RU2139346C1 - Strain of bacterium lactobacillus plantarum showing capability to decrease oxalate level and used for preparing preparations and foodstuffs for prophylaxis and treatment of patients with hyperoxaluria - Google Patents

Abstract

FIELD: biotechnology, microbiology, medicine. SUBSTANCE: invention relates to the strain of lactobacillus showing capability to catabolize oxalate. The strain of lactobacillus Lactobacillus plantarum 421-2 is isolated from the health adult human intestine content. The strain is deposited in Museum of microorganisms of normal human microflora of Moscow NII epidemiology and microbiology at N 151. The strain shows the expressed capability to decrease oxalate level in medium after 72 h of culturing by 69% from the parent level as compared 46% in the strain L. plantarum CS. EFFECT: enhanced effectiveness of the strain. 4 tbl, 1 ex

Description

 The invention relates to biotechnology, in particular to microbiology, and relates to a strain of lactobacilli having the ability to degrade oxalate. The strain can be used for the preparation of drugs and food products, including fermented milk, intended for the prevention and treatment of hyperoxaluria.

 According to available data, 52.7% of men and 18.8% of women living in industrialized countries have an increased content of oxalic acid in their urine, and 5-15% of people, when they reach the age of 70, have an attack of urolithiasis at least once . At the same time, most of the stones (70-80%) found in the urinary system are a complex of oxalic acid and calcium - oxalate-calcium stones. The number of people with a high content of oxalate in their urine is constantly increasing, which gave reason to consider urolithiasis as one of the most common "diseases of civilization."

 It is also proved that the level of oxaluria is largely determined by the state of the normal microflora of the large intestine, in particular the presence in it of a sufficient number of microorganisms that can destroy or bind oxalic acid and calcium, and then remove them from the body with feces. In this regard, one of the promising directions for the prevention of the occurrence of oxalate stones in the kidneys and ureters and for the treatment of urolithiasis is blocking the absorption of oxalic acid from the intestine.

A known bacterial strain Bacillus sp., Capable of complete destruction of oxalate when its concentration in the medium is up to 40 g / l and grows at a temperature of up to 41 ^o C (1). Under aeration conditions at a temperature of 28 ^o C, the strain decomposes 42.6 g / l of oxalate in 40-42 hours. This strain is isolated from garden soil, is intended for wastewater treatment from oxalate and is absolutely unsuitable for use by people.

 A known strain of lactobacilli with the ability to reduce the level of oxalate and used to obtain drugs and food for the prevention and treatment of urolithiasis - Lactobacillus plantarum CS (2).

 This strain is isolated from the intestinal contents of an adult healthy person and is safe for humans. However, for the successful development of the microecological direction of prevention and treatment of urolithiasis, it is necessary to expand, like the rotation of antibiotics in chemotherapy, the arsenal of oxalate-degrading strains suitable for preparing pharmaceutical preparations of functional nutrition products on their basis.

 The purpose of the invention is a strain of lactobacilli with the ability to actively degrade oxalate.

 The strain of lactobacilli Lactobacillus plantarum 421-2 isolated from the intestinal contents of a healthy adult. The strain is deposited in the Museum of microorganisms - representatives of normal human microflora of the Moscow Research Institute of Epidemiology and Microbiology. G.N.Gabrichevsky under N 151.

 Cultural and morphological characteristics of the strain.

 Short fixed sticks, single or arranged in a chain of 2-5 cells. Gram-positive, evenly stained. The size of the cells is 5-10 microns. Spore and capsules do not form. Microaerophile.

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

The temperature optimum for growth is 38-40 ^o C, the optimum pH is 6.6-6.9.

 Biochemical properties.

 The oxidase test is negative. Catalase is negative. Saburo does not grow on meat and peptone agar, in the environments of Endo, Ploskirev.

 It is able to actively develop on liquid nutrient media intended for the cultivation of lactobacilli, and accumulate production biomass by 18-20 hours of cultivation.

 It ferments amygdalin, D-arabinose, L-arabinose, arbitin, galactose, gentobiose, glucose, gluconate, dulcitol, lactose, maltose, mannitol, mannose, A-methyl-D-glycoside, melecitose, melibiosis, ribose, salicin, , sorbose, trehalose, fructose, cellobiose, esculin. Does not ferment glycogen, inulin, xylitol, xylose, rhamnose, raffinose, erythritol, D-turanose. It does not dilute gelatin. According to the results of a detailed biochemical analysis on the API-CHL system, the strain was identified as L.plantarum.

 Comparative biochemical characteristics of the strains of L. plantarum CS (prototype) and L. plantarum 421-2 are shown in table 1.

 The table shows that the compared strains can be distinguished by biochemical properties.

 Strain L.plantarum 421-2 has a high antagonistic activity against Shigella, Proteus, Escherichia coli, Staphylococcus aureus.

 The ratio of strain L.plantarum 421-2 to antibiotics.

