RU2203946C1 - Strain of bacterium lactobacillus acidophilus nv ep 317/402-x "balans-narine" used for preparing antigastritis and antiulcerous fermented-milk product - Google Patents

Abstract

FIELD: biotechnology, medicine, medicinal microbiology. SUBSTANCE: invention relates to the strain of lactobacillus of species Lactobacillus acidophilus that can be used for preparing fermented-milk curative-dietetic foodstuffs. The strain Lactobacillus acidophilus N.V. Ep 317/402-X (author name is "Balans- Narine") is obtained by selection of the parent culture Lactobacillus acidophilus N.V. Ep 317/402 "Narine" and directed selection by the following indices: -antagonistic activity relative to microorganisms causing gastroenteric diseases, in part, pathogenic bacteria Helicobacter pylori causing different forms of gastritis and stomach and duodenum ulcers; - ability to produce useful substances (vitamins, amino acids, antioxidants); - resistance of bacterial cell membrane structures to external effects (acidity, temperature and so on) providing retention of lactobacilli high titer in the storage process. The strain Lactobacillus acidophilus shows more broad spectrum of antagonistic activity, in part, relative to pathogenic bacteria Helicobacter pylori causing different forms of gastritis and stomach and duodenum ulcers and significantly more content of useful substances that are necessary for correction of dysbacteriosis and different gastroenteric diseases. EFFECT: valuable medicinal properties of strain and fermented-milk product. 4 tbl, 5 ex

Description

 The invention relates to biotechnology and medical microbiology and is a strain of lactobacilli of the species Lactobacillus acidophilus, which can be used to produce fermented milk dietetic products.

 The functions of normal microflora in the body are vital and very extensive: synthesizing, regulatory, desensitizing, detoxifying, enzymatic, protective and immunostimulating.

 Preparations and products containing lactobacilli are widely used for the prevention and treatment of dysbiosis and normalization of the intestinal microflora of people who have undergone stressful effects, antibiotic and chemotherapy.

State of the art
Probiotics and medical nutrition products based on strains of lactobacilli of the species Lactobacillus acidophilus are widely used abroad (US patent 3639666; French patent 2622452; Swiss patent 637297; German patent 2421066; Japanese patent 05227946; International application (WO) 93/09793).

 However, the strains of lactobacilli of foreign developers are focused on the creation of mainly therapeutic and prophylactic drugs and dietary supplements in dry form (tablets, capsules). These strains have significant drawbacks if they are used for the preparation of preparations and fermented milk products in liquid form: the short shelf life of these preparations and products, low organoleptic qualities that are not essential for preparations in dry form.

 A known strain of acidophilic bacteria Lactobacillus acidophilus-13 (Aragats), deposited under the number 9606 in the Museum of Cultures of the Institute of Microbiology of the National Academy of Sciences of Armenia (Application for patent of the Russian Federation 94017944, IPC C 12 N 1/20, publ. 03/27/96). The strain is resistant to antibiotics, antiseptic and chemotherapeutic test drugs. The strain was obtained by chemical mutagenic exposure, that is, by introducing lethal doses of chemicals for the strain into the culture medium and further multiplying single survivors of the extreme exposure to lactobacilli.

 However, the delayed biological safety of the acidophilic strain thus obtained and products based on it has not been studied in detail, although it does not have acute toxicity when tested in a clinical setting.

 A known strain of Lactobacillus acidophilus N.V. EP 317/402 "NARINE" AAA, used in the preparation of drugs, dietary and therapeutic products for the treatment of dysbiosis and its consequences (RF Patent 2146454, IPC A 23 C 9/12, publ. March 20, 2000). The strain was obtained by selection from the initial culture of Lactobacillus acidophilus N. V. EP 317/402 "NARINE" by selection for antagonistic activity against intestinal microflora, resistance to broad-spectrum antibiotics.

 However, despite the targeted selection of this strain for antagonistic activity against the intestinal microflora, it does not suppress the activity of pathogenic bacteria such as Helicobacter pylori in the gastrointestinal tract, which cause destruction of the intestinal and gastric mucosa and lead to various forms of gastritis and peptic ulcer diseases stomach and duodenum.

