CZ314192A3 - Method of determining conversion of nitrates to nitrites being carried out by micro-organisms or enzymes thereof in a sample of biological material - Google Patents
Method of determining conversion of nitrates to nitrites being carried out by micro-organisms or enzymes thereof in a sample of biological material Download PDFInfo
- Publication number
- CZ314192A3 CZ314192A3 CS923141A CS314192A CZ314192A3 CZ 314192 A3 CZ314192 A3 CZ 314192A3 CS 923141 A CS923141 A CS 923141A CS 314192 A CS314192 A CS 314192A CZ 314192 A3 CZ314192 A3 CZ 314192A3
- Authority
- CZ
- Czechia
- Prior art keywords
- biological material
- sample
- nitrites
- nitrates
- carried out
- Prior art date
Links
- 150000002826 nitrites Chemical class 0.000 title claims description 12
- 239000012620 biological material Substances 0.000 title claims description 11
- 244000005700 microbiome Species 0.000 title claims description 11
- 238000006243 chemical reaction Methods 0.000 title claims description 9
- 238000000034 method Methods 0.000 title claims description 9
- 150000002823 nitrates Chemical class 0.000 title claims description 8
- 108090000790 Enzymes Proteins 0.000 title claims description 6
- 102000004190 Enzymes Human genes 0.000 title claims description 6
- 239000000523 sample Substances 0.000 claims description 13
- 239000001963 growth medium Substances 0.000 claims description 8
- WZCQRUWWHSTZEM-UHFFFAOYSA-N 1,3-phenylenediamine Chemical compound NC1=CC=CC(N)=C1 WZCQRUWWHSTZEM-UHFFFAOYSA-N 0.000 claims description 6
- 229940018564 m-phenylenediamine Drugs 0.000 claims description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 5
- 229910002651 NO3 Inorganic materials 0.000 claims description 4
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 claims description 4
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 235000013372 meat Nutrition 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 235000015097 nutrients Nutrition 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 238000002835 absorbance Methods 0.000 claims description 2
- 150000001413 amino acids Chemical class 0.000 claims description 2
- 239000012472 biological sample Substances 0.000 claims description 2
- 239000000872 buffer Substances 0.000 claims description 2
- 239000003102 growth factor Substances 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 235000021309 simple sugar Nutrition 0.000 claims description 2
- 235000013343 vitamin Nutrition 0.000 claims description 2
- 239000011782 vitamin Substances 0.000 claims description 2
- 229940088594 vitamin Drugs 0.000 claims description 2
- 229930003231 vitamin Natural products 0.000 claims description 2
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 10
- 230000015572 biosynthetic process Effects 0.000 description 9
- 238000003556 assay Methods 0.000 description 7
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- RUFPHBVGCFYCNW-UHFFFAOYSA-N 1-naphthylamine Chemical compound C1=CC=C2C(N)=CC=CC2=C1 RUFPHBVGCFYCNW-UHFFFAOYSA-N 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 235000010333 potassium nitrate Nutrition 0.000 description 2
- 239000004323 potassium nitrate Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- VEGZHURRCWERMK-UHFFFAOYSA-N benzene-1,3-diamine;hydrochloride Chemical compound Cl.NC1=CC=CC(N)=C1 VEGZHURRCWERMK-UHFFFAOYSA-N 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 229940127554 medical product Drugs 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/12—Nitrate to nitrite reducing bacteria
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/02—Food
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/02—Food
- G01N33/12—Meat; Fish
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Zoology (AREA)
- General Physics & Mathematics (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Toxicology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
Vynález se týká způsobu stanovení konverze dusičnanů na dusitany provedené mikroorganismy nebo jejich enzymy ve vzorku biologického materiálu. Tvorba dusitanů se sleduje zejména v potravinářském průmyslu a ve zdravotnictví, kde tyto dusitany nebo jejich sloučeniny představují látky škodící zdraví.The invention relates to a method for determining the conversion of nitrates to nitrites by microorganisms or their enzymes in a sample of biological material. The formation of nitrites is mainly observed in the food industry and healthcare, where these nitrites or their compounds are harmful to health.
Dosavadní stav technikyBACKGROUND OF THE INVENTION
Dosavadní způsoby stanovení tvorby dusitanů mikroorganismy nebo jejich enzymy se zakládají na postupech, které zejména využívají činidla mající nežádoucí vliv na vlastní tvorbu dusitanů, například činidla inhibující tvorbu dusitanů. Příkladem takových činidel je alfa-naftylamin v silně kyselém prostředí.The prior art methods for determining nitrite formation by microorganisms or their enzymes are based on procedures that mainly utilize agents having an undesirable effect on nitrite formation itself, such as nitrite inhibiting agents. An example of such agents is alpha-naphthylamine in a strongly acidic medium.
