Therapeutic biological product
(57) Abstract:The invention relates to biotechnology and Microbiology, and can be used in veterinary medicine for the prevention and treatment of inflammatory disease and dyspepsia in animals. The preparation includes the biomass of Bacillus subtilis 12 IN - DEPT and the filler in the following ratio, wt.%: biomass of B. subtilis 12 IN-DEPT - living microbial cells (1-2)109in 1 ml of solvent 92-95, filler 5-8. Biologic suppresses the development of veterinary strains of pathogenic microorganisms of the genera: Streptococcus, Esherichia coli, Pseudomonas, Sthaphilococcus, Proteus, Shigella, Hexaera, has a broad resistance to antibiotics and effectiveness in the treatment. 5 table. The invention relates to biotechnology and represents a new therapeutic drug suppressing the development of veterinary strains of pathogenic microorganisms of the genera: Streptococcus, Esherichia coli, Pseudomonas, Sthaphilococcus, Proteus, Shigella, Hexaera and with a broad resistance to antibiotics.Currently, the drugs of probiotics bacteria of the genus Bacillus, which suppress the development of pathogenic microorganisms, are widely used in veterinary medicine for treatment of various diseases of poultry and agricultural the Bacillus, broad antagonism against pathogenic bacteria and fungi, however, rarely observed antagonism against strains of Pseudomonas aeruginosa and Proteus vulgaris (5).The main drawback of these drugs is low biological effectiveness against strains of Pseudomonas, Streptococcus and the impossibility of a combined use with antibiotics for severe infections. To ensure people have already created products from bacilli with antibioticassociated that allows sharing in order to prevent the development of dysbacteriosis (6).Closest to the invention is the preparation of Sublin containing biomass recombinant strain of B. subtilis VKPM B-4759 and the filler, which is used in veterinary medicine for the prevention and treatment of diseases of the gastrointestinal tract, dysbacteriosis (4). However, the drug has low activity against Pseudomonas aeruginosa (4-6 mm stunted growth of the test strains) and not retards the growth of Escherihia coli and Streptococcus, which are often the cause of diseases of farm animals. Also unknown to its sensitivity to antibiotics, which limits its scope with severe forms of chronic inflammatory diseases, LACMA invention - the development of a new drug-probiotic, bacteria of the genus Bacillus, with a broad spectrum of antagonistic activity, including strains of the genera Pseudomonas, Streptococcus, Escherihia coli, the use of which is possible in conjunction with antibiotics.This is achieved by the fact that as the basis of a biological product used strain of Bacillus subtilis 12 IN-DEPT.The strain of Bacillus subtilis 12 IN-DEPT isolated from compost in Ufa district. Refers to the spore-forming aerobic bacteria of the genus Bacillus. Bacteria are gram-positive spore-forming bacilli, polymorphic. Fermented lactose, glucose, sucrose, mannitol, maltose, poorly fermented sorbitol, galactose, arabinose, raffinose, not fermented dulcet. Bacteria are not pathogenic, are characterized by a high antagonistic activity against wide spectrum of pathogenic and conditionally pathogenic microorganisms and resistance to several antibiotics.The strain deposited 7.10.1999 g in the all-Russian state collection of strains of microorganisms, used in veterinary and animal husbandry number of Bacillus subtilis 12V-DEPT. The location of the strain is defined collection of microorganisms, Department of probiotics and bioactive agents (Ugie cells of the strain Bacillus subtilis 12 IN-DEPT and a filler, in the following ratio, wt.%:
Biomass of Bacillus subtilis 12 IN-DEPT - 1109- 2109living microbial cells in 1 ml of solvent 92-95
Filler - 5-8
As a solvent, you can use saline solution, distilled or boiled water. As the filler, the preparation may contain 5% lactose, or 8% of Saharsa-gelatin mixture (7% sucrose and 1% gelatin). The need for filler due to its protective effect for bacterial cells further lyophilic drying of the drug.The drug is characterized by a high antagonistic activity against pathogenic and conditionally pathogenic microorganisms. Antagonistic activity was tested by the method of deferred antagonism (tab. 1).The drug is characterized by being (tab. 2) and resistance to several antibiotics (tab. 3).Example 1. On the basis of a strain of B. subtilis 12 IN-DEPT with typical morphological, cultural, biochemical properties prepared with 5 variants of the drug with different content of microbial cells.Option 1. The product containing wt.%:
Biomass of B. subtilis 12-DEC - 1,0108living microbial cells in 1 ml BR>Biomass of B. subtilis 12-DEC - 5,0 108living microbial cells in 1 ml of physiological solution - 92
Filler - Saharsa gelatin mixture - 8
Option 3. The product containing wt.%:
Biomass of B. subtilis 12-DEC - 1,0109living microbial cells in 1 ml of physiological solution - 93
Filler - Saharsa gelatin mixture - 7
Option 4. The product containing wt.%:
Biomass of B. subtilis 12-DEC - 2,0109living microbial cells in 1 ml of physiological solution - 94
Filler - Saharsa gelatin mixture - 6
Option 5. The product containing wt.%:
Biomass of B. subtilis 12-DEC - 5,0109living microbial cells in 1 ml of saline - 95
The filler is lactose - 5
