Method for evaluation of antioxidant activity of microorganisms
SUBSTANCE: antioxidant activity of a broth culture of microorganisms or a cell suspension of microorganisms grown on a solid nutrient medium is evaluated. An oxidable medium is presented by a lecithin solution, while peroxide initiators are as follows: Staphylococcus aureus cells, ascorbic acid, ferric (II) ions (in the form of an aqueous solution of ferric sulphate). Antioxidant activity is evaluated by an ability to inhibit lipid peroxidation by adding concentrated orthophosphoric acid, and 1% 2-thiobarbituric acid in dimethyl sulphoxide or ethanol (1:1). The stained complex is extracted in n-butanol, and after a butanol phase separated, it is spectrophotometered with regard to transmission unit at 550 nm wherein a reference tray is a tray with n-butanol. It is combined with preparing two controls wherein test tubes containing an oxidation substrate - lecithin are added with known antioxidant activity, while in the other one initiated peroxidation is enabled with no antioxidant added.
EFFECT: derived values of the antioxidant status are calculated in units of antioxidant activity.
The invention relates to Microbiology and clinical laboratory diagnostics and can be used as an option to determine the antioxidant potential of microorganisms in the evaluation of antioxidant status and microecological features of the human body.
Description of the invention
The invention relates to Microbiology and clinical laboratory diagnostics and can be used as an alternative determination of antioxidant potential of microorganisms in assessing the microbial status of the human body. Typical pathological processes, such as hypoxia and inflammation, characteristic and developing in the majority of somatic and infectious diseases, severe injuries and wounds, always accompanied by excessive formation of reactive oxygen species, which leads to the strengthening of processes of lipid peroxidation, are part of cell membranes . A significant contribution in maintaining the redox balance of the body makes normal microflora .
The known method EN No. 2112241, C16G01N 33/48, G01N 33/50 (date of publication of the claims 1998.05.27) determine the concentration of malondialdehyde using thiobarbituric acid (TBA). In this method for analysis using the minimum amount of serum or homogenate (0.2-0.5 ml), Rast is orange TBQ and incubation of the sample is carried out in the presence of Triton X-100, the mixture is stirred with a constant oscillation frequency (120 oscillations / min), the reaction is stopped dihydroquercetin; before determining the optical density of the sample add Trilon B and a mixture of ethanol and chloroform in the ratio (7:3). The method is based on the reaction between malonic dialdehyde (MDA) and thiobarbituric acid, which is at high temperature and acidic pH value proceeds with the formation of colored trimethylboron complex containing one molecule of MDA and two molecules thiobarbiturate acid. The maximum absorption of the complex have at 532 nm. The working solution thiobarbituric acid (TBA) prepared by dissolving weighed TBQ 864 mg in 100 ml of a mixture containing 1% solution of Triton X-100 and 8.2 M solution of ethanol.
However, this method has several drawbacks: it does not take into account the peculiarities of the metabolism of microorganisms to improve the solubility of the lipid fractions and 2-thiobarbituric acid is proposed to use a special reagent (polyoxyethylene-p-(tert-octyl) phenol (Triton X-100)and the reagent that require special conditions of storage and work (trichloromethane). Closest to the claimed method is detection of microorganisms protective actions in the effect of Fenton EN 2279079, C2 G01N 33/483, C12Q 1/02.
In the present method of detection of microorganisms protective actions in the effect of Fe the tone of the studied culture of microorganisms are grown in a liquid nutrient medium, separate the supernatant from the microbial cells by centrifugation, simultaneously prepare the control, representing a liquid nutrient medium, then each of the samples treated with chloroform and separate the supernatant from the chloroform by centrifugation, and then in the experimental sample and the control add test-culture of Staphylococcus aureus risk No. 201108, incubated separating the cells of the test culture by centrifugation and preparing microbial suspension in each of the samples add a solution of hydrogen peroxide and incubated separating the cells of the test culture by centrifugation, add to them a nutrient medium, incubated, measure the optical density of the grown crops in experimental and control samples, determine the degree of growth biomass of the test culture in the experience. The disadvantage of this method is the use as an indicator of a protective effect of microorganisms only the degree of increase in cell mass optical density. Therefore, the method does not take into account the kinetic parameters that occur when you add in the studied system of prooxidants. The method characterizes the duration and relative multistage. The results of the experiment require several stages of incubation, which should be chosen with consideration of the physiology of microorganisms. The considered method is not possible to determine quantitative characteristics, the OTP is concerned with antioxidant potential of microorganisms, members of the microbiocenosis of the person. Determination of protective action of the studied culture in the effect of Fenton, according to this method, it is possible only at presence in the environment of low concentrations of hydrogen peroxide.
