RU2268925C2 - Probiotic lactomicrocicol used in growing and fattening broiler poultry - Google Patents

Abstract

FIELD: biotechnology, microbiology, agriculture.

SUBSTANCE: the probiotic lactomicrocicol comprises microbial biomass of microorganisms of strain Lactobacillus amylovorus BT-24/88 (VKPM, B-6253) and Escherichia coli S-5/98 (VKPM, B-8564) taken in the ratio = 1:1 in the concentration of colony-forming units of each strain in the range (5 x 10⁸)-(5 x 10⁹) CFU in 1 ml of liquid or 1 g of dry preparation. Lactobacillar strain possesses the broad spectrum of antagonistic activity and multiple antibiotic-resistance and Escherichia coli produces microcin B-5/98 suppressing opportunistic and pathogenic microorganisms of genus Escherichia and Salmonella. Using the proposed probiotic in feeding broiler-chickens taken in the dose 0.2 g per 1 kg of dry fodder or 1 l of drinking water provides enhancing their safety, gain of living mass and the yield of production of the 1-st category.

EFFECT: valuable properties of probiotic.

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Description

The invention relates to microbiology and biotechnology and can be used in the microbiological industry in the production of the probiotic lactomycrocycol and in poultry farming in order to increase productivity and improve the quality of broiler chicken meat.

In recent years, probiotics - feed additives that have a beneficial effect on animals and poultry by improving intestinal microbial balance - are increasingly used in poultry farming. Using a probiotic allows you to accelerate the growth of young animals and reduce its waste. They are used for dysbacterioses, for regulating microbiological processes in the digestive tract, for the prevention and treatment of certain digestive disorders of an infectious and alimentary etiology, as substitutes for antibiotics, etc. The effectiveness of probiotics is not inferior to some antibiotics and chemotherapeutic agents, but they do not have a detrimental effect on the microflora of the digestive tract, they do not pollute animal products and the environment, that is, they are environmentally friendly.

In Russia, a bird consortium of VNIISKhM No. 1-32 (ed. St. USSR No. 1398796, A 23 K 1/165, C 12 N 1/00, 1988), a biosan preparation (ed. St. USSR, were tested and proved to be good on birds. No. 1777772, A 23 K 1/165, 1992), probiotics of lactin (Poultry farming, 1992. No. 12. S.9-10), galliferm and enteric acid (Poultry farming. 1996. No. 1. S.25), biological product "Anta- 1 "(RF patent No. 2110927, A 23 K 1/165, A 61 K 35/74, C 12 N 1/20, 1998), preparations STF 1/56 and bifinorm (Poultry farming. 2002. No. 4. P.23 -25), bifidumbacterin and other drugs.

A comparison of the main characteristics of the above drugs indicates that all of them, as a rule, are intended for the prevention and treatment of gastrointestinal diseases, increase the safety and increase in live weight of young animals.

The main disadvantages of these bacterial preparations are: varying application efficiency, relatively low growth stimulation, and most of them do not affect the quality of the products obtained.

The closest analogue is the probiotic lactoamilovorin (RF Patent No. 2054478, C 12 N 1/20, 1996). Distinctive features of the strain Lactobacillus amylovorus BT-24/88 (VKPM-B6253) used for its preparation are: the ability to ferment starch, which other lactobacilli used to prepare probiotics do not possess; resistance to chloramphenicol, tetracycline, streptomycin, kanamycin, rifampicin and polymyxin, as well as to a 1% concentration of baytril (enrofloxacin); the production of broad-spectrum antibiotic substances; high tolerance to adverse intestinal factors (bile, ethanol, phenol); lack of pathogenicity, toxicity, toxigenicity, allergenicity.

