CS247280B1

CS247280B1 - Method of yeast and bacteria quantity quick determination in wine,soft drinks,beer,milk,mineral waters,syrups,drinking,service and waste waters and device for realizatin of this method

Info

Publication number: CS247280B1
Application number: CS144284A
Authority: CS
Inventors: Jan Farkas; Jan Sipos; Jozef Kovac
Original assignee: Jan Farkas; Jan Sipos; Jozef Kovac
Priority date: 1984-03-01
Filing date: 1984-03-01
Publication date: 1986-12-18

Abstract

Rieši sa problém rýchleho zisťovania mikroorganizmov vo vine a iných substrátoch, ako aj zariadenie k prevádzaniu tohoto1 sposoibu, čo umožňuje operatívnu mikrobiologi-ckú kontrolu v závodo-ch. Podstata spočívá v tom, že vzorka vína, alebo iného substrátu, v ktorých sa má zistiť množstvo kvasiniek, přetlačí sa vhodným sposobom cez tlakové filtračně zariadenie s membránou, na ktorej sa zachytia kvasinky. Podfa potřeby přetlačí sa filtrát ešte cez ďalšiu filtrační! membránu, na ktorej sa zachytia přítomné baktérie. Použitá filtračná membrána sa vyberie zo zariadenia, zafarbí a premyje. Bielkovinové částice majú schopnost' zadržať farbivo, v dosledku čoho sú buňky pod mikroskopem dobré viditelné. Přednostou metody je, že za 15 až 30 minút zistí sa přítomnost kvasiniek. Kolonie baktérií ,sú lepšie viditelné, ak sa filtračná membrána položí na agarové médium po dobu 3 až 6 hodin a až po tejto době sa sledujú pod mikroskopom. Využitie předmětného vynálezu umožňuje poznat mikrobiologický stav vína a iných substrátov a operativně riadiť výrobu.The problem of rapid detection of microorganisms is solved in wine and other substrates as well as a device to transfer this 1 sposoib which allows operative microbiological control in the factory. The essence lies in that the sample of wine or other substrate, in which the amount of yeast is to be determined, overpressure through a suitable way through diaphragm pressure filter device on which yeast is captured. According to need the filtrate is forced through the next filtration! the membrane on which they are present bacteria. Filter membrane used is removed from the apparatus, stained and washed. Protein particles have the ability to to hold the dye, as the cells are good visible under the microscope. The preferred method is that in 15 to 30 minutes the presence of yeast is detected. colony bacteria, they are better visible if they are filtering the membrane is placed on the agar medium after for 3 to 6 hours and only after that time are monitored under the microscope. Use of the subject the invention makes it possible to recognize microbiological state of wine and other substrates and operatively to manage production.

Description

Vynález sa týká spůsobu rychlého zistova-nia množstva kvasiniek a baktérií vo vine pre operatívnu mikrobiologická kontrolu výrobkov, ako aj zariadenia k prevádzaniu tohoto spůsobu.BACKGROUND OF THE INVENTION The present invention relates to a method for the rapid detection of a number of yeasts and bacteria in a guilt for the operative microbiological control of products, and to a device for carrying out the method.

Otázkou rychlého zisťovania mikroorganizroov sa zaoberajú viaceré firmy, ako napr. Millipore Corporation, Sartorius, Seitz Werke a iné, k čomu mají aj vyrobené vlastně ultrafiltračné zariadenie. Bolo vyvinuté vakuové membránové zariadenie, ktoré pozostáva z vakuového filtračného zariadenia s 500 mililitroivým lievikom, vákuovej odsávačky, vákuového alebo vodného čerpadla a ďalšieho příslušenstva. Ako uvádzajú Neradí a Kunkee na tomto zariadení je možné zistiť počet kvasiniek v priebehu 15 až 30 minút. Zisťovanie baktérií na tomto zariadení neuvád-z-ajú. Zariadenie nie je u nás přístupné a muselo by sa doviezť.The issue of rapid detection of microorganisms is being addressed by several companies, such as: Millipore Corporation, Sartorius, Seitz Werke and others, for which they have actually produced ultrafiltration equipment. A vacuum membrane device has been developed which consists of a 500 milliliter funnel vacuum filtration device, a vacuum pump, a vacuum or water pump, and other accessories. As stated by Neradi and Kunkee on this device, the yeast count can be measured within 15 to 30 minutes. Detection of bacteria on this device is not reported. The facility is not accessible by us and would have to be imported.

