RU2117703C1 - Multienzyme composition for cattle breeding - Google Patents

Abstract

FIELD: agriculture. SUBSTANCE: multienzyme composition has cellulolytic preparation from Trichoderma viride and amylolytic preparation from Bacillus subtilis. Composition has also the preparation from Trichoderma riesei or Trichoderma viride as a source of fungal celluloses at the following ratio of their activities: fungal xylanase to fungal and bacterial exo-β-glucanase = 1: (3.50-3.70). Composition is used for preparing the mixed feed and full-ration mixtures for agriculture animals. EFFECT: enhanced effectiveness of composition. 14 tbl

Description

 The invention relates to the field of biotechnology, and in particular to the development of biotechnological products - multi-enzyme composition (IEC), which allows to increase the proportion of unpeeled barley introduced into compound feeds or complete mixtures for farm animals and poultry.

 Barley of different technological preparation (peeled and peeled) belongs to traditional fodder crops, but due to some specific features it has limitations in use. For example, the input standards for feed for poultry of different age groups of peeled barley are from 40 to 50%, unpeeled from 15 to 40%. Limitations of barley intake, especially non-peeled, are associated with a high (compared to other grains) fiber content (2.5 times), low energy content, low availability of lysine and the presence of β-glucan (colloidal type polysaccharide), which lead to a decrease in eatability feed, growth rate, nutrient conversion efficiency and sticky droppings.

 In barley, the most important source of energy - starch - is located in the endosperm, surrounded by a cell wall consisting mainly of β -glucan and arabinoxylan. These are specific carbohydrates, characterized by high viscosity, as a result of which highly viscous solutions are formed in the digestive tract of monogastric animals, increasing the volume and mass of the chyme and slowing the speed of the feed, which leads to disruption of digestion processes.

 It should be noted that peeling of crops such as barley and oats can significantly reduce the fiber content, but the content of β -glucans does not change, because they cover starch endosperm.

 A method has been developed to increase the share of unshelled barley in the composition of animal feed (up to 60 - 70%) for farm animals and poultry by using a complex enzyme preparation (MEK), which helps to destroy β -glucans and fiber components and remove problems associated with increased viscosity of intestinal contents.

 Known multi-enzyme compositions (MEK-TSGAP-L and MEK-TsGAP) containing cellulose, β-glucanase, amylase, protease, lysozyme, xylanase, intended for introduction into compound feeds, containing up to 40 - 50% barley 30 - 40% wheat and triticale and 15 to 20% rye. (one).

 The enrichment of feed for broilers with these compositions in doses of 0.05 - 0.1% increases the use of protein in the body of a bird by 4 - 6%, fat by 5 - 7%, energy by 5 - 8% and productivity by 12 - 15%.

 However, the authors recommend the use of compositions in compound feeds containing up to 40 - 50% of barley, which limits their use at a higher content of feed grain (60 - 70%).

 Hoechst (Germany) developed a complex preparation Hostasim C based on Trichoderma reesei to remove β -glucan factor in barley-containing feeds, which makes it possible to use cheaper diets in poultry production (2). The use of Hostazim C, according to the company, ensures the destruction of fiber components in compound feeds containing up to 52% of barley, and the technological method for preparing barley (peeled or not peeled) is not indicated, as in the previous analogue, which is essential for the processes of grain fermentolysis in the digestive tract of animals and birds.

 The authors' task is to create a complex-action preparation effective in the composition of compound feeds containing a higher content of unpeeled barley (60 - 70%), which excludes additional costly pre-treatment of grain husking.

 It is known (3, 4) that for maximum destruction of natural polymers such as cellulose, hemicellulose, lignin, β-glucans, etc., the action of endo- and exoenzymes is necessary, since enzymes of endogenous action predominantly carry out depolymerization of substrate macromolecules (nutrients), and exogenous, mainly, terminal residues of fragments of macromolecule fragments of partially hydrolyzed substrate are cleaved. Moreover, bacteria synthesize mainly endoenzymes, and microscopic fungi - enzymes of exogenous action.