Manufactured variants of the drug was poured into sterile ampoules and freeze-dried. Tested safety the obtained variants of the drug on laboratory animals, specific antagonistic activity against the test cultures of representatives of the different groups of pathogenic and conditionally pathogenic microorganisms, sensitivity to antibiotics.To determine the safety of the contents of the vial were dissolved in 0.5 ml of saline and injected the dose of Perera is, if all mice remained alive for five days of observation, and none of them identified the disease.To determine the specific activity investigated the antagonistic activity of variants of the drug in relation to the test cultures. The study was carried out by the method of deferred antagonism. For this purpose the contents of the vial were dissolved in 1 ml of physiological solution. The obtained suspension was sown by the bar diameter Petri dishes with agar medium Gause N 2. Crops were incubated in a thermostat at a temperature of 37oC for 72 h and Then grown culture was podseval stroke test microorganisms (500 millionth suspension subsistence crops in saline solution). Analysis was performed after 18 hours incubation at 37 ° oC largest zones of no growth of the test cultures. Control of the growth of the test cultures was parallel growing them on plates with agar medium Gause N 2 without the studied culture.Drug sensitivity to antibiotics was determined using the standard discs impregnated with antibiotics. In a Petri dish with IPA poured a 1 ml suspension of the daily culture of the strain of 0.9% sodium chloride solution containing 1109the surface of the seeded agar tweezers played discs with antibiotics, on 5 disks on each Cup. The Cup was kept in a thermostat at a temperature of (371)oFor (182) h, and then measured the zone of growth inhibition around the discs, including the diameter of the disk.The results of the trials are presented in tables 1 to 3.Antagonistic activity of 5 variants of the product are presented in table. 1.From the data table. 1 shows that the optimal number of live microbial cells in 1 dose is 1109-2109cells/ml Further increase in the number of microbial cells does not change significantly antagonistic activity of the drug against the test cultures of microorganisms.A study of the safety of different variants of the drug on white outbred mice when introduced per os 1 dose. Monitoring the weight change of animals was carried out for 5 days. The data obtained are presented in table. 2.The test showed that all animals were alive, their initial weight was increased, there were no signs of the disease.A study of antibiotic resistance of different variants of the drug. The data presented in table. 3.Polucen the study of therapeutic efficacy of the drug, conducted its tests in the livestock complex USC "Alimova" Aktanysh region of Tatarstan. The drug is used for prophylaxis and treatment of calves and piglets from dyspepsia, edema disease. Shown its efficacy and good tolerability.Example 2. Prevention the drug was administered with food and water at a dose of 1 ml per 10 kg of body weight twice a day for 7 days, and then in the same doses 2 times a week for 2 months. 4).To ensure the drug was administered in a dose of 2 ml per 10 kg of body weight 3 times daily for 10 days (table. 5). Other antibacterial drugs in the treatment of drug was not used. Controlling antibiotic was chosen on purpose.Separately observed cases of edematous disease and dyspepsia for prophylactic use of the drug was usually resulted in an easy manner and without loss of weight of the animal. Complications during the application and increase the dose, were observed.Thus, studies of the proposed drug has shown that it is harmless, is characterized by a high antagonistic activity against test strains of pathogenic and conditionally pathogenic microorganisms, has expressed antibioticassociated and has a therapeutic effect.Application: therapeutic drug IP is x pathogenic and conditionally pathogenic microorganisms.Used sources of information
1. Ivanovo A. A., Veprev N. S., Dimireva centuries, Lagunova O. P. a method of obtaining a probiotic for veterinary / RU Patent N 2084233, publ. 20.07.97. - Bull. N 20.2. Smirnov centuries, Reznik, S. R., Sorokulova I. B. and other Preventive biological preparation of sublin / RU Patent N 2035185, publ. 31.01.92. - Bull. N 14.3. Reznik, S. R., Sorokulova I. B., Kunicka C. A. and other Preventive biological sporulant / Patent N 2035186. - EN. - A 61 K 35/66, publ. 20.05.95, bull. N 14.4. Shust A. N., Beliavsky C. A., Alikin Y. C. New therapeutic drug sublin: the efficacy in diseases of calves / Actual problems of veterinary medicine: abstracts. Dokl. 1 Scientific-practical use. the conference actually. wet. the honey. NSAU. Novosibirsk, 1997. - Novosibirsk, 1997. S. 15-16.5. Blinkova L. P. , Semenov, S. A., Butova L., and others Antagonistic activity of freshly isolated strains of bacteria of the genus Bacillus // IMEI. - 1994. - N 5. - S. 71-72.6. Zhirkov I. N., Bratukhin And. And. the Application of probiotic RACES for the correction of dysbacteriosis in calves // veterinary medicine. 1999. - #4. - S. 40-42. Therapeutic biological product, including live microbial mass of Bacillus subtilis and a filler, characterized in that it contains biomass of B. subtilis 12 IN - DEPT when the trail is solvent - 92-95
Filler - 5-8
FIELD: biotechnology, microbiology, medicine.
SUBSTANCE: invention relates to the strain Lactobacillus paracasei CNCM I-2116 used for diarrhea prophylaxis causing by pathogenic microorganisms. Supernatant of this strain culture elicits ability to prevent colonization of intestine with pathogenic microorganisms causing diarrhea also and this strain is designated for preparing agent used for prophylaxis and/or treatment of disorders associated with diarrhea. Agent for oral administration represents therapeutically effective dose of the strain L. paracasei CNCM I-2116 or supernatant of its culture and acceptable foodstuff. Invention provides the enhanced viability of the strain in its applying and effectiveness in prophylaxis of adhesion to intestine cells and invasion to intestine cells of pathogenic microorganisms causing diarrhea.
EFFECT: valuable medicinal properties of strain.
5 cl, 8 dwg, 10 ex