The closest analogue (prototype) is the strain Lactobacillus acidophilus NV EP 317/402 "NARINE" (A. S. USSR 163573, IPC C 12 N 1/20, publ. 04/27/1964). The initial culture was isolated from the feces of a newborn healthy child. The strain is identified and received by L.A. Yerzinkyan at the Institute of Microbiology of the Academy of Sciences of Armenia. Cultivation media: sterilized whole or non-fat milk, sterilized whey. The cultivation temperature is + 37-39 ^o C. Optional anaerobic. The clot formation time is 3-7 hours, depending on the incubation temperature and the amount of yeast (1-3%) added to the milk. The frequency of re-culture - 1 time per month. The number of bacterial cells of the strain in 1 ml of “Narine” starter culture is in the range of 150-250 million. The strain is included in the group of acidophilic bacteria, which are microaerophiles. The minimum growth temperature is above 20 ^o C, optimal 35-39 ^o C, lethal 68-70 ^o C. The maximum acid formation in milk is 300-360 ^o T. This strain produces a significant amount of antimicrobial substances useful for humans, especially for children, (Erzinkyan L.A. Biological features of some lactobacillus bacteria (Izd.

 However, the prototype strain has insufficient antagonistic activity, in particular, does not inhibit the activity of pathogenic bacteria such as Helicobacter pylori in the gastrointestinal tract, which cause destruction of the intestinal and gastric mucosa and lead to various forms of gastritis and peptic ulcer of the stomach and duodenum.

 The technical result of the invention is a strain of lactobacilli Lactobacillus acidophilus, which has a wider spectrum of antagonistic activity, in particular against pathogenic bacteria Helicobacter pylori, causing various forms of gastritis and ulcers of the stomach and duodenum and a significantly higher content of useful substances necessary for the correction of dysbacteriosis and various gastrointestinal intestinal diseases.

The indicated result is achieved by obtaining the strain of Lactobacillus acidophilus NV Ep 317/402-X (author's name "Balance-Narine") by selection of the original culture of Lactobacillus acidophilus NVEp 317/402 "Narine" and directional selection according to the following indicators:
- antagonistic activity against microorganisms that cause gastrointestinal diseases, in particular, pathogenic bacteria Helicobacter pylori, causing various forms of gastritis and ulcers of the stomach and duodenum;
- the ability to produce useful substances (vitamins, amino acids, antioxidants);
- resistance of the membrane structures of bacterial cells to external factors (acidity, temperature, etc.), ensuring the preservation of a high titer of lactobacilli during storage.

 The inventive strain of Lactobacillus acidophilus N.V. Ep 317/402-X "Balance-Narine" was deposited under MCCM B-17 in the Collection of the Interregional Center for the correction of human microcenosis (Novosibirsk).

 The strain Lactobacillus acidophilus N.V. Ep 317/402-X "Balance-Narine" has the following properties.

Cultural and morphological:
Fixed asporogenous rods 2-20 x 0.8-0.9 microns, cells are located singly or in the form of short chains, motionless, gram-positive, spores and capsules do not form.

 Gram stains positively. When stained with methylene blue, distinct metachromatic grains are observed in the cells.

 Homoenzymatic, microaerophiles. Colonies on elective agarized nutrient media are deep, shallow, often form tangles in the form of tangled threads.

Physiological:
The minimum growth temperature is 20 ^o C, optimal + 37-39 ^o C.

 The proposed strain has an increased proteolytic, aroma-forming, antagonistic activity, synthesizes vitamins, amino acids in milk.

Bacterial cells do not form indole and skatole, they do not dilute gelatin, and lactose is fermented vigorously. The strain is non-pathogenic and non-toxic. The temperature range of the growth of the strain is 20-48 ^o C. The physiological range of pH of the vital activity of bacteria of the strain 3.8-6.5. Catalase-negative. It decomposes glucose without gas formation. The logarithmic growth phase in serum or milk medium (skim milk) ends by 8-14 hours with the formation of a uniform clot. Fermentation: lactose, glucose, sucrose, maltose, sorbitol, dulcite, galactose. Does not ferment - mannitol, glycerin. Indole does not form.

 The strain produces a significant amount of harmless antibiotic substances that inhibit the growth and development of both gram-positive (St aureus) and gram-negative (E. coli) pathogenic bacteria, including, without exception, the causative bacteria of dysentery and deserter-like diseases.

 The strain is highly sensitive to antibiotics: benzylpennicillin, streptomycin, erythromycin, carbipicillin, lincomycin, tsifobid, levomycin, doxycycline, furazolidone.

