RU2202224C2 - Method of preparing biologically active fodder additive from vegetable raw material - Google Patents

Abstract

FIELD: fodder production, particularly, methods of preparing biologically active additives for animals, poultry and fish. SUBSTANCE: method involves introduction in basic fodder of biologically active additive prepared by solid-phase or flow-through fermentative process of treatment of cellulose-containing raw material, including cellulose-containing wastes, under optional-anaerobic conditions. For fermentative hydrolysis of vegetable raw material used is preliminarily prepared monoculture or complex of nitrogen-fixing, cellulolytic and associated microorganisms. Complex of microorganisms is separated from sections of digestive tract, and/or from fresh excrements of herbivorous animals. Efficiency of preparation is increased by introduction of additional strains of microorganisms and/or introduction of selenium. EFFECT: increased efficiency of digestion process, higher sorptive properties. 5 cl, 7 tbl, 12 ex

Description

 The invention relates to feed production, in particular to methods for the preparation of feed for mammals, birds and fish, containing digestion enhancing and therapeutic additives, sorbents based on the processing of cellulose-containing raw materials or waste.

State of the art
The issues of fodder production in agriculture, poultry and fish farming, as well as when keeping animals at home and in zoos, are relevant and are addressed in several areas. These include i) the traditional search for optimal feed mixtures using vitamin and other mineral supplements; ii) the search for technical solutions to obtain feed from plant waste; iii) the inclusion in the composition of traditional feed of biological products obtained using biotechnological methods and contributing to the digestion of roughage; iv) the inclusion in the feed of preparations for prevention or treatment; v) the search for new integrated methods that simultaneously solve several problems of feed production and therapeutic prevention.

 Known methods for pre-treatment of coarse cellulosic plant material and waste [1], processing of coniferous waste [2] for their use in feed production.

 The disadvantages of the methods include either high energy consumption when processing straw with steam, or a longer duration (within 23 days) of fermentation of wood waste using mycelium fungus.

 Reducing the processing of roughage is done by using pre-prepared starter cultures based on the contents of the ruminant rumen [3] or by sequentially converting cellulose-containing wastes first under aerobic conditions with at least one type of microorganism, then under anaerobic conditions with other types of microorganisms [4] . Such feeds do not belong to the class of therapeutic and prophylactic, as their main goal is to contribute to the most complete processing of cellulose.

 The invention is known in which an increase in the growth of live weight of cattle is achieved by increasing the processing of roughage and fiber in the digestive tract of animals. For this purpose, an association of microorganisms is isolated from the calf scar, the main criterion of which is the rapid breakdown of cellulose [5]. The resulting association is fed in liquid form in addition to the main feed. This method of feeding will not be highly effective when feeding monogastric animals, since the bulk of the microorganisms will die when passing through the stomach of animals.

 Known methods for the preparation of feeds, in which, as therapeutic and prophylactic preparations that increase the biological value of feed, protein-vitamin-mineral feed mixtures are used [6].

 A known method for the preparation of therapeutic and preventive food for animals, including fish [7], according to which a probiotic based on Bacillus bacteria is introduced into the feed composition.

 Known feed additive for feeding chickens, in which as a therapeutic and prophylactic agent and in order to increase the live weight of broilers, the ANTA-1 biological product is used. The composition of the biological product includes a specially selected composition of 6 types of microorganisms grown on liquid corn extract [8]. A method of obtaining a biological product is associated with the directed selection of strains taken with a specific titer and growth phase for a particular type of substrate. The possibility of using a biological product for the treatment of cellulose-containing waste and its effectiveness for growing other animals is not known.

 Biological products are known that are administered as a feed additive for the treatment of gastrointestinal diseases of animals. The main elements that make up biologics are individual microorganisms or complexes of microorganisms contained, for example, in animal excrement [9].

 Known use of cellulolytic bacteria in feed production [1]. Cellulolytic bacteria were isolated from soil, rotting plants, as well as silage by plating on Hutchinson's agar medium, where straw broth was used as the sole carbon source.

 Closest to the invention under consideration is the method [1]), in accordance with which enzyme hydrolysis of crushed plant material is used to obtain feed by introducing into it a preliminary preparation based on the contents of the caecum of the common vole. The drug is obtained by growing the bacterial flora on a cellulose-containing substrate. After that, the drug is introduced into pre-ground plant material, placed in a nylon bag, and fermented while continuously washing the mixture with running water.