The task of the invention is to improve the known method for determining the antioxidant activity, as well as the possibility of using the proposed method for the determination of antioxidant status in microorganisms that do not produce hydrogen peroxide and/or inactivating its enzymes.
The essence of this method is the determination of the antioxidant status of microorganisms for their ability to block the reaction of lipid peroxidation and/or to inhibit phospholipase.
Method for determination of antioxidant activity of microorganisms is that evaluated the antioxidant activity of broth cultures of microorganisms or the suspension of microorganisms in saline grown on solid nutrient medium. In the proposed method, as the oxidizable medium is a solution of lecithin. It is known that under the action of phospholipases from lecithin cleaved one of the fatty acids, turning it into lysolecithin. This product has a high toxicity in the cells, causing them to necrosis . The proposed method Perek the red oxidation of lecithin is started by adding the cell suspension of microorganisms - producers of phospholipases, isolated from the subject, and in their absence, uses the well-known Museum strain phospholipase activity. Then, on Wednesday sequentially added to an aqueous solution of ascorbic acid, iron ions (II) (in the form of a solution of iron sulfate). After that analyzed the samples incubated for 30 min at a temperature of 42°C, With constant shaking. After that, the sample make a concentrated phosphoric acid to pH 1, and a 1% solution of 2-thiobarbituric acid in dimethyl sulfoxide and ethanol (1:1). The reaction mixture is heated in a water bath at t=70°C for 20 min, cooled in a dark place to a temperature of +4°C and add 3 ml of n-butanol are added, thoroughly mixed, centrifuged at 3 thousand rpm, separate botanology phase and spectrophotometric, given the minimum transmittance at 550 nm. When spectrophotometrically the comparison cuvette cuvette is with n-butanol. In the inventive method uses several controls: control the maximum possible peroxidation reactions (ongoing studies)used for this sample of lecithin-initiated oxidation without antioxidant and control of inhibition of lipid peroxidation, where the specimen using equimolar quantities of substances with known antioxidant activity is updated. To compare the antioxidant status of substances in the hydrophilic phase used an aqueous solution of 6-methyl-2-ethyl-pyridine-3-ol, and substances with lipophilic properties as the reference sample is used tocopherol acetate - oil solution.
To quantify the antioxidant activity is expressed as arbitrary units of antioxidant activity (Ethe AoA)calculated by the formula
Ethe AoA- units of antioxidant activity,
IA- the intensity of the luminous flux (in units of bandwidth), passed through the layer of the absorbing solution of the sample at 550 nm,
IB- the intensity of the luminous flux (in units of bandwidth), passed through the absorbing layer of nutrient solution at 550 nm,
IC- the intensity of the luminous flux (in units of bandwidth), passed through the layer of the absorbing solution control with induced oxidation at 550 nm,
10 is the conversion factor in units of volume,
IE- the molar ratio trimethylboron complex in units of bandwidth 0,413 mmol·cm-1,
ID- the intensity of the luminous flux (in units of bandwidth), passed through the layer of the absorbing solution control with known antioxidant at 550 nm.
Culture of Staphylococcus aureus isolated from Ki is echnolo microbiocenosis of the subject, grew up on a meat-peptone agar at 37°C for 48 hours and Then suspended in sterile 0.9% sodium chloride solution and standardized by the turbidity standard to 109microbial cells in 1 ml
Isolated from feces of the patient's strain of Bifidobacterium breve, were grown in liquid nutrient medium, Blaurock. On the day of the studies prepared solutions: lecithin 0.1%of ascorbic acid 0,M, iron sulfate 0.07 m in bidistilled, svejeprokipachena and cooled to room temperature water.