Výše uvedené nevýhody odstraňuje sposob podfa vynálezu, ktorého podstata spočívá v tom, že sa vzorka vína, piva, mlieka, nealkoholických nápojo-v, alebo minerálnych vůd a iných substrátov podrobí tlakovej filtrácii najprv cez membránu s priemerom pórov 1,2 μΐη a potom cez membránu s priemerom pórov 0,3 μΐη, membrány sa napustia farbivom, ktoré sa vlaže na bielkovinové částice, prebytočné farbivo sa vymyje a vyfarbené kvasinky a baktérie sa na membránách spočítajú pod mikroskopom. Rýchla metoda na zisťovanie kvasiniek a baktérií vo< vine a ostatných substrátoch je teda založená na dvojitej filtrácii s rozdielnymi pórami prietoku filtračnej membrány, vyfarbeni-a mikroorganizmov zachytených na membránách a na mikroskopickom vyhodnotení.The above disadvantages are overcome by the method according to the invention, which consists in subjecting a sample of wine, beer, milk, soft drinks or mineral leads and other substrates to a pressure filtration first through a membrane with a pore diameter of 1.2 μΐη and then through a membrane with a pore diameter of 0.3 μΐη, the membranes are impregnated with a dye, which is pulverized into protein particles, the excess dye is washed out and the stained yeast and bacteria are counted on the membranes under a microscope. Thus, a rapid method for detecting yeast and bacteria in vine and other substrates is based on double filtration with different pores of filtration membrane flow, staining and microorganisms trapped on the membranes, and microscopic evaluation.

Vynález sa dalej týká zariadenia k prevádzaniu uvedeného spůsobu, ktoré pozostiava z telesa- 1 filtra a viečkia 2 filtra, imedzi ktorými je vložená filtračná doska 3 s filtr-a-čnoiu imembránou 4, pričom vo- vlečku 2 filtra je vytvořený otvor 5 n-a pripojenie tlakového- dávko-vača B. Celé zariadenie je n-asadené na vh-odnú zbernú -nádobu 7.The invention further relates to a device for carrying out said method, which comprises a filter body 1 and a filter cap 2, between which a filter plate 3 with a filter-like membrane 4 is inserted, wherein a filter aperture 2 is provided for connection of the pressure feeder B. The whole apparatus is n-mounted on a suitable collection container 7.

Pri stanovení sa postupuje tak, že sa sterilně odo-berie vzorka vína, alebo iného substrátu, výhodné injekčnou striek-ačkou, táto sa nasadí ιη-a -otvor 5 vlečka 2 filtra a jemným tlakom n-a, piest injekčnej striekačky přetlačí sa obsah vína, alebo iného substrátu cez membránu 4 s pórami 1,2 μΐη, n-a ktorej sa zachytia kvasinky a potom- cez membránu 4 s pórami 0,3 «m, na ktorej sa zachytia baktérie.The assay is performed by sterile removal of a sample of wine or other substrate, preferably via a syringe, which is fitted with a filter opening 5 of the filter spigot 2, and gently presses on the plunger of the syringe to squeeze the wine content. or another substrate through a 1.2 μ sη pore membrane 4 on which yeast is retained and then through a 0.3 µm pore membrane 4 on which bacteria are retained.

Filtračně zariadenie sa opatrné rozoberie a vyberie sa filtračná membrána. Filtr-ačná membrána sa položí n-a gázu napuštěnu farbivom, s výhodou Ponceau S, v Petriho miske a nechá sa působit 4 minúty. Farbivo Ponceau sa připraví vždy čerstvé tak, že sa rozpustí 0,9 g Ponceau S v 13,4 g kyseliny trichlóroctavej a 13,4 g kyseliny sulfos-alíicylo-vej v 72,3 g sterilnej destilovanej vody, čím s-a získá 100 g upraveného< farbiva. Filtra-čná membrána sa prenesie do ďalšej Petriho- misky s 5 % ro-ztokom kyseliny octovej, kde sa nechá působit 60 sekúnd.Carefully disassemble the filter device and remove the filter membrane. The filter membrane is placed n-a gauze impregnated with a dye, preferably Ponceau S, in a Petri dish and allowed to act for 4 minutes. The Ponceau dye is always fresh prepared by dissolving 0.9 g of Ponceau S in 13.4 g of trichloroacetic acid and 13.4 g of sulfosalicylic acid in 72.3 g of sterile distilled water to give 100 g of treated <dyes. The filter membrane was transferred to another petri-dish with a 5% acetic acid solution for 60 seconds.

Pri tomto sa farbivo vyplaví z nebielkovinových častíc. Bielkovinové časti-ce farbivo zadržia, to znamená, že buňky zostanú červene sfarbené, v důsledku čoho je ich možné pod mikroskopom vidieť. Filtr-ačná membrána sa prenáša tofkokrát, dokial' sa farbivo úplné nevyplaví a filter zostane biely až růžový.In this case, the dye is washed out of the non-protein particles. Protein particles retain the dye, i.e. the cells remain colored red, which makes them visible under the microscope. The filter membrane is transferred three times until the dye is completely washed out and the filter remains white to pink.