 The disadvantages of this method are the conduct of the process by bacteria isolated from only one source, and possibly not the most effective, as well as the implementation of fermentation under flowing conditions. The latter requires additional energy costs associated with mixing, ensuring the flow of water. The process is accompanied by a significant consumption of water. In addition, waste water is generated as a waste product.

SUMMARY OF THE INVENTION
The technical result of the invention is to increase the digestibility of feed when introducing into the diet of farm animals, poultry, fish, domestic animals biologically active feed additives, as well as reducing the cost of feed mixtures.

 To achieve a technical result in the preparation of a biologically active feed additive based on plant materials using one culture or a mixture of cultures of microorganisms, the selection of bacterial flora is carried out from various sections of the digestive tract or excrement of herbivorous animals, while the efficiency of microorganisms can be increased by introducing at least one additional strain of microorganisms and / or additional introduction of trace elements.

 An additional aspect of the invention relates to the fact that the selection of bacterial flora is carried out from various parts of the digestive tract of animals: the oral cavity, or goiter, or the pancreas, or different parts of the intestine, for example, the cecum or colon, or excrement.

 The effectiveness of the drug can be improved by introducing at least one additional strain of microorganisms selected from the group of microorganisms belonging to the genus: Bacillus, Clostridium, Lactobacillus, Streptococcus, Micrococcus, Ruminococcus, Proteus, Enterobacter, Escherichia, Klebsiella, Pseudomonard, Pseudomonard, Pseudomonard, Thermomonospora.

 To increase the effectiveness of the drug, a trace element, such as selenium, can be included in it.

 Obtaining biologically active feed additives is carried out in the following way.

 Samples from the digestive tract or feces of herbivorous animals are taken, which include microorganisms. Isolation of at least one culture or association of microorganisms is carried out by incubating the isolated samples in a nitrogen-free medium containing cellulose. The association of microorganisms may additionally include at least one separate strain of microorganisms belonging to the genera Bacillus, Clostridium, Streptococcus, Micrococcus, Ruminococcus, Proteus, Enterobacter, Escherichia, Klebsiella, Pseudomonas, Micromonospora, Thermoactinces. The resulting complex of microorganisms is maintained by propagating it in appropriate media. The plant material or its waste is pre-crushed and mixed with the resulting complex of microorganisms.

A biologically active product is obtained by subsequent fermentation of the mixture by flow or solid-phase method under optional anaerobic conditions at a temperature of 15-70 ^o C.

 For the introduction into industrial production, it seemed necessary to select an association of microorganisms that is characterized by high rates of growth, cellulolytic activity, population resistance and competitive ability, as well as resistance to various adverse factors.

 The task was to isolate strains of microorganisms producing cellulolytic enzymes that meet these requirements.

 An association is isolated from one or more species of herbivorous animals. The selection of the bacterial flora is carried out: i) from one part of the digestive tract; ii) from several parts of the digestive tract; iii) from samples of excrement; iv) from the digestive tract and from samples of excrement, iiv) from the digestive tract and / or from samples of excrement in combination with collection strains. In cases where microorganisms are isolated from different parts of the digestive tract, depending on the type of herbivorous animals, microorganisms are isolated from the contents of the animal's oral cavity (including gland secrets), and / or goiter, and / or pancreas, and / or intestines, for example, cecum, colon. The excrement group includes feces and cecotrophs.

 To increase the efficiency of processing roughage with the help of isolated microorganisms, it is preferable to select species of herbivorous animals that, in accordance with feeding conditions, produce the necessary complex of bacteria. For this purpose, animals are preliminarily kept on a diet, which includes at least 20% and not more than 40% (by weight) of roughage. To increase the nitrogen-fixing microorganisms in the complex, it is advisable to use feeds with a minimal amount of protein nitrogen, for example, dandelion leaves, apples, etc. in the last phase of feeding.

 Of the animals, it is preferable to use representatives of monogastric mammals (rabbits, hares, pikas, damans, hutia, etc.) or polygastric mammals (sloth, elk, wild species of rams and goats, camel, alpaca, etc.). Wild animals must be quarantined. And, if they are rare species, then methods of selecting microorganisms that do not injure animals are used, for example, only samples of fresh excrement are taken.

