RU2176668C1 - Strain of bacterium lactobacillus acidophilus nv ep 317/402 "narine" tnci used for preparing curative-prophylactic preparations for intestine microflora normalization - Google Patents

Abstract

FIELD: biotechnology, microbiology. SUBSTANCE: invention relates to the strain of lactobacillus showing ability to produce interferon in human unicellular cells and to effect on physiological and immunological processes in body. The strain Lactobacillus acidophilus N.V.EP 317/402 "Narine" TNCi is deposited in VKPM at N B-8017. The strain is obtained by of many years selection of the parent culture L. acidophilus N.V. 317/402 "Narine". The strain is selected by its antibiotic activity to pathogens of gastroenteric diseases, resistance to antibiotics, vitamin producing capacity, growth rate and so on. Ferment of the strain L. acidophilus N.V.EP 317/402 "Narine" TNCi can be used for preparing dietetic and curative foodstuffs or for direct use in nutrition, as medicinal agent for treatment and prophylaxis of dysbacteriosis and its complications. EFFECT: strain indicated above, enhanced effectiveness of preparations. 4 dwg, 3 tbl, 2 ex

Description

 The invention relates to biotechnology and relates to a strain of lactobacilli, which has the ability to produce interferon, to influence physiological and immunobiological processes in the body. The strain can be used for the preparation of drugs and dietary fermented milk products designed to normalize the intestinal microflora.

 The causes of dysbacteriosis are known: these are various infectious diseases, taking antibiotics, chemo-, hormone- and radiation therapy, psycho-emotional stress and adverse environmental conditions, social factors, alcoholism, etc.

 They directly or indirectly contribute to the imbalance of the microbiocenosis of the body, and, first of all, the intestinal microflora.

 For the effective treatment of dysbiosis, it is necessary, if possible, to eliminate its causes and restore the microflora.

 Thus, the urgent task is to identify new strains of lactobacilli and the development of drugs and food products based on them for the prevention and restoration of normal intestinal microflora function, eliminate dysbiosis and its consequences.

 Promising strains of lactobacilli of this species are known that are used in the development of drugs and lactic acid products that contribute to the regulation of intestinal microbiocenosis and nonspecific immunostimulation for intestinal dysbiosis of various ecologies (V. M. Korshunov. “The problem of regulation of intestinal microflora.” Journal of Microbiology, Epidemiology and Immunology M. M. 1995, N 3, pp. 48-52).

 A known strain of Lactobacillus acidophilus VKM B-2020 D-antibiotic-resistant antagonist of intestinal infections. The strain is recommended as the basis for the development of antagonistic biological agents for the prevention and treatment of intestinal diseases caused by pathogenic and conditionally pathogenic microflora. It can be used to create bacterial preparations for the correction of microfunctional disorders of the gastrointestinal tract (RU 2063436 C1, C 12 N 1/20, 08.02.94).

 A known strain of lactobacilli Lactobacillus acidophilus-13 (Agaraz), which positively affects the regenerative processes in the intestine in the treatment of gastrointestinal diseases, is used to correct dysbacteriosis of a different type and nature in newborns and adults.

The strain is used in the manufacture of a medical and dietary food product that helps to suppress the growth of pathogenic forms of microorganisms and normalizes the intestinal microflora due to the high limit acidity of the strain of the order of 400 ^o T (RU 2103354, C 12 N 1/20, 01/27/98).

 A known bacterial strain Lactobacillus acidophilus-92 n.v. (Ani) used to correct dysbiosis in gastrointestinal diseases and in patients with colon cancer (RU 2103354, C 12 N 1/20, 01/27/98).

 A known bacterial strain Lactobacillus acidophilus NV EP 317/402 "Narine" AAA, used in the preparation of drugs, dietary and therapeutic products for the treatment of dysbiosis and its consequences (RU, 2146454, A 23 C 9/12, A 61 K 35/74 C 12 N 1/20, 03/20/2000).