Tento typ činidel ovlivňuje konverzi dusičnanů na dusitany a určitým způsobem zkresluje výsledky stanovení. Někdy prostředí, ve kterém dusitany vznikají, obsahuje látky, které s dusitany reagují rychleji než s použitým analytickým činidlem, takže i v tomto případě dochází ke zkreslení výsledků vzhledem k tomu, že takové analytické činidlo nezachytí skutečný stav tvorby dusitanů panující ve stanovovaném vzorku. Zbývající stanovení tvorby dusitanů na základě stanovení úbytku dusičnanů jsou velmi náročná a nákladná.This type of reagent affects the conversion of nitrates to nitrites and somehow distorts the results of the assay. Sometimes the nitrite-forming environment contains substances that react more quickly with the nitrites than with the analytical reagent used, so that even in this case the results are distorted because such analytical reagent does not capture the actual nitrite formation state prevailing in the sample being determined. The remaining determination of nitrite formation based on the determination of nitrate loss is very demanding and costly.
Podstata vynálezuSUMMARY OF THE INVENTION
Výše uvedené nedostatky jsou podstatnou měrou odstraněny způsobem stanovení konverze dusičnanů na dusitany provedené mikroorganismy nebo jejich enzymy ve vzorku biologického materiálu, jehož podstata spočívá v tom, že se vzorek biologického materiálu s obsahem dusičnanů přítomných nebo dodaných do vzorku nebo až do kultivačního prostředí kultivuje buď samotný nebo v kultivačním prostředí v přítomnosti m-fenylendiaminu nebo jeho soli v koncentraci 0,01 až 10 g.l 1 vzorku biologického materiálu a případně kultivačního prostředí při teplotě 1 až 70 °C po dobu až 14 dnů, načež se vizuálně nebo fotometricky vyhodnotí absorbance kultivovaného vzorku biologického materiálu nebo kultivované směsi vzorku biologického materiálu a kultivačního prostředí.The above-mentioned deficiencies are substantially eliminated by the method for determining the conversion of nitrates to nitrites by microorganisms or their enzymes in a biological material sample, which consists in culturing either a nitrate-containing biological material present or added to the sample or into the culture medium either alone. or in a culture medium in the presence of m-phenylenediamine or a salt thereof at a concentration of 0,01 to 10 gl 1 of the biological material sample and, optionally, a culture medium at a temperature of 1 to 70 ° C for up to 14 days. biological material or cultured mixture of biological material sample and culture medium.
Uvedená kultivace se výhodně provádí v přítomnosti pufru s optimální hodnotou pH pro konverzi dusičnanů na dusitany.Said cultivation is preferably carried out in the presence of a pH optimum buffer for the conversion of nitrates to nitrites.
Uvedená kultivace může být rovněž výhodně provedena v přítomnosti živin a/nebo růstových faktorů mikroorganismů, jakými výhodně jsou jednoduché cukry, pepton, masový výtažek, kvasničný autolyzát, aminokyseliny a vitaminy.Said cultivation can also advantageously be carried out in the presence of nutrients and / or growth factors of microorganisms, such as preferably simple sugars, peptone, meat extract, yeast autolysate, amino acids and vitamins.
Vznikající dusitan se při způsobu stanovení podle vynálezu váže na m-fenylendiamin za vzniku snadno identifikovatelné sloučeniny absorbující světlo. Výhodou tohoto stanovení je rychlost uvedené reakce, která je vyšší než rychlost reakce dusitanů s ostatními konkurenčními látkami, které se obvykle nachází ve stanovovaném vzorku biologického materiálu. Dalšími výhodami způsobu stanovení podle vynálezu je levnost, jednoduchost a nízká toxicita stanovení. Významnou výhodou stanovení je i nízký inhibiční účinek m-fenylendiaminu na mikroorganismy nebo jejich enzymy provádějící konverzi dusičnanů na dusitany, takže je možné sledovat tvorbu dusitanu v přirozených podmínkách. Vzhledem k tomu, že m-fenylendiamin má nízký inhibiční účinek i vůči ostatním mikroorganismům, lze stanovení provádět i v přítomnosti takových doprovodných mikroorganismů.The nitrite formed in the assay according to the invention binds to m-phenylenediamine to form an easily identifiable light-absorbing compound. The advantage of this assay is the rate of said reaction, which is higher than the rate of reaction of nitrites with other competing substances usually found in the biological sample to be determined. Further advantages of the assay method of the invention are the cheapness, simplicity and low toxicity of the assay. An important advantage of the assay is the low inhibitory effect of m-phenylenediamine on microorganisms or their enzymes converting nitrates to nitrites, so that nitrite formation can be monitored under natural conditions. Since m-phenylenediamine has a low inhibitory effect against other microorganisms, the assay can also be carried out in the presence of such accompanying microorganisms.