 This invention does not limit the list of species of herbivorous animals living in different climatic zones and adapted to digest coarse plant feed.

 For food produced in the European Region, from the group of wild animals, it is preferable to use representatives of widespread and pest-related green-eating rodents, for example, red forest, gray dark, common gray, East European voles, hares, etc. Capercaillies are most promising from birds , black grouse, partridge. From reptiles, turtles, iguanas are promising.

 It is preferable to select separately the sources of allocation of groups of microorganisms for different types of animals, birds, fish. In particular, for birds, pigs, fur animals, it is preferable to use complexes of bacteria from animal donors: green-eating rodents, hare-like, damans, etc. For agricultural ruminants, for example cows, groups of microorganisms from marsupials, not-toothed (in particular, sloth) are preferably used and wild ruminants.

 Highly active nitrogen-fixing cellulolytic complexes of intestinal bacteria are isolated from the contents of the cecum and colon of the forest red vole, gray voles - dark, ordinary, Eastern European, rabbit. In a rabbit, in addition, a complex of bacteria is isolated from cecotrophs. In rodents - from soft feces. In a sloth, a complex of bacteria is isolated from fresh feces.

 The isolation of a self-sustaining complex of microorganisms from herbivorous animals is carried out by incubating it in at least one nitrogen-free medium containing cellulose as the sole carbon source.

 As one example, which is not limited by the choice of media, the following are examples of using standard Ashby medium in a modified form using ground pulp as the sole carbon source. These examples do not limit the use of other nitrogen-free media known from literature and patent sources.

Example 1. Samples, which include one type of microorganism, for example, Bacillus subtilis B-8130, isolated from the cecum of capercaillie, are incubated in Ashby liquid nitrogen-free medium containing crushed filter paper up to 2-15% as the sole carbon source (weight / volume), or another cellulose-containing fine-grained substrate, for example, unbleached pulp, crushed husks of sunflower seeds, millet, buckwheat, rape, crushed coconut fruit. Incubation is carried out at a temperature selected from 18-70 ^o C. For the incubation, optional anaerobic conditions are used. The incubation process is carried out in a period from 5 to 72 hours. The development of microorganisms is judged by microscopic control with an increase in x1000 smears of cultural contents stained in the flame of the burner stained with carbolic genzian violet, as well as cellulase activity (at least 90 units / g of dry fermentation product determined by the viscometric method).

Example 2. An association of microorganisms is obtained from the red-field vole pre-stomach, then the resulting complex is propagated in a larger volume, for which the complex is transferred to Ashby liquid medium, in which 2% sucrose and up to 5% pulped pulp are added. It is possible to add 0.2% yeast extract to this mixture. Incubation is carried out at a temperature selected from the range of 18-70 ^o C, most preferably at a temperature selected from the range of 35-37 ^o C. For incubation, optional anaerobic conditions are used, for which they form a high layer of the fermentable mixture or conduct the process in filled at least than on 3/4 containers closed with caps, etc. The incubation process is carried out for a period of 5 to 48 hours.

Example 3. A monoculture or a complex of microorganisms is grown, as shown in the first and second examples, and at the same time at least one of the bacterial strains (for example, Klebsiella pneumoniae VKPM B-8049) is propagated at a temperature of 35-37 ^o C in optional anaerobic conditions from 24 to 48 hours on Ashby solid medium with 2% sucrose. For the subsequent formation of a complex of grown microorganisms, the Klebsiella microorganism is washed off from Ashby solid medium and the indicated wash is added to Ashby liquid medium with the grown complex of microorganisms on cellulose.

 Example 4. In the industrial preparation of the drug, it is possible to use an association of bacteria designed for a certain type of plant material. For example, use the association of bacteria isolated from the cecum of the common vole, consisting of Streptococcus mitis VKPM B-8048, Klebsiella pneumoniae VKPM B-8049, Proteus mirabilis VKPM B-8047, Bacillus macerans VKPM B-8050, for enzymatic treatment of straw, seed husks sunflower, rice, millet, buckwheat, coconut fruit, etc. This association was deposited in the All-Russian collection of industrial microorganisms on November 28, 2000. In the process of incubation of the association of microorganisms on nitrogen-free media with a single carbon source - crushed cellulose due to the self-organization of microorganisms, the numerical ratio of different forms of microorganisms entering the association in an effective amount sufficient for the life of the association is established.