The well-known strain of Lactobacillus acidophilus nv 317/402 "Narine" IN-MI-9602 has a slow acid-forming ability, the maximum acid formation of 360 ^o T. The strain produces a significant amount of harmless to children and adults antibiotic substances that inhibit the growth and development of both gram-positive and gram-negative bacteria .

Long-term observations showed the absence of phagolysis in the strain Lactobacillus acidophilus nv 317/402 Narine. The fermented milk product is prepared by fermenting whole milk at (28-40) ^o C. Coagulation occurs in 5-8 hours, the acidity of the product is 60-90 ^o T. The specified product is suitable as a starter culture, as well as a therapeutic and prophylactic agent for diseases of the gastrointestinal tract . Use the product in medical and veterinary practice (SU 163573, C 12 N 1/20, 1964).

The product obtained using the strain Lactobacillus acidophilus nv 317/402 "Narine", acidity not higher than 90 ^o T does not provide a person with the whole complex of amino acids, vitamins necessary for the correction of dysbiosis and various gastrointestinal diseases.

The aim of the invention is to identify a new strain of Lactobacillus acidophilus nv 317/402 "Narine" TNSi that meets the increased requirements for modern drugs and therapeutic products, resistant to a number of antimicrobial agents, exhibiting high acid-forming activity up to 380 ^o T and pronounced antagonistic properties in relation to opportunistic microorganisms with high vitamin-forming ability, increased productivity of α- and γ-interferon in the human body. All these factors play a significant role in antiviral and anticancer protection, affect physiological processes in the body.

The strain Lactobacillus acidophilus nv 317/402 "Narine" TNSi was obtained by long-term selection of the original culture of Lactobacillus acidophilus nv 317/402 "Narine" and targeted selection according to the indicators: antibiotic activity to pathogens of the gastrointestinal diseases, antibiotic resistance, breeding dynamics of the breeding dynamics (by the number of viable bacteria in 1 ml of strain), vitamin-forming ability. When cultured at a temperature of (38 ± 1) ^o C for 24-36 hours, the maximum number of viable bacteria is achieved, which remains for 2.5 days.

Selection was carried out by traditional passivation of the initial strain nv 317/402 "Narine" on dairy media without the introduction of artificial chemicals - mutagens. Variations of the passaged strain were selected as an important technological breeding trait that spontaneously changed during cultivation (by 100–105 passage) on the basis of an increase in the total density of lipoprotein structural membranes of cells of strain nv 317/402 “Narine”. The stability of membrane structures increases the viability of the cells of the strain at maximum acidification of the nutrient medium (up to 300-500 ^o T), i.e., under adverse conditions for the strain. The increased stability of the membrane structures increases the competitiveness of the strain also when symbiotic cultivation with other lactic acid bacteria - producers of acids (bactericins) and when the cells pass the acidic gastric “barrier” strain in case of oral administration. This is especially important for lyophilized forms of bacteria.

The method of small-angle X-ray scattering (SAXR) was chosen by measuring the total density of lipoprotein structures of the strain cells (qualitative integrated analysis of the internal structure of cells). This is a physical method - an analogue of X-ray diffraction analysis; it does not require any disintegration, destruction of cells, and for the study of a fairly small number of cells washed in saline in the form of a suspension, i.e. allows you to evaluate the truly native structure of cells. The analysis was performed on a Siemens small-angle X-ray diffractometer. The radiation wavelength is λ = 0.154 nm, in the range of angles 0.016 <h <0.056, where h = 4πsinθ / λ. The temperature of the samples is 20 ^o C.

 Lyophilized preparations of strain n.v. were taken as samples. 317/402 "Narine" TNSi produced by FSUE NPO "Virion" ser. 237 and the original strain n.v. 317/402 "Narine" ser. 1.

 The informational content of the SAXM method as a whole has previously been used in other laboratories to study the stability of cell membranes during freeze drying, especially their lipoprotein constituents. Thus, Thomson et al. In the study of rat liver mitochondrial membranes by the SAXS method found that at a moisture content of 20-30%, existing changes occur in the lipid part of the membranes, their reorganization with further dehydration (Shipley GG Recent X-ray diffraction studies of biological membranes.- Biological membranes (London - New York), 1973, vol. 2, p. 1 - 89). Knutton et al., While dehydrating the erythrocyte membrane fraction, found the destruction of membranes in the lipoprotein and lipid fractions using the SAXD method (Knutton S., Finean JB, Coleman R. Low angle X-ray diffraction and electron-microscop stadies of isolated erytrocyte membrane. - J. Cell Sci ., 1970, vol. 7, N 2, p. 357-371).