 It is sensitive to amoxicillin, ampicillin, doxycycline, carbenicillin, chloramphenicol, lincomycin, oxacillin, oleandomycin, penicillin, rifampicin, erythromycin.

 Resistant to vancomycin, kanamycin, carbenicillin, nalidixic acid, neomycin, polymyxin, ristomycin, streptomycin and tetracycline.

 Table 2 presents comparative data on sensitivity to antibiotics of strains of L. plantarum CS and L. plantarum 421-2.

 The data presented in Table 2 indicate the same sensitivity of the compared strains of lactobacilli to antibiotics, with the exception of sensitivity to lincomycin.

 The duration of coagulation of milk with strain L.plantarum 421-2 with at least 2% sourdough is 10-12 hours. The results of a comparative study of the time of fermentation of milk with strains of L. plantarum CS and L.plantarum 421-2 are presented in table 3.

 Thus, as follows from table 3, the proposed strain of lactobacilli fermented milk faster, regardless of the dose of yeast.

 Harmlessness.

The strain L.plantarum 421-2 was harmless when tested on white mice by introducing bacteria per os at a concentration of 10 ^10-12 CFU / ml for 2 weeks, as well as with eye application and application to the skin.

 Example 1. Determination of the ability of strain L.plantarum 421-2 to reduce the content of oxalate in the medium.

Strain L.plantarum 421-2 was grown in liquid medium MRS-2 at 38 ^o C for 18 hours, then the culture was seeded with a loop on Petri dishes with dense medium MRS-2 and cultivated under the same conditions. Then, the grown bacteria introduce it into the polysynthetic API medium of the following composition (g / l):
Polypepton - 10
Yeast extract - 5
Twin 80 - 1
K ₂ HPO ₄ - 2
Sodium Acetate - 5
Ammonium Citrate - 2
MnSO ₄ • 4H ₂ O - 0.05
MgSO ₄ • 7H ₂ O - 0.2
Bromcresol red - 0.17
Sodium Oxalate - 20
Distilled water - up to 1000
pH - 6.9
Seeds are added to the prepared medium in an amount of 10% of the total volume with a final concentration of bacteria in this medium of 10 ⁷ CFU / ml.

 As control use the same nutrient medium without making seed.

Incubation is carried out at 38 ^o C under anaerobic conditions for 72 hours.

Then the samples are centrifuged for 15 min at 12,000 rpm in order to prepare for the determination of oxalate content in the culture medium by gas chromatography. 0.4 ml is added to 1 ml of the supernatant. 50% H ₂ SO ₄ , 2 ml of CH ₃ OH and 100 ml of a 1% solution of fumaric acid. Samples in a tightly closed vial are incubated at 60 ^° C. for 30 minutes, then 1 ml of H ₂ O and 0.5 ml are added. chloroform, shake well and centrifuged. For further research, a layer of chloroform is taken. 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 Chromatopac C-R3A plasma-ionization detector, a 2 m long glass column and an inner diameter of 2 mm. Column filled with SP 1200/1% H ₃ PO ₄ on Chromosorb-WAW.

The temperature of the detector is 260 ^o C, the temperature of the evaporator is 250 ^o C. The feed rate of the carrier gas, nitrogen, is 30 ml / min.

 The results of determining the ability of strains of L.plantarum 421-2 and L.plantarumCS to reduce the level of oxalate are presented in table. 4.

 Incubation was carried out under anaerobic conditions, the determination of oxalate - by gas chromatography.

 As follows from table 4, the strain L.plantarum 421-2 is much more active in comparison with the strain of the prototype destroys oxalate. So, the strain L.plantarum 421-2 reduces the level of oxalate in the medium by 72 hours of cultivation by 69% from the initial level against 46% for the strain L.plantarum CS. At the same time, the content of sodium oxalate in the control solution remained unchanged during the entire incubation period.

 The ability to reduce the level of oxalate strain L.plantarum 421-2 stably retains with prolonged passage on nutrient media for cultivation of lactobacilli.

 Thus, the strain Lactobacillus plantarum 421-2 has a pronounced ability to actively reduce the level of oxalate, grow well on nutrient media of different compositions for the cultivation of lactobacilli and accumulate production biomass by 18 hours of cultivation. With this in mind, as well as the well-established fact of the safety of the strain, it can be used for the preparation of pharmacopeia drugs and food products intended for the correction of specific metabolic disorders in the human body.

 LIST OF REFERENCES.

 1. USSR author's certificate N 1331889 "Bacillus sp. Bacterial strain used for wastewater treatment from oxalate." Declared 05.11.85, IPC C 12 N 1/20.

 2. RF patent N 2061040 "The bacterial strain Lactobacillus plantarum, with the ability to reduce the level of oxalate and used for the preparation of drugs and food products for the prevention and treatment of hyperoxaluria. Priority 05/23/94. IPC C 12 N 1/20.

Claims (1)

 The bacterial strain Lactobacillus plantarum, deposited in GKNM N 151, with the ability to reduce the level of oxalate and used to prepare drugs and food products for the prevention and treatment of hyperoxaluria.

Images