In the experimental tube was added 0.5 ml of a solution of lecithin, 0.3 ml of the bacterial substrate, investigated the antioxidant activity - .breve, and 0.8 ml of ascorbic acid solution. The reaction of free radical formation was initiated by addition of 0.8 ml of a solution of iron sulfate. The tubes were placed in a thermostat at a constant shaking for 30 min In the quality control reference antioxidant used a 0.3 ml solution emoxipin. As a control of the maximum possible peroxidation used the test tube with the sample of lecithin, where a process has been initiated in the absence of an antioxidant. The investigated tubes were placed in a thermostat at 30 min, at a temperature of 42°C, With constant shaking. After that, in the analyzed samples had consistently contributed 0.2 ml of concentrated phosphoric acid and 0.8 is l 1% solution of 2-thiobarbituric acid. The resulting reaction mixture was heated in a water bath at t=70°C for 20 min, cooled in a dark place to a temperature of +4°C and with thorough stirring, was added 3 ml of n-butanol. The resulting emulsion was centrifuged at 3 thousand rpm and gently separated botanology phase, which was spectrophotometrically on the spectrophotometer SF-2000, taking into account the unit of transmission at 550 nm. As the cell comparisons used a cell with n-butanol. Substituting these values into the formula, calculated that for the analyzed strain Bifidobacterium breve antioxidant activity was 3,03 Ethe AoA.
Sources of information
1. Shanin VY Clinical pathophysiology. Saint-Petersburg.: "Special literature", 1998, 569 S.
2. Shenderov B.A. Medical microbial ecology and functional nutrition 3 so / Bassendean. - M.: the Verge, 2001, Vol.3, 288 S.
The method of quantitative determination of antioxidant activity of microorganisms is that evaluated the antioxidant activity broth cultures of microorganisms or suspension of microorganism cells grown on solid nutrient medium, characterized in that as the oxidizable medium is a solution of lecithin, and as initiators of lipid peroxidation: sequentially added to a suspension of Staphylococcus aureus cells in physiological solution, the solution ASKO is benovoy acid, iron ions (II) (in the form of an aqueous solution of iron sulfate), incubated for 30 min at a temperature of t=42°C, with constant shaking, then zachisliaut to pH 1 by adding concentrated phosphoric acid and 1%solution of 2-thiobarbituric acid in dimethyl sulfoxide and ethanol (1:1), heated in a water bath at t=70°C for 20 minutes, cool, add 3 ml of n-butanol are added, thoroughly mixed, centrifuged at 3 thousand rpm, separate botanology phase and spectrophotometric, given the unit of transmission at 550 nm, where as the cell comparison using a cuvette containing n-butanol, and controls use of the test tube in which the substrate oxidation - lecithin in one added substance with known antioxidant activity, and the other initiated by peroxide oxidation is carried out without addition of antioxidant.
SUBSTANCE: invention relates to field of medicine, namely to oncology. To diagnose cancer and potential resistance of malignant cells to hypoxia, prepared is cell imprint or suspension of cells, for which conditions of hypoxia are created and spectro-flowmetric analysis of said cells in dynamics of development of process of photodestruction of NAD(P)H fluorescence is performed. Conclusion about presence of tumour cells and their potential resistance to hypoxia is made by increase of NAD(P)H fluorescence intensity in conditions of darkness. Conditions of hypoxia are created by placement of cell suspension or cell imprint between two glasses for cytologic analysis. Spectro-flowmetric analysis is carried out in the range 440-470 nm, with length of excitation wave 365-370 nm.
EFFECT: method makes it possible to diagnose cancer and potential resistance of malignant cells to hypoxia.