 In Fig.1 shows a comparative x-ray SAXR of two strains, and in Fig. 2 - calculated functions of interatomic distances in the cell particles of both strains of P (r). Corrections for background scattering with 0.9% sodium chloride solution (saline), beam collimation, and smoothing were performed in the X-ray diffraction pattern of the SAXS, and smoothing was performed (Svergun DI, Feigin L.A. X-ray and neutron small-angle scattering. Nauka, Moscow, 1986 )

 Figure 1, which shows the normalized X-ray diffraction patterns of the SAXS samples, it is seen that the x-ray differs significantly and significantly. Similar differences can be seen on the functions P (r) calculated from the experimental SAXS data (Fig. 2).

Cells of strains nv 317/402 "Narine" TNSi (ser. 237) and nv 317/402 "Narine" (ser. 1) were also comparatively studied by the method of Murray in the active state. From lyophilized samples ser. 237 and ser. 1, laboratory starter cultures (uterine culture) were made under the same conditions in sterile skim milk. After milk fermentation for 3-4 hours at a temperature of (38 ± 1) ^o C with an acidity of 100 ^o T, samples were taken for analysis by the SAXS method.

 Previously, the cells of both samples were washed in saline and besieged on a T-51 centrifuge for 10 min under the same conditions. Background scattering was subtracted from the X-ray diffraction patterns of the analyzed samples and the points were smoothed. The measurement error of the x-ray did not exceed 1%.

 In the table. 1 and in FIG. Figure 3 shows the normalized values of the SAXS intensity from samples ser.1 and ser.237 in linear and logarithmic form. From the table. 1 and FIG. Figure 3 shows that the initial sections of the radiographs practically coincide, but with the numbers of measurement points from 8 to 19, the deviations of the normalized SAXS intensities reach 2-3%, which exceeds the maximum measurement error by 2–3 times. This allows us to make a general conclusion about the comparative analysis of sulfur samples. 1 and ser.237: the total internal structure of Narine cells in these samples is significantly different from each other, which indicates that these are different strains.

 A steeper descending curve from sample ser. 237 indicates that the internal structure of cell membranes is more condensed and homogeneous (possibly more holistic), homogeneous. At the same time, the "elevation" of the curve from the sample is sulfur. 1 indicates the presence in the cell membranes of either more loose, "untwisted" structures, or the presence of a noticeable heterogeneity of the cells themselves.

 Comparative data on the antagonistic activity of the proposed and known strains are shown in table 2.

 The inventive strain has an increased antagonism with respect to the above representatives of enteropathogenic intestinal microflora. It has a higher growth rate than the original strain, which is a positive point for the production of drugs.

 L. acidophilus n.v. 317/402 "Narine" TNSi was deposited in the All-Russian collection of industrial microorganisms VKPM B-8017 and the collection of microorganism cultures of the Federal State Unitary Enterprise NPO "Virion" under the number V-20, as intended for the preparation of the dry preparation "Narine".

Cultural, morphological and biochemical characteristics of the strain
The cells are rod-shaped, usually of regular shape 2.2 • 0.8-0.9 microns with rounded ends. Located singly or in short chains. Gram-positive, do not form a dispute. Optional anaerobic. Colonies on agarized media are deep, shallow, convex, opaque, unpigmented. Often form balls in the form of tangled threads or cotton lumps. Acid resistant. Bacteria multiply by binary halving.