The use of lactoamilovorin in the cultivation of broiler chickens provides the following biological effects: inhibition of Escherichia, Salmonella and hemolytic bacteria in the intestine; increase the safety of broilers from 88.9% to 90% at the age of 49 days; increase in live weight of broilers by 120 g or by 7.8%; improved appetite and increased feed intake by 3.25%; reduction of feed costs per 1 kg of live weight gain by 5.4%; increase in the yield of carcasses of the 1st category from 66.9% to 69.5% (Poultry. 1999. No. 6. P.32-33).

However, despite the rather high parameters of the efficiency of broiler cultivation using lactoamilovorin, it is not yet possible to conclude that the potential of probiotics in broiler production has been fully exhausted. Therefore, the search for new highly effective strains of bacteria and their associations providing good health, high productivity and product quality remains necessary.

The objective of the invention is to obtain a biological product (probiotic) for broiler poultry with enhanced antagonistic activity against potential intestinal pathogens of bacteria of the genera Escherichia and Salmonella, which can increase the safety and productivity of poultry, as well as increase the output of the 1st category.

The technical result of the invention is to create a new probiotic with enhanced antagonistic activity and a high potential for increasing bird productivity.

The inventive new probiotic lactomycrocyclic for growing and feeding broiler poultry contains a composition of strains of Lactobacillus amylovorus BT-24/88 and Escherichia coli S 5/98, taken in a 1: 1 ratio at a concentration of colony forming units of each strain within 5 × 10 ⁸ -5 × 10 ⁹ in 1 ml of liquid or 1 g of dry preparation.

The strain Lactobacillus amylovorus BT-24/88 was isolated using traditional microbiological techniques from the chyme of the cecum of a healthy pig. The strain is deposited in VKPM under N B-6253 and is characterized by the following features;

Cultural and morphological characters.

The cells of the strain are of medium length with rounded ends of the rods, located in smears singly in pairs and in short chains. Stationary, do not form a dispute. Gram stains positively.

MPA colonies with glucose and tryptose agar (39 ° C, 72 h) are small, round, convex, smooth, and transparent.

On a dense medium with bactofoc and whey, round, convex, white, opaque colonies with a diameter of 0.5-1 mm are formed.

In a liquid modified medium of Rogoza with cabbage broth, tubes suspended in the broth and fixed on the walls, but easily falling off flakes and a gray-white precipitate, which is easily stirred up by shaking, are formed.

On glucose MPB, slight turbidity, small flakes fixed on the tube wall, gray-white precipitate.

Physiological and biochemical characteristics.

Optional anaerobic.

The optimum temperature is 37-39 ° C. It grows at 45 ° C, but not at 15 ° C. It remains viable after an hour and a half warming up at 60 ° С and 65 ° С.

It grows well in the range of pH 5-7.

Gas does not form from glucose.

Catalase-negative.

Nitrate does not reduce.

Milk coagulates.

Methylene blue (0.01 and 0.1%) restores.

It grows in environments with 2, 4 and 6% sodium chloride; 20 and 40% of bile; 2, 4 and 6% ethanol; 0.2 and 0.4% phenol.

It has amylolytic activity.

The acid is produced from glucose, cellobiose, galactose, lactose, maltose, mannose, salicin, trehalose, esculin, turanose and starch. Arabinose, gluconate, mannitol, melecitosis, melibiosis, raffinose, rhamnose, ribose, sorbitol, sucrose, xylose, inulin, sorbose, nutrosa and pectin are not fermented.

The final products of glucose fermentation are lactic and acetic acids, as well as ethanol.

The strain produces broad-spectrum antibiotic substances that inhibit bacteria of the genera Staphylococcus, Micrococcus, Streptococcus, Bacillus, Escherichia, Pseudomonas, Salmonella and some species of Lactobacillus.

The strain is resistant to chloramphenicol, tetracycline, streptomycin, kanamycin, rifampicin and polymyxin.

The strain is non-pathogenic, does not show toxic and toxigenic properties.

A new strain of Escherichia coli S 5/98 - producer of microcin B5 / 98, isolated using traditional microbiological methods from feces of an adult pig. The strain is deposited in VKPM under No. B 8564 and is characterized by the following features.

