SK278700B6 - Method for quatification of conversion of nitrates to nitrites accomplished by microorganisms or their enzymes in a biological material sample - Google Patents
Method for quatification of conversion of nitrates to nitrites accomplished by microorganisms or their enzymes in a biological material sample Download PDFInfo
- Publication number
- SK278700B6 SK278700B6 SK3141-92A SK314192A SK278700B6 SK 278700 B6 SK278700 B6 SK 278700B6 SK 314192 A SK314192 A SK 314192A SK 278700 B6 SK278700 B6 SK 278700B6
- Authority
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- Slovakia
- Prior art keywords
- sample
- biological material
- nitrates
- nitrites
- conversion
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- 239000012620 biological material Substances 0.000 title claims abstract description 21
- 150000002826 nitrites Chemical class 0.000 title claims abstract description 14
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 12
- 150000002823 nitrates Chemical class 0.000 title claims abstract description 12
- 238000000034 method Methods 0.000 title claims abstract description 11
- 244000005700 microbiome Species 0.000 title claims description 10
- 108090000790 Enzymes Proteins 0.000 title claims description 6
- 102000004190 Enzymes Human genes 0.000 title claims description 6
- 239000000203 mixture Substances 0.000 claims abstract description 5
- 150000003839 salts Chemical class 0.000 claims abstract description 4
- 239000001963 growth medium Substances 0.000 claims description 12
- WZCQRUWWHSTZEM-UHFFFAOYSA-N 1,3-phenylenediamine Chemical compound NC1=CC=CC(N)=C1 WZCQRUWWHSTZEM-UHFFFAOYSA-N 0.000 claims description 8
- 229940018564 m-phenylenediamine Drugs 0.000 claims description 8
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 5
- 238000002835 absorbance Methods 0.000 claims description 4
- 235000015097 nutrients Nutrition 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
- 235000019319 peptone Nutrition 0.000 claims description 3
- 150000001413 amino acids Chemical class 0.000 claims description 2
- 239000000872 buffer Substances 0.000 claims description 2
- 239000003102 growth factor Substances 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
- 238000005375 photometry Methods 0.000 abstract 1
- 230000000007 visual effect Effects 0.000 abstract 1
- 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 6
- 241000894006 Bacteria Species 0.000 description 4
- 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
- 229910002651 NO3 Inorganic materials 0.000 description 3
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 239000000126 substance Substances 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
- VEGZHURRCWERMK-UHFFFAOYSA-N benzene-1,3-diamine;hydrochloride Chemical compound Cl.NC1=CC=CC(N)=C1 VEGZHURRCWERMK-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
- 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
- 230000002411 adverse Effects 0.000 description 1
- 239000007864 aqueous solution Substances 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
- 238000007824 enzymatic assay Methods 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
- 238000002816 microbial assay Methods 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
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- 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)
Abstract
Description
(57) Anotácia:(57) Annotation:
Spôsob stanovenia konverzie dusičnanov na dusitany spočíva v tom, že sa vzorka biologického materiálu s obsahom dusičnanov prítomných alebo dodaných do vzorky alebo až do kultivačného prostredia kultivuje buď samotná alebo v kultivačnom prostredí v prítomnosti m-fenyléndiamínu alebo jeho soli v koncentrácii 0,01 až 10 g.l'1 vzorky biologického materiálu a prípadne kultivačného prostredia pri teplote 1 až 70 °C počas až 14 dní, následne sa vizuálne alebo fotometrický vyhodnotí absorbancia kultivovanej vzorky biologického materiálu alebo kultivovanej zmesi vzorky biologického materiálu a kultivačného prostredia.A method for determining the conversion of nitrates to nitrites is to sample a biological material containing or supplied to the sample or into the culture medium either alone or in the culture medium in the presence of m-phenylenediamine or a salt thereof at a concentration of 0.01 to 10 g -1 samples of the biological material and, optionally, the culture medium at 1 to 70 ° C for up to 14 days, then the absorbance of the cultured biological material sample or the cultured mixture of the biological material sample and the culture medium is visually or photometrically evaluated.
Oblasť technikyTechnical field
Vynález sa týka spôsobu stanovenia konverzie dusičnanov na dusitany uskutočnenej mikroorganizmami alebo ich enzýmami vo vzorke biologického materiálu. Tvorba dusitanov sa sleduje najmä v potravinárskom priemysle a v zdravotníctve, kde tieto dusitany alebo ich zlúčeniny predstavujú látky zdraviu škodlivé.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 in the health sector where these nitrites or their compounds are substances harmful to health.
konverziu dusičnanov na dusitany, takže sa môže sledovať tvorba dusitanu v prirodzených podmienkach. Vzhľadom na to, že m-fenyléndiamín má nízky inhibičný účinok i proti ostatným mikroorganizmom, môžu sa stanovenia uskutočňovať i v prítomnosti takýchto sprievodných mikroorganizmov.the conversion of nitrates to nitrites so that nitrite formation can be monitored under natural conditions. Since m-phenylenediamine has a low inhibitory effect against other microorganisms, assays can also be carried out in the presence of such accompanying microorganisms.