Lactobacilli of this strain are homoenzymatic microaerophiles with facultative anaerobic type of respiration, heterotrophic type of nutrition. The strain has a fermentation type metabolism, sugar-classical. Ferments milk for 8-14 hours at (38 ± 1) ^o C with the formation of a uniform clot. It ferments lactose, glucose, sucrose, maltose, mannose, sorbitol, dulcite, galactose without the formation of gas. Does not ferment mannitol, glycerin. Does not form indole. Nitrate does not reduce. It does not dilute gelatin. Does not produce catalase. The logarithmic growth phase in the dairy medium ends by 8-14 hours.

 The strain produces vitamins A, B, C, enzymes, nicotine, choline, contains minerals (table. 3).

When the strain is introduced into milk at a temperature of (38 ± 1) ^o C for 8 hours, the number of microbial cells and the acidity of the milk gradually increase. Then the growth rate of these indicators increases. The number of viable lactobacilli reaches a maximum value by 36 hours of cultivation and is 10 ¹¹ KOE / ml. This value remains constant up to 2.5 days, after which a decrease is noted, complete cell death occurs on the 5th day.

The fastest increase in acidity is observed 10-12 hours after the introduction of the strain, in the future, the increase in acidity continues and reaches a maximum value of 350-400 ^o T by 3-5 days. The acidification curve (Fig. 4) does not have a downward end, because acidity after reaching the maximum value remains at this level with further milk exposure at (38 ± 1) ^o C. Maintaining a high titer of lactobacillus strain (10 ¹¹ CFU / ml) for 60 hours against the background of an increase in acidity to 400 ^o T is explained by the fact that high-density membrane structures of the cells of the proposed strain are resistant to the damaging effect of acids.

 The strain is lyophilized in a culture medium with the addition of sucrose-gelatin-milk environment.

The resistance of the strain to antibiotics
It is unstable to the following antibiotics: benzylpenicillin, streptomycin, erythromycin, carbenicillin, lincomycin, tsifobid, chloramphenicol, doxycycline, furazolidone.

 Weakly resistant to: gentamicin, tetracycline, oletetrin, kefzol, rifampicin, ampicillin, canomycin, nitroxoline, fusidine, cloforan, polymyxin, ampiox.

 Highly resistant to: sulfadimethoxine, sulfadimesin, biseptol, toravide, sulfalene.

 The resistance of the strain to antibiotics allows its use in bacterio- and antibiotic therapy.

 Example 1

To obtain starter culture, sterilized milk or pasteurized milk is inoculated with a culture of L. acidophilus nv 317/402 "Narine" TNSi at the rate of 2% of the total amount of milk. The mixture is incubated for 28 hours at a temperature of (38 ± 1) ^o C. The starter culture is cooled to 6 ^o C, maintained at this temperature for 8 hours. The acidity of the resulting clot is 190 ^o T. The number of living microbial cells is 10 ¹¹ CFU / ml of liquid starter culture.

 The resulting sourdough can be used as a bacterial preparation with high breeding properties for dietary and medicinal food products or for direct consumption. This lyophilized product is used as a medicine for the treatment and prevention of dysbiosis and its consequences.

 Example 2

For the preparation of the Narine fermented milk product, 2% of the starter strain of L. acidophilus nv 317/402 Narine TNSi is added to whole milk cooled to a temperature of (38 ± 1) ^o C. The mixture is stirred and kept at a temperature of 40 ^o C for 6 hours or at room temperature (18-20) ^o C for 8 hours until a clot with an acidity of 80-90 ^o T, then placed in a refrigerator at a temperature of (4-6) ^o C for 2 hours. The quality of the prepared drink is maintained for 2 days at a temperature of (4-6) ^o C.

 The nutritional value of the Narine product is 30 kcal per 100 g of product.

 The drug is based on a strain of L. acidophilus n.v. 317/402 "Narine" TNSi, passed clinical trials at the Department of Pediatric Infections at the Siberian Medical State University. The effectiveness of the drug “Narine” in complex therapy was studied in 147 children aged 3 months to 14 years with chronic diseases of the digestive system. The first group consisted of children suffering from gastroduodenitis, duodenal ulcer, group 2 - children with chronic intestinal dysfunction, irritable bowel syndrome, dyskinesia of the gastrointestinal tract (GIT), group 3 - children with skin diseases ( eczema, neurodermatitis, atopic dermatitis). The control group consisted of children with comparable pathology who are on complex therapy without the use of the drug "Narine".