 The strain is weakly sensitive to antibiotics: gentamicin, tetracycline, oletetrin, kefzola, rifampicin, ampicillin, kanamycin, nitroxaline, fusidine, cloforan, polymyxin, ampiox.

 It is insensitive to antibiotics: sulfodimethoxin, sulfadimezin, biseptol, toravid, sulfalene.

 The antagonistic activity of the strain, expressed in millimeters of zones of inhibition of growth of pathogenic Flexner test strains and Sonne strain, Proteus, enteropathogenic Escherichia coli, Staphylococcus aureus, is at least 15 mm.

 In whole milk with a SOMO content of 8-10%, the dynamics of growth and acid formation are as follows.

 Cultivation medium: whole cow's milk, normalized or skimmed (reverse).

Cultivation temperature: 37-39 ^o C.

 Incubation time: 5-7 hours, depending on the incubation temperature, the amount of yeast introduced into milk is 1.5-2%.

 Maintaining the strain.

Storage Methods:
- in a liquid nutrient medium at + 5-7 ^o C;
- lyophilized culture at + 5-7 ^o C;
- in liquid nitrogen at minus 193 ^o C.

Storage media:
- whole sterilized milk;
- sterilized skim milk;
- whey sterilized.

 Frequency of transfers 1 time in 2 months.

 Strain Lactobacillus acidophilus N.V. EP 317/402-X "Balance-Narine" can be used in bacterial starter cultures, as well as for the preparation of medical and dietary products.

 The selection of the proposed strain was carried out by traditionally passaging the initial strain 317/402 on a milk-whey medium without introducing any artificial chemical mutagen substances, and therefore the strain does not have a potential hazard.

 Example 1. The method of selection of cells of a strain of Lactobacillus acidophilus N.V. Ep 317/402-X "Balance-Narine" on antagonistic activity against pathogenic bacteria Helicobacter pylori.

The adhesion of Helicobacter pylori bacteria to the walls of the gastrointestinal tract was modeled on their known ability to attach to cancer cells (adenocarcinomas) of the intestine, for example, to a monolayer of HT29 cells. For this, HT29 cells were cultured on defatted glasses at 37 ^° C in Dulbecco's minimal modified medium (DMEM) containing 10% galactose and dialyzed calf plasma in an atmosphere of 10% CO ₂ and 90% air and used until the 2nd division.

Helicobacter pylori bacteria are cultured separately on Mueller-Hitton plates in 10% sheep blood for 72 hours at 37 ^{° C.} in an atmosphere of 5% O ₂ , 10% CO ₂ and 85% nitrogen. Bacteria are scraped and collected from the plates and washed in saline. Helicobacter pylori cells are applied to a monolayer of HT29 cells in an amount of 10 ⁶ cells / cm ² and incubated for 2 hours at 37 ^° C followed by washing of non-adherent cells. The number of Helicobacter pylori cells attached to HT29 cells is determined by a urease test. The intensity of the reaction is read from a spectrophotometer at a wavelength of 550 nm.

Plates with monolayers of Helicobacter pylori bacteria adhering to HT29 cells are placed in the cell suspension of the studied lactobacillus strain Lactobacillus acidophilus NV Ep 317/402-X Balance-Narine and incubated for 1 hour, followed by washing the plates with monolayers and testing for. A cell suspension based on the inventive strain of lactobacilli Lactobacillus acidophilus NV Ep 317/402-X "Balance-Narine" is able (after prolonged selection) to reduce the content of attached Helicobacter pylori cells to the monolayer of HT29 cells from 10 ⁶ cells / cm ² to 10 ² cells / cm cm ² .

 This confirms that the inventive strain of Lactobacillus acidophilus N.V. Ep 317/402-X "Balance-Narine" and its metabolic products have antagonistic activity against pathogenic bacteria Helicobacter pylori, causing gastritis or gastric ulcer, and thus has anti-gastritis and antiulcer properties.

 Example 2. As a result of the selection of the strain, the authors selected variations of the passaged strain that spontaneously changed during cultivation (passage 110-115), based on an increase in the total density of lipoprotein structures of the cell membranes of strain 317/402. The stability of membrane structures increases the viability of the cells of the strain at maximum acidification of the nutrient medium (up to 300 - 350 deg.T), i.e. under adverse environmental 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 producing acid and when the cells pass the acidic gastric “barrier” strain in case of oral administration.

 This is especially important for lyophilized forms of bacteria.

