher than the control sample by 30.9% and 59.2%, respectively, then indicators of haylage obtained using the drug "Bioamide-3" and using the drug "MicroLife" - 0.086 and 0.081 rubles. respectively, which is higher than the control sample by 21.1% and 14.1%.

Thus, the use of the biological preparation “MicroLife” for canning alfalfa is the most effective compared to the use of known biological preparations.

The declared biological preparation "MicroLife" for canning alfalfa has a positive effect on the overall nutritional value of the resulting haylage, the concentration and ratio of organic acids, the development of desirable physiological groups of microorganisms in the finished feed, as well as their “aerobic stability” during harvesting. At the same time, the dry matter content is higher by 2.0%, crude protein by 0.34% and crude fat by 0.27% compared to the control. In terms of energy value, this difference was 0.22 MJ or 5.2%.

The cost per unit of metabolizable energy and crude protein is lower by 3.6 and 1.8%, respectively, compared to the values of the control sample.

Thus, the claimed biological preparation “MicroLife” for canning alfalfa ensures high quality of the resulting feed during the entire period of aging with high energy and protein value, a short readiness period, and also guarantees “aerobic stability” when it is removed and fed to farm animals.

The declared biological preparation "MicroLife" for preserving alfalfa biomass expands the arsenal of preparations for preserving difficult-to-silage crops and can be actively used in the modern production of canned feed, dairy and beef cattle breeding.

Claims (1)

Composition for canning alfalfa for the production of feed for farm animals, characterized by the fact that it consists of strains with the following concentrations of CFU/ml: rod-shaped Lactobacillus plantarum VKPM V-5337 - up to 3.5 * 10 ¹⁰ CFU/ml and coccal form of Lactococcus lactis B-4989 – up to 3.5*10 ¹⁰ CFU/ml, Enterococcus faecium B-4489 – up to 1*10 ¹⁰ CFU/ml, propionic acid bacteria Propionibacterium freudenreichii B-9653 – up to 1*10 ¹⁰ CFU/ml, as well as Bacillus subtillis B-4506 – up to 1*10 ¹⁰ CFU/ml, taken in equal volume fractions.

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