3 cl, 6 dwg, 1 ex
SUBSTANCE: method involves preparation of a patient's blood serum sample by drying, grinding and suspending in Vaseline oil; then the sample is examined by infrared spectroscopy in 1200-1000 cm-1 to determine a peak height of absorption bands with maximums 1150, 1130, 1125 cm-1 that is followed by the calculation of the relation of the peak heights with maximum 1150 cm-1 to the peak height with maximum 1130 cm-1 in males and the relation of the peak height with maximum 1150 cm-1 to the peak height with maximum 1125 cm-1 in females. The blood serum sample immediately follows the performed growth surgery, and thereafter in progression, preferentially monthly. If observing increase of the relation of the peak height with maximum 1150 cm-1 to the peak height with maximum 1130 cm-1 in a male patient and the relation of the peak height with maximum 1150 cm-1 to the peak height with maximum 1125 cm-1 in a female patient by the value of 0.5±0.015 as compared to the relation value derived from the previous sample examination, the recurrent cerebral malignant growth is stated.
EFFECT: use of the method enables stating the postoperative recurrent cerebral malignant growth at the early stages.
2 cl, 3 tbl, 3 ex
SUBSTANCE: method for mineral recovery from human connective tissue by low-temperature tissue ignition involving drying of connective tissue kept in 10% formalin in the air for 2-3 days, sample suspension, tissue ignition in porcelain crucibles in an ignition muffle at temperature 450°C for 10-12 hours, suspension of the prepared ignited residue.
EFFECT: method enables effective mineral recovery from human connective tissue.
5 dwg, 1 ex
SUBSTANCE: patient's prostate secretion microscopy and microbiological analysis of sperm are conducted prior to and after three sessions of low-intensity infra-red laser therapy. Further, a final diagnosis of category II, category IIIA or category IIIB chronic prostatitis is stated.
EFFECT: higher differential diagnostic accuracy in chronic prostatitis forms which have essentially different pathogenesis and therapy and improved therapeutic results.
SUBSTANCE: in patients with bronchial asthma performed is iionophoresis with 5% acetylcholine solution in course of laser Doppler fluometry, time of rise and restoration of dopplerogramme on skin of left forearm in point located on medial line 4 cm higher than base of styloid processes of ulnar and radial bones.
EFFECT: basing on obtained data type of microvascular endothelium response is determined as normoreactive-stable, normoreactive-incremental, normoreactive-decremental, hyperreactive-stable, hypereactive-incremental, hypereactive-decremental, hyporeactive-stable, hyporeactive-decremental ot hyporeactive-decremental type of microvascular endothelium response.
10 dwg, 9 ex
SUBSTANCE: reagent for thrombocyte measurement containing Nile blue hydrosulphate as a reagent for thrombocyte staining. A reagents kit for thrombocyte measurement containing Nile blue hydrosulphate as a first reagent, and a buffer solution as a second reagent. A method for thrombocyte measurement wherein Nile blue hydrosulphate is used as the first reagent, and the buffer solution - as the second reagent. A reagent for thrombocyte measurement containing Nile blue and an acid as a staining agent for thrombocyte staining. The reagents kit for thrombocyte measurement containing Nile blue as the first reagent, and a buffer substance as the second reagent. The method for thrombocyte measurement wherein Nile blue and the acid are used as the first reagent, and the buffer substance - as the second reagent.
EFFECT: reagents described above provides higher accuracy of thrombocyte evaluation.
13 cl, 15 dwg, 10 tbl, 48 ex
SUBSTANCE: histologic examination of transverse section is performed through all the layers of arachnoid cyst walls with previous alignment of the piece in paraffin on the edge and study the state of lining and connective-tissue membrane. In case the wall of the cyst is covered in flat arachnoendothelium with hyperchromatic nucleus, the cyst is regarded the non-proliferating, the prognosis is favorable. In case the wall of the cyst is covered with round-shaped arachnoidendothelium with the zones of amplified proliferation of arachnoidendothelium in form of multirowed layer and/or vertical cells alignment, it is regarded as proliferating - the prognosis is unfavorable.
EFFECT: morphologic prediction of children arachnoid brain cyst progression.
1 dwg, 2 dwg, 1 tbl, 2 ex
SUBSTANCE: invention relates to field of medicine, namely to hepathology and pediatrics. In order to predict exacerbations of chronic viral hepatitis B in teenagers, blood test is performed. By means of bioluminescent method determined is activity of lymphocyte enzymes in peripheral blood of patients: glucose-6-phosphate dehydrogenase (G6PDG) and lactate dehydrogenase (NADN-LDG), ratio of activities is calculated. If value of obtained index is equal or is higher than 1.15 exacerbations are predicted, if index value is lower than 1.15, absence of exacerbations is predicted.