Premytá filtračná membrána položí sa na podložně sklo a může sa stanovovat počet kvasiniek pod mikroskopom pri 400 až 600násobnom zvačšení.The washed filter membrane is placed on a glass support and the yeast count can be determined under a microscope at 400-600 fold magnification.

Baktérie můžu sa dobré pozorovat tak, že filtračná membrána sa po vybratí z filtr-ačného zariadenia položí na agar-ové médium -n-a 3 až 6 hodin. Vytvořené kolonie sú poto-m lepšie viditelné.The bacteria can be observed by placing the filter membrane on the agar medium for 3-6 hours after removal from the filter device. The colonies formed are therefore more visible.

Pri mikrobiologickej kontrole stabilizovaných vín v-o ffiašiilach piamacnu uvádzianej rýchlo-metódy sa v niektorých vzorkách pod mikroskopom nenašli žiadne buňky a v niektorých našli sa len ojedinělé buňky kvasiniek a baktérií. Pri celkovom hodnotení m-ikrobiologickej stability musí sa brat do úvahy a hodnotit počet buniek na 1 liter. Všeobecne sa uvádza, že na 1 liter může byť přítomných do- 100 buniek. Vo vine, ktoré sa stabilizuje kyselinou sorbov-ou, může byť počet až do 500 buniek.In the microbiological control of stabilized wines in p-flasks presented by the fast method, no cells were found in some samples under the microscope and in some only isolated cells of yeast and bacteria were found. In an overall assessment of m-icrobiological stability, the number of cells per liter must be considered and evaluated. It is generally stated that up to 100 cells may be present per liter. Up to 500 cells may be present in the wine which is stabilized with sorbic acid.

Pri mikrobiologickej kontrole vín zo- sudov, ktoré boli pri vizuálnom h-odnotení číře a iskrivé, -našlo- s-a velké množstvo buniek kvasiniek i baktérií.In the microbiological control of wines from barrels which were visually clear and sparkling, a large number of yeast and bacteria cells were found.

Vyriešená rýchla metóda na zisťovanie přítomnosti kvasiniek a baktérií je vhodná ma kontrolu- mikrobiologickej stability vína, piv-a, mlieka, sirupov, nealkoholických nápojov, minerálnych vůd a iných tekutin v různých stádia,ch výroby a najma pire-d flašovaním a dá s-a využit v závodoch potravinářského priemyslu. Jej přednostou je, že za 15 až 30 minút zistí sa prítomno-sť kvasiniek. Kolonie baktérii sú lepšie viditelné, ak sa filtračná membrána položí na agarové médium po- dobu 3 až 6 hodin a až po tejto době sa sledujú po-d mikroskopom.The solved fast method for detecting the presence of yeast and bacteria is suitable for controlling the microbiological stability of wine, beer, milk, syrups, soft drinks, mineral leads and other liquids at various stages, production and bottling, and can be used in food processing plants. Its advantage is that the presence of yeast is detected in 15 to 30 minutes. Bacteria colonies are better visible when the filter membrane is placed on agar medium for 3 to 6 hours and only after this time is monitored by microscope.

Zariadenie k prevádzaniu spůsobu je možné vyrobit a používat za vyu-žitia iba tuzemských materiálov- a pomůcok.The process apparatus can be manufactured and used using only domestic materials and aids.

Claims

SUBJECT

INVENTION

1. A method for rapidly detecting the amount of yeasts and bacteria in wine, beer, milk, soft drinks, syrups, mineral waters, drinking, service and waste water, characterized in that a sample of the test substrate is first subjected to pressure filtration through a membrane with a pore diameter of 1.2 µm and then through a membrane with a pore diameter of 0.3 µm, the membranes are impregnated fairly, which binds to the proteinaceous particles, the excess dye is washed out and the stained yeast and bacteria are counted on the membranes! under the microscope.

2. Method according to claim 1, characterized in that the bacteria are allowed to propagate on the membrane for 3 to 6 hours on an agar nutrient medium before staining.

3. Method according to claim 1, characterized in that the coloring agent is a food-red color and as the eluting agent a solution of acetic acid in water at a concentration of 5% by weight.

Device according to claim 1, characterized in that it comprises a filter body (1j), a filter cap (2) and between which a filter plate (3j) with a filter membrane (4j) is inserted, wherein in the filter cap (2) an opening (5) is provided for connecting the pressure dispenser (6j).

Device according to Claim 4, characterized in that the pressure dispenser (6) is a syringe.

1 sheet of drawings