 The aim of the invention is the creation of a complex action drug balanced by the presence of enzymes of fungal and bacterial origin, effectively breaking down cellulose derivatives - β-glucans, xylans, arabinoxylans and other barley grains, which forms the basis of industrial feed (up to 60 - 70%).

 To develop a complex preparation, fungal cellulases from the most productive strains of Trichoderma reesei (Tr.reesei) and Trichoderma viride (Tr.viride), differing in different activity levels of β-glucanases and xylanases, as well as a bacterial preparation from Bacillus subtilis (Bac.subtilis) were used ), which produces, along with amylase, β-glucanase enzymes (Table 1).

 Characterization of the preparations was carried out according to methods adopted in the microbiological industry (3, 5, 6, 7, 8, 9).

 This goal is achieved by the fact that enzyme cellulolytic preparations with exo-β-glucanase content of 450 - 800 units / g, endo-β-glucanase 300 - 500 units / g and xylanase at the level of 700 - 900 units / g are used as MEK components. an enzyme preparation from Bac.subtilis with an exo-β-glucanase content of at least 200 u / g and endo-β-glucanase at a level of 50-100 u / g. The use of highly active preparations with the presence of the necessary enzymes creates the prerequisites for the development of a complex preparation containing a system of fungal and bacterial cellulases with a certain ratio of glucanases and xylanases that can effectively destroy natural barley grain polymers.

Из таблицы 1 видно, что целловиридин Г20х (образцы 2, 3) по уровню β -глюканаз и ксиланазы значительно уступает препарату целловиридин-5000 при одном и том же уровне общей целлюлолитической активности. Следует отметить, что существенным преимуществом препарата из Tr.reesei (образцы 4, 6) и Tr. viride (образец 1) является наличие высокого уровня активности эндо- β -глюканазы, катализирующей процесс деполимеризации вязких ячменных субстанций.Thus, a multi-enzyme composition is proposed, characterized in that it contains a cellulolytic preparation of Trichoderma reesei or Trichoderma viride and amylolytic from Bacillus subtilis in the following ratio of enzyme activities

From table 1 it can be seen that celloviridin G20x (samples 2, 3) in terms of β-glucanases and xylanases is significantly inferior to celloviridin-5000 with the same level of total cellulolytic activity. It should be noted that a significant advantage of the drug from Tr.reesei (samples 4, 6) and Tr. viride (sample 1) is the presence of a high level of activity of endo-β-glucanase, which catalyzes the process of depolymerization of viscous barley substances.

 The regularity of the proportionality of the activities of endo- and exoenzymes is related to the advantages of the drug from Tr.reesei, which is explained by the specific properties of the producer and directed biosynthesis. In this regard, a drug obtained mainly on the basis of Tr.reesei was chosen to create an IEC. A drug from Tr.viride can be used provided that it contains β-glucanase of at least 450 eg / g.

 Amylosubtilin GXx was used as a source of bacterial enzymes.

 To assess the effectiveness of the proposed multi-enzyme composition was made 5 samples of IEC with different ratios of enzymes.

 Samples were prepared on an Erveka laboratory setup by dry mixing, with a stirring frequency of 30 rpm for 30 minutes.

 The characteristics of the IEC samples are presented in table 2.

 Sample IEC TsGAP (IEC-1) in terms of enzymatic activities and their ratios corresponds to the closest analogue (1), samples IEC-2, IEC-3, IEC-4 correspond in terms of the ratio of fungal xylanase and β-glucanase to the composition of the compositions, samples IEC- 5 and IEC-6 in terms of the ratio of enzyme activities that determine the essence of the invention, differ from the claimed solution.

 The advantages of the proposed solution compared to the closest analogue are presented in examples 1, 2, 3, 4, 5.