Příklady provedení vynálezuDETAILED DESCRIPTION OF THE INVENTION
Příklad 1Example 1
K zakvašené, mladinovými bakteriemi kontaminované mladině s obsahem 20 mg NO, .1 se přidá 1% vodný roztok m-fenylenJ __ 1 diaminhydrochloridu ve výsledné koncentraci 0,25 g.l ' a mladina se kultivuje po dobu 5 dní při teplotě 10 °c. Jako slepý pokus se použije stejný vzorek bez přídavku m-fenylendiaminhydrochloridu. Rozdíl absorbance při 450 nm obou zfiltrovaných vzorků po kultivaci je mírou tvorby dusitanů.To the fermented wort contaminated with wort containing 20 mg of NO.1, a 1% aqueous solution of m-phenylene-1-diamine hydrochloride was added at a final concentration of 0.25 g.l -1 and the wort was cultured for 5 days at 10 ° C. The same sample was used as a blank without addition of m-phenylenediamine hydrochloride. The difference in absorbance at 450 nm of both filtered samples after culture is a measure of nitrite formation.
». Příklad 2 * 2 ml várečných kvasnic se zalije 8 ml citrátového pufru s pH 5,0, načež se přidá dusičnan draselný ve výsledné koncentraci 150 mg NO^ .1 1 a m-fenylendiamin ve výsledné koncentraci — i „ „ o». Example 2 * 2 ml brewer's yeast was quenched with 8 ml citrate buffer pH 5.0 before the addition of potassium nitrate at a final concentration of 150 mg NO-.1 1 and m-phenylenediamine in a final concentration of - i "" o
0,25 g.l a směs se kultivuje po dobu 24 hodin pn teplote 37 C Červenohnědé zbarvení je důkazem tvorby dusitanů bakteriemi, přítomnými jako nečistota v kvasnicích.0.25 g.l and the mixture was cultured for 24 hours at 37 ° C. The red-brown color is evidence of nitrite formation by bacteria present as an impurity in yeast.
Příklad 3Example 3
Na plotnu živné půdy obsahující 0,5 % masového výtažku, 0,3 % peptonu, 0,5 % kvasničného autolyzátu, 0,5 % glukózy,For a culture medium containing 0.5% meat extract, 0.3% peptone, 0.5% yeast autolysate, 0.5% glucose,
0,5 % dusičnanu draselného, 0,1 % m-fenyldiaminu a 2,0 % agaru se zaočkuje vzorek s obsahem bakterií. Po 48 hodinové kultivaci při teplotě 37 °C se přítomnost bakterií tvořících dusitany prokáže tvorbou kolonií s hnědými zónami.0.5% potassium nitrate, 0.1% m-phenyldiamine and 2.0% agar were seeded with a sample containing bacteria. After cultivation at 37 ° C for 48 hours, the presence of nitrite-forming bacteria is demonstrated by the formation of colonies with brown zones.
Způsob průmyslové využitelnosti vynálezuMethod of industrial applicability of the invention
Vynález může být využit při kontrole produkce potravinářských a zdravotnických výrobků, při výrobě selektivních živných půd pro stanovení mikroorganismů produkujících dusitany a při sta novení obsahu dusičnanů mikrobiálními nebo enzymatickými testy a při výrobě činidel pro takové testy.The invention can be used to control the production of food and medical products, to produce selective nutrient media for the determination of nitrite-producing microorganisms and to determine the nitrate content by microbial or enzymatic tests and to produce reagents for such tests.
Claims (3)
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SK3141-92A SK278700B6 (en) | 1992-10-15 | 1992-10-15 | Method for quatification of conversion of nitrates to nitrites accomplished by microorganisms or their enzymes in a biological material sample |
| CS923141A CZ280631B6 (en) | 1992-10-15 | 1992-10-15 | Method of determining conversion of nitrates to nitrites being carried out by micro-organisms or enzymes thereof in a sample of biological material |
| DE19934331889 DE4331889A1 (en) | 1992-10-15 | 1993-09-20 | Detection for the conversion of nitrate into nitrite - uses 1,3-phenylene di-amine in a biological sample, useful in the food and sanitary industry |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS923141A CZ280631B6 (en) | 1992-10-15 | 1992-10-15 | Method of determining conversion of nitrates to nitrites being carried out by micro-organisms or enzymes thereof in a sample of biological material |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CZ314192A3 true CZ314192A3 (en) | 1995-11-15 |
| CZ280631B6 CZ280631B6 (en) | 1996-03-13 |
Family
ID=5370623
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CS923141A CZ280631B6 (en) | 1992-10-15 | 1992-10-15 | Method of determining conversion of nitrates to nitrites being carried out by micro-organisms or enzymes thereof in a sample of biological material |
Country Status (3)
| Country | Link |
|---|---|
| CZ (1) | CZ280631B6 (en) |
| DE (1) | DE4331889A1 (en) |
| SK (1) | SK278700B6 (en) |
-
1992
- 1992-10-15 SK SK3141-92A patent/SK278700B6/en unknown
- 1992-10-15 CZ CS923141A patent/CZ280631B6/en not_active IP Right Cessation
-
1993
- 1993-09-20 DE DE19934331889 patent/DE4331889A1/en not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| SK314192A3 (en) | 1995-11-08 |
| SK278700B6 (en) | 1998-01-14 |
| DE4331889A1 (en) | 1994-04-21 |
| CZ280631B6 (en) | 1996-03-13 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| IF00 | In force as of 2000-06-30 in czech republic | ||
| MK4A | Patent expired |
Effective date: 20001015 |