Doterajší stav technikyBACKGROUND OF THE INVENTION
Doterajšie spôsoby stanovenia tvorby dusitanov mikroorganizmami alebo ich enzýmami sú založené na postupoch, ktoré využívajú hlavne činidlá s nežiaducim vplyvom na samotnú tvorbu dusitanov, napríklad činidlá inhibujúce tvorbu dusitanov. Príkladom takýchto činidiel je ct-naftylamín v silno kyslom prostredí. Tento typ činidiel ovplyvňuje konverziu dusičnanov na dusitany a určitým spôsobom skresľuje výsledky stanovení. Niekedy obsahuje prostredie, v ktorom vznikajú dusitany, látky, ktoré s dusitanmi reagujú rýchlejšie ako s použitým analytickým činidlom, takže i v tomto prípade dochádza k skresleniu výsledkov, vzhľadom na to, že takéto analytické činidlo nezachytí skutočný stav tvorby dusitanov, ktorý existuje v stanovovanej vzorke. Ostatné stanovenia tvorby dusitanov na základe stanovenia úbytku dusičnanov sú veľmi náročné a nákladné.The prior art methods for determining nitrite formation by microorganisms or their enzymes are based on procedures that mainly use agents with an adverse effect on nitrite formation itself, such as nitrite inhibiting agents. An example of such agents is α-naphthylamine in a strongly acidic environment. This type of reagent affects the conversion of nitrates to nitrites and somehow distorts the results of the assay. Sometimes a nitrite-forming environment contains substances that react more quickly with nitrites than the analytical reagent used, so even in this case the results are distorted, since such analytical reagent does not capture the true nitrite formation state that exists in the sample being determined . Other determinations of nitrite formation based on the determination of nitrate depletion are very demanding and costly.
Podstata vynálezuSUMMARY OF THE INVENTION
Uvedené nedostatky sa podstatnou mierou odstránia spôsobom stanovenia konverzie dusičnanov na dusitany uskutočnenej mikroorganizmami alebo ich enzýmami vo vzorke biologického materiálu, ktorého podstata spočíva v tom, že sa vzorka biologického materiálu s obsahom dusičnanov prítomných alebo dodaných do vzorky, alebo až do kultivačného prostredia kultivuje buď samotná, alebo v kultivačnom prostredí v prítomnosti m-fenyléndiamínu alebo jeho soli v koncentrácii 0,01 až 10 g.ľ1 vzorky biologického materiálu a prípadne kultivačného prostredia pri teplote 1 až 70 °C počas až 14 dní, potom sa vizuálne alebo fotometrický vyhodnotí absorbancia kultivovanej vzorky biologického materiálu alebo kultivovanej zmesi vzorky biologického materiálu a kultivačného prostredia.These deficiencies are substantially eliminated by determining the conversion of nitrates to nitrites by microorganisms or their enzymes in a sample of biological material, which consists in culturing either a sample of the nitrate-containing biological material present or supplied to the sample or into the culture medium either alone. or in a culture medium in the presence of m-phenylenediamine or its salt at a concentration of 0,01 to 10 g / l of a sample of biological material and, if appropriate, a culture medium at 1 to 70 ° C for up to 14 days, then absorbance is assessed visually or photometrically a cultured biological material sample or a cultivated mixture of biological material sample and culture medium.
Uvedená kultivácia sa výhodne uskutočňuje v prítomnosti pufra s optimálnou hodnotou pH na konverziu dusičnanov na dusitany.Said cultivation is preferably carried out in the presence of a pH optimum buffer for the conversion of nitrates to nitrites.
Uvedená kultivácia sa taktiež môže výhodne uskutočniť v prítomnosti živín a/alebo rastových faktorov mikroorganizmov, akými sú výhodne jednoduché cukry, peptón, mäsový výťažok, kvasničný autolyzát, aminokyseliny a vitamíny.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.