Table 1 shows the growth dynamics of strains of Lactobacillus acidophilus N ₁ V ₁ Ep 317/402 (prototype) and 317/402-X "Balance-Narine" (the claimed strain) in a milk growth medium at a temperature of + 40 ^o C.

 Analysis of table 1 shows that the claimed strain in comparison with the prototype most fully uses the growth resources of a relatively poor growth medium (skim milk without growth factors), has higher growth and technological characteristics compared to the prototype strain, namely: reduced ripening time by 4 hours, the titer in the finished product increased by an order of magnitude, and after 96 hours of cultivation, the biological activity of the claimed strain was higher by one order of magnitude. In addition, the inventive strain in comparison with the prototype has a high activity of acid formation, which contributes to the accumulation of volatile acids in a concentrate of lactobacilli, and, therefore, increased antagonistic activity of products obtained using this strain.

 Example. 3. The study of the amino acid composition of biomass prepared on the basis of the strain L. acidophilus 317/402-X "Balance-Narine."

 Studies of the amino acid composition of biomass prepared on the basis of the L. acidophilus 317/402-X "Balance-Narine" strain were carried out on a US Biotronik amino acid analyzer.

Experiment Conditions
1. Sample preparation: precipitation of the total protein with sulfosalicylic acid; protein removal by centrifugation.

2. The determination of the content of free amino acids in the sample was carried out on a Biotronik LC 5001 amino acid analyzer with the following parameters:
- Column 3.2x385 mm "Biotronik resin 2710";
- Mobile phase: a sequence of five citrate buffer solutions of varying acidity and ionic strength;
- Flow rate: buffer - 0.3 ml / min, reagent - 0.55 ml / min;
- Sample volume: 50 μl;
- Quantification using a calibration solution of amino acids "Hamilton"(USA);
- Detection: post-column staining with a solution of ninhydrin in DMSO, L _vis - 570 nm.

 The results of the analysis of the amino acid composition of the biomass of bacteria L. acidophilus 317/402-X "Balance-Narine" are shown in table 2.

 In the biomass of bacteria based on L. acidophilus 317/402-X "Balance-Narine" the content of free amino acids is increased and reaches about 14.7%, of which the proportion of essential amino acids reaches 50.01%.

 Table 2 shows that the increased amount of essential amino acids from the total amount of free amino acids, especially such as valine - 8.3%, leucine - 13.4%, lysine - 7.7%, and methionine - 3.1%, branched chain amino acids has an important physiological significance, since these amino acids are most actively involved in protein synthesis processes. Thus, a lack of valine leads to impaired coordination of movements, a lack of leucine causes changes in the kidneys and thyroid gland, a lack of lysine leads to disruption of blood formation. In the bifidobacteria liquid concentrate, the arginine content is increased to 7.7%. An increased arginine content is biologically important, since free arginine helps stimulate the body’s immune system, in addition, it has antioxidant properties, which has a beneficial effect on increasing the shelf life of bifidobacteria liquid concentrate.

 Despite the increased utilization of free amino acids by L. acidophilus 317/402-X "Balance-Narine" strain during bacterial growth in a milk growth medium enriched with growth factors, a sufficiently large amount of free amino acids remains in the final product - 418.39 mg / l, especially irreplaceable. In addition, the set of free amino acids is physiological, as evidenced by the ratio of amino acid concentrations of methionine / isoleucine, equal to a coefficient of 0.83, quite close to a coefficient of 1 (optimal physiological ratio).

 Example 4. The study of the antagonistic activity of the strain of Lactobacillus acidophilus N.V. Yer 317/402-X "Balance-Narine."

 Antagonistic activity of the strain of Lactobacillus acidophilus N.V. Ep 317/402-X "Balance-Narine" was studied in vitro by co-cultivation with pathogenic test strains in the zone of growth inhibition of test microbes by the method of bar codes (table 3).

 Antagonistic activity of the strain L. acidophilus N.V. Ep 317/402-X "Balance-Narine" and its colonization activity were most fully studied in vivo in the organism-population model - laboratory mice in a chronic experiment.

 - The studies were conducted on tribal mice of both sexes of the ICR colony for 3 years on the 18th generation of random breeding in the amount of 900 females and 450 males.

 - The preparation of lactobacilli was made on the basis of the strain L. acidophilus N. V. Ep 317/402-X "Balance-Narine" on a dairy medium with the addition of growth factors.