 Example 5. A method of obtaining a biologically active additives.

 In the present invention, a ground substrate containing cellulosic fibers is subjected to an enzymatic hydrolysis process. The substrates are selected from the group of: i) plant materials grown on land or in the aquatic environment; ii) cellulose-containing waste (husks of sunflower seeds, buckwheat, millet, oats, rapeseed, corn cobs, chopped rye, wheat straw, unbleached pulp, coconut fruit, etc.); iii) waste from medicinal plants; iv) bark, leaves and sawdust of trees, for example, with a pronounced therapeutic focus (oak, eucalyptus, sea buckthorn, lemongrass, etc.); v) combinations of items i), ii), iii), iv).

 To carry out a more efficient process of enzymatic hydrolysis, cellulose-containing raw materials or wastes are pre-crushed into small fractions, from 60 to 85% of the sizes of which do not exceed 1 mm.

Fermentation is carried out in a solid-phase manner, for which crushed raw materials and / or waste are used. Water is added to the air-dry substrate to a final moisture content of 50-70%, then 2 to 10% (vol.%) Of inoculum containing at least 10 ⁹ cells / ml is added, mixed with crushed raw materials and / or waste and placed in optional anaerobic conditions at a temperature of 18-70 ^o C. Optional anaerobic conditions are created using, for example, closed plastic bags or sealed plastic, metal or wooden containers or specially designed containers. The fermentation process is carried out in the temperature range from 18 to 70 ^o C for 5-48 hours until the cellulase activity in the substrate will be at least 90 u / g of dry fermentation product.

 The introduction of selenium into the reaction mixture in organic form in the form of a DAFS-25 drug in a regulated dose increases the activity of endoglucanases by 30%.

 The obtained biologically active additive is used in the preparation of various types of feed. Its amount is from 1 to 10% of the total amount of feed, by weight, depending on the type and age of the animal for which this supplement is used. It is possible to replace a biologically active additive with an appropriate proportion of the individual components of the feed. The feed may be prepared in dry or wet form, or in suspension. When preparing the feed in dry form, it is preferable to granulate the feed by known methods after adding the additive and mixing. When preparing the feed in the form of a suspension, it is necessary to take into account the periods at which the additive retains its biological activity.

 In order to increase the biological activity of the feed, the microelement selenium can be added to the obtained non-granulated feed in an inorganic or organic form, for example, a 0.5-1.0% DAFS-25 solution in vegetable oil, in a dose authorized for use. DAFS-25 can also be introduced into the fermentable mixture before starting the process of obtaining a feed additive. Food using DAFS-25 is used for adults and young animals.

 Example 6. A self-sustaining complex of microorganisms is isolated from the feces of an ordinary vole. This complex is treated with rye straw and the composition of the drug is determined.

 Example 7. A self-sustaining complex of microorganisms is isolated from the soft feces of an ordinary vole. This complex is treated with husk of sunflower seeds and rye straw and the binding of metal ions is determined due to the sorbent properties of the drug. The data on the binding of metal ions are given in tables 2 and 3.

 Example 8. Test the biologically active additives in black ferrets. The preparations are given to males of the black polecat as an additive to the feed (1 g of the drug per head per day) for 10 days. Control - males who did not receive feed additives. The condition of all animals is normal, behavior is normal, litter is unchanged.

Example 9. The results of feeding young ferrets standard food with 1% of the preparation of rye straw with DAFS-25
Example 10. The results of feeding young ferrets with standard food with 1% of the preparation from the husks of sunflower seeds with DAFS-25.

 Example 11. The results of feeding adult rats with compound feed with 5% and 10% supplementation from husk of sunflower seeds with DAFS-25. The results of consumption and digestibility by laboratory rats of feed and feed additives (a preparation from husk of sunflower seeds with DAFS-25) are shown in Table 6.

 In the experiments, 18 rats were used, the duration of feeding experiments was 20 days, and the balance experiment was 3 days. From the experiment it follows that husk from the drug was digested by rats by almost 50%, even with the introduction of 10% of the drug.

