RU2061040C1 - Strain of bacterium lactobacillus plantarum showing capability to decrease oxalate level and used for preparing preparations and foodstuffs for hyperoxaluria prophylaxis and treatment - Google Patents

Abstract

FIELD: microbiology. SUBSTANCE: strain Lactobacillus plantarum CS (Collection number 152 of the State Microorganism Collection) shows capability to decrease oxalate level, develop on the nutrient media of the different composition and accumulate the productive biomass. Strain is isolated from intestine of health adult man. EFFECT: isolation of the strain indicated above. 2 tbl

Description

 The invention relates to microbiology, and relates to a strain of bacteria of the destructor of oxalate, which can be used for the preparation of medicinal products and products of medical and dietary purposes with a specific therapeutic effect.

 Among urological diseases, one of the leading places is urolithiasis. The most common stones (59% of all cases) are calcium salts of oxalic acid oxalates.

 Recent studies have shown that the spectrum of clinical syndromes and pathological conditions, the etiopathogenesis of which can be associated with a change in the composition and functions of the normal microflora inhabiting the host’s body, is currently quite wide and tends to increase.

 One of the promising directions for the prevention of oxalate kidney and ureter stones is blocking the absorption of oxalic acid from the intestine. In this regard, the data of foreign scientists on the participation of intestinal microflora in the metabolism of oxalic acid deserve attention. This process involves some types of microorganisms, mainly soil ones, capable of degradation and modification of oxalate: Pseudomonas, Mycobacterium, Alcaligenes, Streptomyces, Clostridium, etc.

 Back in the early 80s, American scientists made attempts to isolate microorganisms from the gastrointestinal tract of animals that are responsible for the degradation of oxalate in the abomasum of ruminants. As a result, they selected obligate-anaerobic non-spore forming oxalate-degrading bacteria with biological characteristics different from all other previously known microorganisms capable of utilizing oxalic acid salts. Later, similar oxalate-degrading anaerobic microorganisms were also isolated from the intestines of other animals and humans and were called Oxalatobacter formigenes. Data on the distribution of oxalate-degrading microorganisms in the human intestine is limited, although all researchers dealing with this issue have confirmed the presence of such microorganisms in human feces. Data were also obtained on the possibility of creating an animal model to detail the role of anaerobic oxalate-degrading bacteria in the metabolism of dietary oxalate in mammals. In recent years, a series of studies has been conducted to study the biochemistry of the transformation of oxalic acid and its salts by anaerobic O. formigenes. It was also established that the number of such anaerobic bacteria is directly dependent on the concentration of oxalates entering the intestinal tract with food. In humans, the number of such bacteria can reach significant numbers. According to Allison M. J. et al., It is these bacteria that are responsible for the degradation of 50-100 mg of oxalic acid in the digestive tract daily.

 It is necessary to emphasize the fact that the results of research in this area are far from their practical use.

 The purpose of the invention is a strain of lactobacilli with oxalate-degrading activity.

 The strain Lactobacillus plantarum CS was deposited in the Museum of microorganisms of representatives of normal human microflora of the Moscow Research Institute of Epidemiology and Microbiology named after G.N. Gabrichevsky under N 152.

 The strain Lactobacillus plantarum CS is isolated from the intestinal contents of a healthy adult.

 The strain is characterized by the following cultural and morphological characteristics: short motionless gram-positive rods, uniformly stained, single or arranged in a chain of 2-5 cells, the size of cells is 5-10 microns, spores and capsules do not form, microaerophile.

 On a solid MRS medium it forms flat, opaque, grayish, convex, rounded, with even edges of a colony 1.5-2 mm in size after 48 hours of growth, the consistency is oily, no pigment. The smear is located in random clusters and separate chains. Granularity and other inclusions are absent.

Optimum temperature: ^about 38-40, pH 6.4-6.6.

 Biochemical properties.

 The oxidase test is negative. Catalase is negative. On the environments of Endo, Ploskireva Saburo does not grow.

 It is able to actively develop on liquid nutrient media of various compositions intended for the cultivation of lactobacilli and accumulate production biomass by 18-20 hours of cultivation. It ferments L-arabinose, ribose, galactose, glucose, fructose, mannose, mannitol, sorbitol, amygdalin, arbitin, esculin, salicin, celobiose, D-turanose, gluconate, maltose, trehalose, lactose, melibiosis, sucrose, raffinozito, melozito, : does not ferment erythritol, D-arabinose, xylose, inulin, sorbose, ramnose, dulcitol, xylitol, does not dilute gelatin.

 According to the results of a detailed biochemical analysis on the API-CHL system, the strain was identified as Lactobacillus plantarum.