 - The preparation of lactobacilli was used at a dose of 0.1 ml per head per day for 1 month daily.

 - Bacteriological studies of feces of mice were carried out in accordance with the guidelines "Intestinal dysbiosis and methods for its laboratory diagnosis."

 - Surveillance of survival, weight of mice was carried out.

 - Based on the data on the number of corpus luteum and implantation sites of live and dead embryos, the post- and preimplantation mortality of the embryos was determined.

The results of a bacteriological study of young generation 1, received from parents who received a liquid preparation of lactobacilli, showed that there was not only a significant decrease in the level of pathogenic and conditionally pathogenic microflora, but also an increase in bifidobacteria and lactobacilli almost equally from 10 ⁵ to 10 ⁸ - 10 ⁹ microbial bodies in 1 g of feces (Table 4).

 Bacteriological studies of the feces of breeding young stocks of the next generations showed the presence of a formed barrier effect of gastro-intestinal microflora in relation to pathogenic microorganisms such as Salmonella, Staphylococcus aureus, Clostridium digficile, Candida, Proteus, etc. fungi. This is the result of the intestinal colonizing ability of mice strain L. acid NV Yer 317/402-X "Balance-Narine." The earlier formation of the microflora of newborns through the correction of the microflora of the parents provided good body protection and the ability to resist infection from the outside.

 Example 5. Obtaining a therapeutic and dietary anti-gastritis anti-ulcer dairy product based on the starter culture of lactobacilli L. acidophilus N.V. Yer 317/402-X "Balance-Narine."

Lactobacillus starter culture, prepared on the basis of a new strain L. acidophilus NV Ep 317/402-X "Balance-Narine", was used for the preparation of fermented milk products of therapeutic and dietary nutrition. As a basis, milk is used, which is normalized by the mass fraction of fat. Then the milk is subjected to "deep" pasteurization at a temperature of 92 ± 2 ^o C with holding for at least 15 min and cooled to a fermentation temperature of 35 ^o C. Milk is fermented in fermented milk beverage tanks with a cooled jacket equipped with special stirrers for uniform mixing of the milk clot. In milk make industrial sourdough based on liquid sourdough of lactobacilli (strain L. acidophilus NV Ep 317/402-X "Balance-Narine") in an amount of 2-5% by weight of the normalized mixture. The resulting mixture is fermented at a temperature of 38 ± 1 ^o C for 4-5 hours. The end of the fermentation is determined by the formation of a sufficiently strong clot and acidity, which should be no more than 100 ^o T. Upon completion of the fermentation, the product is cooled to a temperature of 25-30 ^o C, feeding ice water into the inter-space of the tank. Then the clot is stirred for 10-15 minutes. The mixed clot is immediately fed to the bottling, and then cooled to a temperature of 6 ± 2 ^o C in a refrigerated chamber in a packaged form. The shelf life of a fermented milk product packaged in containers with airtight capping is 5 days.

 Experimental animal studies, the prevention and treatment of sick people in a hospital and on an outpatient basis show that a fermented milk product made on the basis of the inventive strain has high antagonistic activity, anti-gastritis and antiulcer properties and an increased shelf life with a high titer, which allows it to be used as a drug for the treatment of gastritis and ulcers of the gastrointestinal tract, immunocorrection of the body; treat bacterial and viral dysbiosis, dyspepsia and dysentery without antibiotics.

 Thus, it follows from the foregoing that a new, selected strain of lactobacillus Lactobacillus acidophilus N. V. Ep 317/402-X "Balance-Narine" is possessed for patenting, which exhibits a stable manifestation of the above properties upon subsequent passage of the museum culture of the strain on the Blaurock liver medium. In total, the claimed strain passed 57 passages without changing the selected properties.

The proposed strain of Lactobacillus acidophilus NV Ep 317/402-X "Balance-Narine" compared with the strain of the prototype has the following advantages:
- increased the biosynthetic activity of the strain and its growth activity in the utilization of free amino acids of milk culture media, which increased by 9-13 times in relation to most free amino acids;
- the safety of live lactobacilli in the milk culture medium increased by one order of magnitude;
the strain has higher therapeutic and prophylactic properties, in particular, has targeted anti-gastritis and antiulcer properties.

Claims (1)

 The bacterial strain Lactobacillus acidophilus N. V. Ep 317/402-X MCCM B-17 (the author's name is "Balance-Narine") used for the preparation of anti-gastritis and antiulcer sour milk product.

Images