 Example 1. The effectiveness of the claimed composition in the destruction of β -glucan of peeled barley (table 3). The content of β -glucan in barley was determined by the method recommended by R. Ermakova (ten).

 The results of the table. 3 confirm the advantages of the claimed composition on the efficiency of hydrolysis of β-glucan: the degree of destruction of β-glucan in options 3, 4, 5 is 14.80 - 16.15% higher than in option 1 using an analogue. Samples IEC-5 and IEC-6, in which the ratios of XA and exo-β-GLA are outside the scope of the claimed solution, are inferior to options 3, 4, 5. The increase in the degree of hydrolysis of β-glucan in options 6, 7 is 8.10% and 5.20%.

 Example 2. The effectiveness of the claimed composition in the degradation processes of barley grain polysaccharides and barley-based compound feeds was evaluated by the method based on the sequential determination of carbohydrate hydrolysis products using CSN reagent (11, 12). From the analysis of the presented data (Table 4), it follows that the hydrolysis of barley grain carbohydrates proceeds with greater intensity in options 3, 4, 5 (IEC-2, IEC-3, IEC-4) than in option 2 with the closest analogue and options 6, 7 (IEC-5, IEC-6). The associated increase in the number of reducing sugars is explained by the synergistic effect of β-glucanase, xylanase in optimal proportions and amylase, moreover, the availability and attackability of starch grains by amylases is due to the initial enzymatic destruction of non-starch polysaccharides enveloping starch-containing substances in the grain. In variants -2, 5, 6, where the ratio of KsA and exo-β-GLA differs from the declared ones, the indicators of hemicellulose and cellulose reduction are inferior to the corresponding values in optimal variants.

 So, the hemicellulose content in options 3, 4, 5 is reduced by 1.26 - 1.52%, against 0.45% (the closest analogue) - 1.07 - 1.02% (options 6, 7); the decrease in cellulose content is respectively 0.45 - 0.57% versus 0.14 - 0.31 - 0.35%.

 IEC samples were introduced into the composition of barley-wheat type feed in the amount of 0.1 - 0.2% (depending on the value of exo - β-GLa) to the feed weight. Compound feed included in %%: barley - 69.7; soybean meal - 12.0; skimmed milk powder - 2.0; fishmeal - 3.0; wheat bran - 5.0; feed yeast - 3.0; animal feed fat - 2.0; defluorinated phosphate - 1.2; chalk - 0.9; table salt - 0.9; premix KS-3.

 1 kg of feed contained: feed units - 1.23; exchange energy - 13.60 MJ; crude protein - 182.00 g; lysine - 9.6 g; methionine + cystine - 5.9 g; crude fiber - 35.6 g; calcium - 9.7 g; phosphorus - 7.7 g; iron - 122.5 mg; copper - 88.0 mg; zinc - 120.5 mg; Manganese - 92.0 mg; cobalt - 0.55 mg; iodine - 1.55 mg; vitamins: A - 59500 ME; D - 6000 ME; E - 42.0 mg; B1 - 3.0 mg; B2 - 15.2 mg; B3 - 17.5 mg; B4 - 1.4 mg; B5 45.0 mg; B12 - 34.0 mg.

 The results of the fermentolysis of the feed are presented in table 5.

 In the data table. 5, illustrating the effectiveness of the enzyme analysis of barley-wheat feed, a trend is observed that is similar to the process of hydrolysis of barley polysaccharides (Table 4). The results on an increase in sugar content and a decrease in the amount of carbohydrates in options 3 to 7 exceed the values of the corresponding indicators for the closest analogue (option 2).

 The maximum increase in the content of reducing sugars by 4.59–4.77%, due to the enzymatic destruction of hemicelluloses, cellulose, and starch, was noted in variants with the optimal (claimed) ratio of XA and exo-β-gluconase (var. 3, 4, 5). This indicator is inferior to optimal and amounts to 2.97 - 3.07%.