 The resulting preparations contain: i) microfractional cellulose fibers; ii) a complex of cellulolytic and nitrogen-fixing bacteria, as well as concomitant forms of bacteria. Enriched with nitrogen and bacteria metabolism products, the preparation containing cellulose has high sorption properties.

 The drug has high biological activity, since high temperature is not used during its production, which leads to the preservation of a complex of cellulolytic, nitrogen-fixing and related microorganisms, which additionally helps digestion. In addition, during the passage of the components of the supplement through the gastrointestinal tract, toxic components and waste are removed from the body. This is due to the binding of toxic components and slag to a developed surface with sorption properties.

 Example 12. Feeding animals with a preparation of needles obtained using a nitrogen-fixing cellulolytic culture of B.subtilis GL.

 Dwarf rabbits and guinea pigs located in the vivarium A. N. Severtsov RAS, for 34 days was kept on a diet in which 25% of the bran (for rabbits) and hercules (for guinea pigs) were replaced with the same amount of needles.

The preparation was obtained by solid-phase fermentation of chopped needles using a culture of Bacillus subtilis B-8130, isolated from the cecum of the capercaillie and deposited in VKPM, and dried at 60 ^° C. The animals fed the usual food were controlled. The feeding results are given in table.7.

 The experiment demonstrated the positive effects of biologically active feed additives on the body of dwarf rabbits and guinea pigs.

LIST OF REFERENCES
1. Aybazov O.A. and others. Feed based on cellulose-containing crop waste and a method for its production. RU 2050142 Int. C1, A 23 K 1/12, 12.20.1995.

 2. Wolf V.V. et al. Method for the production of protein feed from wood waste RU 2092073 Int. C1, A 23 K 1/12, 10/10/1997.

 3. Yudin Yu.I. and others. A method of preparing feed from plant materials. SU 1287829 Int. C1, A 23 K 1/165, 07.02.87.

 4. Lizak Y. Process of bio-conversion of industrial or agricultural cellulose containing organic wastes into a proteinaceous nutrition product US 5952020, US C1, 426/8, 09/14/1999.

 5. Nikolicheva T.A. and other Association of microorganisms for feeding young cattle. SU 1671693 Int. C1, C 12 P 39/00, 08/23/1991.

 6. Drevko R.F. and others. A method of preparing a protein-vitamin-mineral feed mixture. RU 2082300 Int. C1, A 23 K 1/16, 06/27/1997.

 7. Yukhimenko L.N. and others. The method of preparation of feed. RU 2140165 Int, C1, A 23 K 1/00, 10.27.1999.

 8. Al-Nuri Ya.M. Biological product "ANTA-1" - feed additive for broiler chickens RU 2110927 Int. C1, A 23 K 1/165, 05.20.1998.

 9. Reinhart G. A. Pet food product containing fermentable fibers and process for treating gastrointestinal disorders US 5616569, US C1, 514/54, 04/01/1997.

 10. Saubenova MG and others. The use of cellulolytic bacteria in feed production. Adj biochem. and a microbe. (1998), v. 34, 1, pp. 91-94.

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Claims (5)

1. The method of obtaining biologically active feed additives from plant materials, including grinding plant materials, selecting bacteria cultures, obtaining seed and enzymatic hydrolysis, characterized in that the selection of bacteria cultures is carried out from various sections of the digestive tract of herbivorous animals: the oral cavity, or goiter, or the pancreas, or intestines, or excrement, enzymatic hydrolysis is carried out by a monoculture or an association of microorganisms, or a complex consisting of an association and microorganisms and at least one additional strain of microorganisms and / or additional microelement at a temperature of 18-70 ^o C for 5-48 hours to obtain cellulosic activity of the additive of not less than 90 units / g of dry fermentation product.  2. The method according to claim 1, characterized in that the intestinal sections for the selection of bacterial cultures are selected from the group: cecum or colon.  3. The method according to claim 1, characterized in that at least one additional strain of microorganisms is selected from the group of microorganisms belonging to the genera: Bacillus, Clostridium, Lactobacillus, Bifidobacterium, Streptococcus, Micrococcus, Ruminococcus, Proteus, Enterobacter, Escherichia, Klebsiella, Pseudomonas, Brevibacterium.  4. The method according to claim 1, characterized in that selenium is used as a trace element.  5. The method according to claim 1, characterized in that the enzymatic hydrolysis is carried out by a solid-phase method.

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