EFFECT: method makes it possible to predict exacerbations of chronic viral hepatitis B in teenagers with clinical-biochemical remission with high accuracy.
4 ex, 2 tbl
SUBSTANCE: formation of a risk group of neoplastic disorders in a cervical epithelium is carried out by IF staining of a cervical epithelium smear with using a monoclonal antibody reacting with a sialylated conformational glycopeptide antigen determinant as a part of MUC1 followed by with cell finish staining with chromogenic nuclear stain. The sample is examined by alternated fluorescent light and transmission light. Each cell with a depolarised stained surface membrane detected by fluorescent light is examined in transmission light. If the cells with the depolarised stained surface membrane detected by fluorescent light show cytological symptoms of dyscariosis, and/or proliferative parabasal cell complexes with no symptoms of dyscariosis show depolarised stained surface membranes or granular stained cytoplasm, a patient is subsumed to the risk group of the presence of neoplastic disorders in the cervical epithelium.
EFFECT: higher analysis accuracy.
SUBSTANCE: LAKK-02 apparatus is used to evaluate a capillary blood flow reserve in two iontophoresis sampled taken from skin of a left forearm in a point along a median line 4 cm higher than styloid process of ulna and radius. An endothelial function coefficient EFC is calculated by formula: where AC BFR is the capillary blood flow reserve in response to acertylcholine; SN BFR is the capillary blood flow reserve in response to sodium nitroprusside. If the EFC value is lower than 1.0, a disturbed microvascular endothelium vasoregulation is stated.
EFFECT: use of the invention allows simplifying diagnostics of the microvascular endothelium status in patients with bronchial asthma.
SUBSTANCE: method involves collection of adenoviruses in a permissive cell culture of line HEK-293, collection of these cells, release of recombinant viruses due to cell destruction by re-frosting, and purification. It involves the use of the recombinant capside-modified adenovirus with modified capside protein pIX carrying a polysaccharide-binding domain specified in a group consisting of a cellulose-binding domain, a dextran-binding domain. The recombinant capside-modified adenoviruses are implanted on the polysaccharide carrier specified from a group consisting of a cellulose carrier for binding with the cellulose-binding domain, the dextran carrier - for binding with the dextran-binding domain. The mixture is incubated. It is followed by the process of purification; for this purpose the polysaccharide carrier with the adenoviruses is settled by centrifugation, while the prepared supernatant is removed. The settled polysaccharide carrier with the adenoviruses is washed in washing buffers, then the adenoviruses are eluted from the polysaccharide carrier. The prepared suspension is separated by centrifugation. The virus-containing supernatant is selected and stabilised.
EFFECT: invention provides preparing the biologically active, chromatographically pure and concentrated adenovirus preparation and reducing a purification time.
15 cl, 4 dwg, 2 tbl, 4 ex
SUBSTANCE: composition contains the strains Lactobacillus fermentum 57A deposited under No. B/00007, Lactobacillus plantarum 57B deposited under No. B/00008 and Lactobacillus gasseri 57C deposited under No. B/00007 in relation 0.5:0.5:1.
EFFECT: use of the given composition provides recovered disturbed balance of female vaginal microflora, leads to reducing a risk of bacterial vaginosis, ensures an immunomodulatory effect.
8 tbl, 4 dwg
SUBSTANCE: diagnostic RN2/57-human A(H7N2) influenza virus strain is produced by cross breeding of the sterile equine influenza virus A/horse/Prague/1/1956(H7N7) and the cold-adapted vaccine strain A/17/duck/Potsdam/86/92(H5N2) on the basis of the attenuation donor A/Leningrad/134/17/5 7(H2N2). The strain contains neuraminidase of the subtype 2 influenza virus A/Leningrad/134/17/1957(H2N2) and hemagglutinin of equine influenza virus A/horse/Prague/1/1956(H7N2). The RN2/57-human A(H7N2) strain replicates actively in growing chicken embryos at optimum temperature 33°C that enables collecting the viral material to be purified and concentrated.