Vznikajúci dusitan sa pri spôsobe podľa vynálezu viaže na m-fenyléndiamín a vznikajú ľahko identifikovateľné zlúčeniny absorbujúce svetlo. Výhodou tohto stanovenia je rýchlosť uvedenej reakcie, ktorá jc vyššia ako rýchlosť reakcie dusitanov s ostatnými konkurenčnými látkami, ktoré sa zvyčajne nachádzajú v stanovovanej vzorke biologického materiálu. Ďalšími výhodami spôsobu stanovenia podľa vynálezu je to, že stanovenie je lacné, jednoduché a málo toxické. Významnou výhodou stanovenia je i nízky inhĺbičný účinok m-fenyléndiamínu na mikroorganizmy alebo ich enzýmy, ktoré uskutočňujúThe nitrite formed in the process of the invention binds to m-phenylenediamine to form easily identifiable light absorbing compounds. The advantage of this determination is the rate of said reaction, which is higher than the rate of reaction of nitrites with other competing substances usually found in the sample of biological material to be determined. Another advantage of the assay method of the invention is that the assay is cheap, simple and of low toxicity. Another important advantage of the assay is the low inhibitory effect of m-phenylenediamine on microorganisms or their enzymes
Príklady uskutočnenia vynálezuDETAILED DESCRIPTION OF THE INVENTION
Príklad 1Example 1
Do zakvasenej, mladinovými baktériami kontaminovanej mladine s obsahom 20 mg NO3'.ľ', sa pridá 1 % vodný roztok m-fcnyléndiamínhydrochloridu vo výslednej koncentrácii 0,25 g.ľ1 a mladina sa kultivuje počas 5 dní pri teplote 10 °C. Ako slepý pokus sa použije rovnaká vzorka bez pridania m-fenyléndiamínhydrochloridu. Rozdiel absorbancie pri 450 nm oboch sfiltrovaných vzoriek po kultivácii je mierou tvorby dusitanov.To a fermented wort bacterium contaminated with wort containing 20 mg NO 3 '-1', a 1% aqueous solution of m-phenylenediamine hydrochloride is added at a final concentration of 0.25 g.l -1 and the wort is 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 cultivation is a measure of nitrite formation.
Príklad 2 ml pivovarníckych kvasníc sa zaleje 8 ml citrátového pufra s pH 5,0, potom sa pridá dusičnan draselný vo výslednej koncentrácii 150 mg NOj'.ľ1 a m-fenyléndiamín vo výslednej koncentrácii 0,25 g-ľ1 a zmes sa kultivuje počas 24 hodín pri teplote 37 °C. Červenohnedé zafarbenie je dôkazom tvorby dusitanov baktériami, prítomnými ako nečistota v kvasniciach.EXAMPLE 2 ml of brewer's yeast is quenched with 8 ml of citrate buffer pH 5.0, then potassium nitrate at a final concentration of 150 mg NO @ -1 and 1 m-phenylenediamine at a final concentration of 0.25 g @ -1 are added and the mixture is cultured. for 24 hours at 37 ° C. The reddish-brown color is evidence of nitrite formation by bacteria present as an impurity in yeast.
Príklad 3Example 3
Na platňu živnej pôdy obsahujúcej 0,5 % mäsového výťažku, 0,3 % peptónu, 0,5 % kvasničného autolyzátu, 0,5 % glukózy, 0,5 % dusičnanu draselného, 0,1 % m-fenyléndiamínu a 2,0 % agaru sa zaočkuje vzorka s obsahom baktérií. Po 48 hodinovej kultivácii pri teplote 37 °C sa prítomnosť baktérií tvoriacich dusitany preukáže tvorbou kolónií s hnedými zónami.For a nutrient plate containing 0.5% meat yield, 0.3% peptone, 0.5% yeast autolysate, 0.5% glucose, 0.5% potassium nitrate, 0.1% m-phenylenediamine and 2.0% Inoculate a sample containing bacteria. After cultivation at 37 ° C for 48 hours, the presence of nitrite-forming bacteria was demonstrated by the formation of colonies with brown zones.
Priemyselná využiteľnosťIndustrial usability
Vynález sa môže využiť pri kontrole produkcie potravinárskych a zdravotníckych výrobkov, pri výrobe selektívnych živných pôd na stanovenie mikroorganizmov produkujúcich dusitany a pri stanovení obsahu dusičnanov mikrobiálnymi alebo enzymatickými testami a pri výrobe činidiel na také 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 assays and to produce reagents for such assays.
Claims (3)
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 |
|---|---|
| SK314192A3 SK314192A3 (en) | 1995-11-08 |
| SK278700B6 true SK278700B6 (en) | 1998-01-14 |
Family
ID=5370623
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| 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 |
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 |
| CZ314192A3 (en) | 1995-11-15 |
| DE4331889A1 (en) | 1994-04-21 |
| CZ280631B6 (en) | 1996-03-13 |
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