 Example 4. One of the essential indicators of the effectiveness of IEC is the decrease in the viscosity of aqueous polymers in the body of the animal, which helps to eliminate the stickiness of the litter, and thereby normalize the digestive processes. The effectiveness of IEC in the process of liquefying barley-based compound feeds was evaluated by changing the viscosity of aqueous extracts from compound feed fermented by IEC samples. The process of fermentation of extracts was carried out according to 7 options. The dosage of MEK was 0.1 - 0.2% of the dry weight of the feed.

 The results are presented in table. 6. From the data it follows that the decrease in viscosity to the greatest extent - by 29.3; 31.0 and 32.3 rel. % - noted as a result of fermentation of feed using the inventive composition MEK in optimal ratios (var. 3-5) of enzymes. The use of IEC-4 and IEC-5 also helps to reduce viscosity by 21.3 - 19.7 rel. %, which exceeds the effect of the closest analogue (var. 2), but inferior to the results in optimal variants. According to experts, a 30% decrease in the viscosity of barley β-glucan in solution is a necessary condition for the classification of enzyme preparations intended for use in diets (high in barley) for farm animals and poultry (13).

 Further, in examples 5, 6, 7, the effectiveness of the claimed composition is presented in the composition of compound feeds with a high content of barley used in feeding laying hens, broiler chickens and growing piglets.

 Example 5. Compound feed for feeding laying hens had the composition shown in table 6A.

 IEC tests were carried out under experimental conditions of the experimental farm VNIITIP on 4 groups of poultry, 100 animals each.

The effectiveness of fermented feed was evaluated by the main zootechnical indicators:
- livestock safety,%;
- egg production on the average laying hen, pcs .;
- feed costs for 10 eggs and 1 kg of egg mass;
- digestibility of feed nutrients.

 As follows from table 7, the safety of the stock of chickens over the period of the experiment was high and did not depend on the factors studied (departure for Marek’s disease).

 The egg production of chickens in options 2 and 3 was 200.03 and 197.71 pcs., Which exceeded the egg production in the control version and in option 4, which differs from the claimed one.

 The advantage of the claimed composition is manifested in the analysis of these feed costs per 10 eggs: if in optimal options the costs are 1.50-1.52 kg, then in the control (version 1) this figure is 1.67 kg, which exceeds the cost of feed all 3 other groups of birds. A similar pattern was noted in the data on feed costs per 1 kg of egg mass.

 The reduction in feed costs in the claimed variants is explained by the best absorption of nutrients, which is associated with high availability and digestibility of nutrients and removal of β -glucan factor.

 So, in options 2 and 3, the value of this indicator for fiber is 37.66 - 36.42% versus 27.97% (var. 1) and 34.41% (var. 4). The digestibility of protein in optimal variants (var. 2, 3) is 89.86% and 89.35%, which exceeds the value of this indicator in var. 1 and 4. Due to this, the use of nitrogen and calcium increases, which is a positive factor in mineral metabolism.

 Example 6. The effectiveness of the IEC when feeding broiler chickens.

 Recipes of experimental feed are presented in table. eight.

 The effectiveness of IEC is presented in table. nine.

 As follows from the presented zootechnical indicators for broiler chickens that received mixed feed with IEC samples, the best results were obtained in versions with IEC-2 and IEC-4: data on average live weight (1 goal) (1760 g and 1558 g), average daily growth (27.0 g and 12.5 rel.%), feed costs per 1 kg of growth (2.52 kg and 2.94 kg) confirm the advantages of the claimed IEC composition over the closest analogue (version 1) and option (4) other than stated.

 Example 7. The effectiveness of the IEC in the diets for feeding piglets up to 60 days of age and raising pigs from 60 to 120 days was tested in a pilot farm VIZH.

 Used feed of the following composition: The results of evaluating the effectiveness of IEC are presented in table. 11, 12.