 The results showed that the appointment of the drug "Narine" allowed to normalize bowel function in a shorter time, i.e. contributed to the restoration of microflora, suppression of the growth of pathogenic and opportunistic microorganisms, regulation of the local immunity system and enzymatic functions of the gastrointestinal mucosa, and also to eliminate skin symptoms in patients with food allergies.

 The drug "Narine" was used in the treatment of 34 children with gastrointestinal diseases, including infectious (20 children) and allergic (14 children) geneses. The control group consisted of 20 children with a comparable gastrointestinal pathology who received complex therapy without the Narine drug. The results of observations showed that for the normalization of microbiocenosis and the functions of the intestinal mucous membranes, the use of the drug “Narine” and medical nutrition products based on it is indicated. The acidic environment created by living microbial cells promotes high adhesion and vegetation of bifidobacteria in the intestine, contributing to the manifestation of their antagonistic properties against gram-negative bacteria.

 At the Department of Obstetrics and Gynecology of SSMU, the effectiveness of the use of the drug "Narine" for newborns was studied. The duration of use was determined by the days the child was in the hospital. The results of observations showed that the drug "Narine" has an effective effect on the formation of a normal microbial biocenosis of the intestines of newborns, because prevents intestinal colonization by opportunistic enterobacteria. At the same time, a prophylactic effect was noted in relation to skin diseases (dermatitis, exudative diathesis), and oral mucosa (stomatitis, thrush).

 It should be noted that in newborns treated with Narine in the maternity ward, intestinal diseases (intestinal dysfunctions, enterocolitis) were recorded less frequently than in the control group and the number of children without any illness during the first year of life increased.

 In the lipid center of the clinic of the Institute of Therapy of the Siberian Branch of the Russian Academy of Medical Sciences, a study was conducted of the effect of the drug Narine on the level of blood lipoproteins in patients with atherosclerosis. For this, we used the rapid method for determining changes in the fractional composition of blood lipoproteins by the small-angle X-ray scattering method and the contrast method. Within 7 weeks in 11 people receiving Narine, the fractional composition of blood plasma lipoproteins was studied. In 7 of them, before the start of treatment, the fractional composition of blood plasma lipoproteins had a different degree of deviation from the norm in the fractions of high density lipoproteins and low density lipoproteins. In 4 people, the fractional composition of lipoproteins in the blood plasma was normal, they made up the control group.

 Blood was examined weekly before taking Narine, while taking Narine and for 2 weeks after taking Narine.

 Studies have proven the effectiveness of the drug "Narine", made on the basis of the inventive strain. In the treatment and prevention of atherosclerosis, Narine can be used as an independent therapeutic product. After 7 days of use, even a critical (high) level of low density lipoprotein concentration returned to normal without expensive chemical preparations.

 In addition, within 3 months of using the Narine drug, changes in the condition of patients with a diagnosis of atherosclerosis, hypertension of the 2nd degree were studied. Patients who used Narine gradually were able to abandon chemical preparations, as bouts of tachycardia, angina pectoris disappeared, blood pressure was established 160/100.

 In addition, the fermented milk product "Narine" was tested in veterinary medicine (ZAO "Lebedev"). We studied its effect on the survival and productivity of calves. Studies have shown that the use of sour milk "Narine" helps to increase the percentage of survival of calves, increase productivity. An increase in the concentration of bifidobacteria in the feces indicates a positive clinical result of taking Narine for the treatment of dysbacteriosis and diseases associated with a violation of the normal intestinal microflora of animals. Based on test data, Narine is recommended for use in animal husbandry for the health of calves.

 Thus, the inventive strain can be used to treat dysbiosis and diseases associated with a violation of the normal intestinal microflora in humans and animals.

Claims (1)

 The bacterial strain Lactobacillus acidophilus N.V.EP 317/402 "Narine" TNSi VKPM B-8017 used in the preparation of therapeutic and prophylactic preparations for the normalization of intestinal microflora.

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