Cultural and morphological characters.

The cells of the strain are represented by sticks with rounded ends, negatively stained by Gram. In smears are located in the form of individual cells and random clusters. Spore and capsules do not form.

Dark red with a characteristic metallic luster of the colony are formed on the Endo medium.

On the fourth tryptose agar (1/4 of the nutrients from the official prescription) grow round, convex, with a smooth edge, up to 2 mm in diameter, shiny, translucent, gray-white colonies.

On a nutrient broth with glucose, turbidity and a gray-white precipitate at the bottom of the tube.

Physiological and biochemical characteristics.

The optimum temperature is 37-39 ° C. It grows at 15 ° C and 45 ° C. Dies after 30 minutes of exposure at 60 ° C.

The strain is tolerant to phenol (0.6%), ethanol (2%), grows well in an environment with 20% bile. It produces gas from glucose, does not form hydrogen sulfide, gives a positive reaction with methyl red and a negative Voges-Proskauer. It grows on Simmons citrate agar, but does not use acetate and malonate as an energy source; phenylalanine and urea does not hydrolyze.

It ferments with the formation of acid arabinose, lactose, maltose, mannitol, glucose, xylose, trehalose, sorbitol and does not ferment sucrose, inositol, dulcite and salicin.

The strain is sensitive to oxacillin, chloramphenicol, streptomycin, gentamicin, rifampicin, neomycin, erythromycin, tetracycline, kanamycin, vancomycin, clindamycin, clarithromycin, ampicacin, as well as nalidixic acid and fusidine.

The strain produces microcin B 5/98.

Microcin B 5/98 has a molecular weight of less than 10 kDa. Its production decreases or is absent during cultivation of the producer on rich nutrient media and increases on depleted (minimal) nutrient media. It is inactivated by subtilisin E, a metalloproteinase from you. mesentericus, protolychetrem and pronase, but resistant to thermolysin and trypsin.

No plasmids were found in the strain, i.e. genes that determine microcin production and immunity to it are localized on the bacterial chromosome.

The strain has a wide spectrum of antagonistic activity and suppresses 69-100% Escherichia and 42.6-80% of wild-type Salmonella isolated from the intestines of livestock, pigs, chickens and cats. Active against colicinogenic strains of E. coli. Among Salmonella, it inhibits Salmonella give, S. bovis morbipicans 988, S.dublin 42, S.london 1446, S.gaminare, S.derby, S.amager 2399, S.rostock, S.readiry, S.enteritidis 41997. antagonism against bacteria of the genus Klebsiella.

The strains included in the probiotic are grown separately. The drug can be used in liquid and dry forms. It is advisable to mix the liquid cultures of the strains making up the lactomycrocycol before use, the dry biomass of L.amylovorus BT-24/88 and E.coli S5 / 98 are combined into the preparation immediately after preparation. Liquid and dry preparations of lactomycrocycols are stored at a temperature of + 4-6 ° C.

Example 1. For the cultivation of the strain L.amylovorus BT-24/88 prepare a dairy environment. For this purpose, 10% skimmed milk powder is added to tap water and, after its dissolution, the medium is poured into bottles of heat-resistant glass or pumped into a fermenter and sterilized at 1 atm (120 ± 1 ° C) for 30 minutes. The prepared medium is cooled to 35-38 ° C and inoculated with the daily culture of the strain L. amylovorus BT-24/88 in milk medium at the rate of 5-10% by volume of the medium. Crops are incubated at 38 ± 1 ° C for 24 hours and the resulting product is used to feed the bird or prepare a dry preparation.

Liquid culture is released in hermetically sealed glass bottles or plastic cans.

A dry preparation is obtained by lyophilization of a milk culture together with a protective medium, stabilizers and fillers.