 Consideration of the data table. 11 allows us to judge the advantage of the proposed composition of the IEC (version 1, 2) over the closest analogue (version 1) and a comparison sample (version 4), which is confirmed by a number of zootechnical indicators: an increase in the average daily growth of piglets (both groups) to 105.3 - 113.0 rel. % compared with control (var. 1); reduction of feed costs from 100% in control (var. 1) to 97.4 rel. % (gr. 1) - 90.9 - 92.9 rel. % (column 2).

 The results of the table. 12 supplement the data on the effectiveness of the IEC in the feed composition: increasing the degree of digestibility of the main components of the feed - organic substances: crude protein, crude fat, crude fiber, BEV in the claimed options (2, 3) - IEC-2, IEC-3 compared with the control IEC-1 (version 1) provides the same options for reducing feed costs and increasing growth (table. 11).

 Example 8. An experiment on a comparative evaluation of the effectiveness of the processing methods (enzymatic and mechanical) of peeled barley was carried out during the cultivation of piglets 13 - 104 days. age against the background of compound feeds SK-3 (from 13 - 42 days), SK-4 (from 43 - 60 days), SK-5 (from 61 - 104 days). Compound feeds had the composition shown in table. thirteen.

 The experimental results are presented in table. 14.

 The data table. 14 convincingly prove that the introduction of IEC in compound feeds with peeled barley (60 - 48%) eliminates the need for mechanical processing of peeled barley - peeling grain. The values of the main zootechnical indicators - the average daily gain and feed consumption per 1 kg of growth in the versions with IEC exceed the corresponding indicators in the variant with peeled barley (control-1) and even in the variant with peeled barley (control-2).

 The best results are provided by the claimed composition IEC-3. When growing piglets aged 13 to 60 days. the average daily increase increases by 14.9% against control-1 (peeled barley) and by 8.7% against control-2 (peeled barley), and feed costs are reduced, respectively, to 86.5%. IEC-1 (the closest analogue) is inferior in efficiency to the claimed one, which is expressed in lower average daily growth rates - 108.5 - 102.2% and feed saving - 91.2 - 99.5%.

 Thus, the proposed multi-enzyme composition allows, first of all, to increase, in comparison with the closest analogues, the level of unpeeled barley in compound feeds up to 60 - 70% for farm animals and poultry, as well as to exclude the peeling operation from the technological process of preparing feed raw materials, which is superior in cost 2 - 2.5 times the introduction of IEC. The use of feed using the proposed composition provides an improvement in zootechnical indicators: increase in live weight, increase in egg production, livestock safety, digestibility of feed nutrients and, in addition, partially or completely replace valuable feed grain (wheat, corn) with unpeeled barley.

Literature
1. Feed industry, 1996, N 1, p. 32.

 2. Information materials of the company "Hoechst" (Germany).

 3. Bioorganic chemistry, 1980, v. 6, N 8.

 4. Enzyme preparations in the food industry. M. Food Industry, 1975, p. 76 - 78, 325 - 351.

 5. TUop IEC-CX-2, 1996

 6. J. biochem., 1985, v. 22, p. 177 - 180.

 7. TUop IEC-CX-1, 1994.

 8. GOST 20264.4-89.

 9. GOST 20264.2-88.

 10. The enzyme and alcohol industry, 1983, N 6, p. 21-22.

 11. Ermakov A.I. Methods of biochemical studies of plants. - L .: Kolos, 1972.

 12. Research methods for animal feed, organs and tissues. - M .: Kolos, 1969.

 13. J. of Animal and Feed Science, 1992, v. 1, p. 65 - 70.

Claims (1)

 A multi-enzyme composition for animal husbandry containing cellulolytic and amylolytic preparations, characterized in that it contains a cellulolytic preparation from Trichoderma reesei or Trichoderma viride and an amylolytic preparation from Bacillus subtilis with the following ratio of enzyme activities: fungal xidanase: fungal and bacterial exo-β-glucan : (3.50 - 3.70).

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