EFFECT: invention may be used for detection neuraminidase antibodies of the N2 influenza virus in serums of experimental animals and volunteers with the use of solid-phase sialydase activity neuraminidase inhibition test.
1 dwg, 2 tbl
SUBSTANCE: method provides the preparation of a nutrient medium and/or a mycobaterial culture to be exposed to the electromagnetic field for 30 min. An electromagnetic field source presented by the KFS-7 apparatus is placed at 3 cm from the nutrient medium and/or the mycobaterial culture.
EFFECT: invention provides accelerating growth of the slow-growing tuberculosis mycobacteria.
3 tbl, 3 ex
SUBSTANCE: disclosed is an E.coli strain which produces a recombinant protein p30 of the African swine fever virus. The strain is homogeneous, stable during passage and culturing in liquid and solid culture media and is resistant to chloramphenicol.
EFFECT: invention can be used to produce a recombinant protein p30 of African swine fever virus for diagnostic purposes.
1 tbl, 3 ex
SUBSTANCE: method consists in cultivation of the microorganism Clostridia acetobutylicum with the butirate kinase coding gene buk being deleted in the appropriate culture medium containing a carbon source, and n-butanol extraction from the culture medium. The microorganism used in the method can also contain inactivated mutations in endogenous genes coding a polypeptide with lactate dehydrogenase activity, a polypeptide with phosphotransacetylase or lactate kinase activity and have attenuation in the gene coding a polypeptide with hydrogenase activity.
EFFECT: invention provides preparing high n-butanol yield from the inexpensive carbon substrate, such as glucose or other sugars under action of the genetically stable Clostridia acetobutylicum.
10 cl, 7 tbl, 8 ex
SUBSTANCE: Enterococcus durans I-26 strain possessing high antagonist activity is deposited in the Russian National Collection of Industrial Microorganisms (RNCIM), registration No. VKPM B-10093 and may be used in preparing such fermented milk products as kefir, cottage cheese, fermented baked milk.
EFFECT: invention enables producing the strain possessing high antagonist activity and high milk ripening rate.
SUBSTANCE: invention refers to biotechnology and dairy products industry. The Lactobacillus gallinarum I-37 strain possessing high antagonist activity is deposited in the Russian National Collection of Industrial Microorganisms (RNCIM), registration No. VKPM B-10133 and may be used in preparing such fermented milk products as kefir, acidophilus milk, acidophilus yeast milk, acidophilus butter.
EFFECT: invention enables producing the strain possessing high antagonist activity and high milk ripening rate.
SUBSTANCE: Lactobacillus gallinarum G-37 strain possessing high antagonist activity is deposited in the Russian National Collection of Industrial Microorganisms (RNCIM), registration No. VKPM B-10132 and may be used in preparing such fermented milk products as kefir, acidophilus milk, acidophilus yeast milk, acidophilus butter.
EFFECT: invention enables producing the strain possessing high antagonist activity and high milk ripening rate.
SUBSTANCE: Lactobacillus paracasei 3-37 strain possessing high antagonist activity is deposited in the Russian National Collection of Industrial Microorganisms (RNCIM), registration No. VKPM B-10092 and may be used in preparing such fermented milk products as kefir, acidophilus milk, acidophilus yeast milk, acidophilus butter.
SUBSTANCE: Yersinia pseudotuberculosis 634 bacterial strain is recovered from a patient suffering pseudotuberculosis, deposited in the collection of the Russian Science and Research Antiplague Institute Microbe, No. KM 214 and is applicable as a test strain for differentiation of Yersinia pseudotuberculosis bacteria genetic group IA. The characteristic of the strain is the content of chromosomal gene of the Y.pseudotuberculosis YPMa/YPMc (urtA/C) superantigen and the YAPI (pilPQ) Yersinia adhesive pathogenicity island gene determined by PCR method.
EFFECT: invention shall enable epidemiologic study of sporadic and group cases of pseudotuberculosis: specifying an infection source in a hotbed of the disease and identifying transmission factors, as well as monitoring strain circulation at various territories.
2 dwg, 1 tbl