Example 2. For the cultivation of E. coli S 5/98 strain, a nutrient broth or MPB is prepared, to which 0.2% glucose is added. The pH of the medium is adjusted to 6.8-7.2, poured into test tubes, flasks, heat-resistant bottles or pumped into a fermenter and sterilized at 0.5 atm for 30 minutes.

The daily culture of E. coli S 5/98, which is added at the rate of 5-10% to the volume of medium, is used as seed material. Crops are incubated at 38 ± 1 ° C for 24 hours. The grown liquid culture is either Packed in suitable containers (bottles, polyethylene canisters), or used to prepare dry biomass.

To prepare a dry preparation, the microbial biomass is separated from the culture fluid (separation, filtration) and suspended in a protective medium (1/20 of the original volume) containing phosphates, sucrose and skim milk, the suspension of biomass in a protective medium is brought to a creamy consistency by adding dry powder skimmed skim, mix thoroughly, cool to + 2-4 ° C and carry out L-drying in a vacuum dryer directly from the liquid state.

The dried mass is crushed, standardized by the addition of fillers (starch, sucrose, lactose, dry return) and Packed in plastic bags.

Example 3. Obtained in examples 1 and 2, the probiotic lactomycrocycol is used when growing broiler chickens as an additive to the main diet.

In the scientific and production experiment, 4 groups of chickens of 20 goals each were formed. The birds of the control group received only the main diet, and chickens of the 2nd and 3rd groups per 1 kg of compound feed were added 0.1 g and 0.2 g of dry lactomycrocycol, respectively. Broilers of the 4th group received a probiotic with drinking water at the rate of 0.2 g / l. The experiment lasted 49 days.

Studies have shown that lactomycrocycol does not have adverse effects on the body of the bird and that no pathological changes were detected during its slaughter. The use of the drug had a stimulating effect on broiler growth. So, the live weight of 49-day broilers of the 3rd and 4th groups was 1767 g and 1963 g and exceeded the control (1690 g) by 4.6% and 16.2%, respectively. At the same time, a significant improvement in product quality was observed, and the output of carcasses of the 1st category from 74% in the control increased to 78.3; 91.3 and 91.9% in the 2nd, 3rd and 4th groups, respectively (table). From the data obtained, it is obvious that giving lactomycrocycols with drinking water provides the maximum productive effect.

Production tests of lactomycrocycols for broiler chickens conducted at ZAO Orenburgskaya Poultry Farm on a livestock of 13360 heads showed that giving an effective dose of the drug increased the safety of young animals by 5% and the average live weight of one head by 0.27 kg. The economic effect amounted to 78.4 thousand rubles.

Thus, the probiotic lactomycrocycol proposed for broiler poultry breeding can increase the safety of young animals, increase its productivity and yield of products of the 1st category.

Table Indicator Groups one 2 3 four Number of animals, goal at the beginning of the experiment twenty twenty twenty twenty at the end of the experiment 19 twenty 19 19 Preservation,% 95 one hundred 95 95 The live weight of one head at 49 days of age, g 1690 1697 1767 1963 % one hundred 100,4 104.6 116.2 The total live weight of the group, kg 32.11 33.94 33.57 37.30 Carcass weight, kg 1.18 1.19 1.24 1.40 Slaughter yield,% 69.8 70.1 70,2 71.3 Meat yield, kg total 22.42 23.80 23.56 26.60 including 1st category 16.60 18.63 21.50 24.45 % 74 78.3 91.3 91.9 2nd category 5.82 3.17 1.06 2.15 % 26 13.3 4,5 8.1 non-standard - 2 one -

Claims (1)

A probiotic used for growing and feeding broiler birds, characterized in that it contains a microbial biomass of strains of Lactobacillus amylovorus VKPM B-6253 and Escherichia coli VKPM B-8564, taken in a 1: 1 ratio, with a concentration of colony forming units of each strain within 5 × 10 ⁸ -5 × 10 ⁹ in 1 ml of liquid or 1 g of dry preparation.