 Attitude to antibiotics: Sensitive to erythromycin, carbenicillin, oleandomycin, chloramphenicol, oxacillin, amoxicillin; moderately resistant to rifampicin, penicillin, doxycycline, ampicillin. Resistant to neomycin, polymyxin, tetracycline, kanamycin, ristomycin, streptomycin, nalidixic acid, vancomycin, lincomycin. Duration of milk coagulation is 10-12 hours. High antagonistic activity against Escherichia coli, Staphylococcus aureus, Shigella, Proteus. Harmless: Harmless when tested on white mice by administering per os.

 PRI me R 1.

Strain Lactobacillus plantarum CS grown in liquid MRS medium-2 at 38 ^{° C} for 18 hours, then the culture was seeded loop on Petri dishes with MRS-4 medium and cultivated under the same conditions. After 18 hours, a microbial suspension of the culture is prepared for inclusion in the semi-synthetic nutrient medium API of the following composition, g / l: Polypeptone 10 Yeast extract 5 Tween-80 1 K ₂ NRA ₄ 2 Acetate Na 5 Ammonium citrate 2 MnSO ₄ · 4H ₂ O 0, 05 MgSO ₄ · 7H ₂ O 0.2 Bromocresol red 0.17 Oxalate Na 20 Distilled water up to 1000 pH 6.9-7.0
Seed is introduced into the experimental semi-synthetic nutrient medium in an amount of 10% of the total volume with a final concentration of bacteria in this medium of 10 ⁷ CFU / ml. Incubation was carried out at 38 ^{° C} in microaerophilic conditions for 72 h.

As control use the same nutrient medium without microorganisms. To determine the oxalate content in the culture medium by gas chromatography, the samples are pre-centrifuged for 15 minutes at 12,000 rpm. Then, 0.4 ml of 50% H ₂ SO ₄ , 2 ml of CH ₃ OH and 100 ml of a 1% solution of fumaric acid are added to 1 ml of the supernatant. After incubation at 60 ^about for 30 min in a vial with a tightly closed stopper, add 1 ml of H ₂ O and 0.5 ml of chloroform. Shake well and centrifuge. A layer of chloroform is taken for research. Quantitative determination of oxalate in the medium is carried out by gas-liquid chromatography on a 104: Pye Unicam chromatograph (England) with a Shimadzu Data Processor Cromatopac C-R3A flame ionization detector, a glass column 2 m long and 2 mm inner diameter. Column filled with Sp 1200 / I / H ₃ PO ₄ on Chromosorb WAW. Detector temperature 260 ^{° C} evaporator temperature of 250 ^C. The feed rate of the carrier gas (nitrogen), 30 mL / min. The results of determining the ability of various strains of lactobacilli to reduce the level of oxalate are presented in table. one.

 As can be seen from the table. 1, the strain of Lactobacillus Plantarum CS reduces the level of Na oxalate in the in vitro experiment after 72 hours by 46% from the initial level. At the same time, the content of Na oxalate in the control solution, without adding the test strain, remained at the same level. The strain Lactobacillus plantarum CS stably retains the ability to reduce the level of oxalate during prolonged passage on nutrient media for cultivation of lactobacilli.

 PRI me R 2. Oxalate-reducing activity of the strain and the initial concentration of oxalate in the experimental nutrient medium. The conditions of the experiment are the same as in example 1, add a different concentration of sodium oxalate. After incubation, according to the above methodology, the final concentration of oxalate in the samples is determined and the percentage reduction in its level is calculated (Table 2).

 In the table. Figure 2 shows the oxalate-lowering activity of the strain L.plantarum CS depending on the initial concentration of Na oxalate in the experimental nutrient medium over time.

 Analysis of the results is carried out after 24, 48 and 72 hours by comparing the content of oxalate in the control experimental samples.

 The oxalate-lowering ability of the strain L.plantarum CS little depends on the initial concentration of oxalate Na in the medium. At a lower concentration in the first day, the decrease in oxalate in the medium is more active, although in the next two days this difference is almost leveled.

 Thus, taking into account the ability of the strain Lactobacillus plantarum CS to actively reduce the level of oxalate, to develop on nutrient media of different compositions and to accumulate production biomass by 18 hours of cultivation, it can be used for the preparation of pharmacopeia preparations and food products of targeted action for the treatment and prevention of hyperoxaluria, including urolithiasis.

Claims (1)

 The bacterial strain Lactobacillus plantarum is deposited in GKNM N 152, which has the ability to reduce the level of oxalate and is used to obtain drugs and food products for the prevention and treatment of hyperoxaluria.

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