Patent Application: US-201414780651-A

Abstract:
the invention deals with chromogenic media which are suitable for the selective growth and identification of one or more species of yeast . the subject of the invention is the method that enables us to identify and determine the cell count of brettanomyces / dekkera and zygosaccharomyces yeasts . besides , the subjects of invention are also the use of the method in wine and / or food industry and the stocks for conducting the experiment .

Description:
the present inventors unexpectedly found during the creation of the invention that if azure - ii - eosinate or other chromogenic paint that contains chemically similarly structured molecules is added to a selective medium — which is suitable for growing yeast , but blocking the growth of saccharomyces — then in the medium we get , we can identify brettanomyces species among the appearing colonies , with the help of azure - ii - eosinate . 1 . on this medium , the brettanomyces colonies are painted pink , which are visible for the eyes 2 . the pink colonies are fluorescent in ultraviolet light , which confirms the separation of the species . with the help of this method , the brettanomyces / dekkera colonies can be easily distinguished from other wild yeasts that are able to grow on a selective medium , by looking at them . the identification that is based on visibility , not only makes the identification process much easier , but it also does not require experience and scent samples , furthermore , it makes scenting samples unnecessary , which bears the possibility of spreading the infection . the reason why it is more specific to brettanomyces / dekkera species than other methods , is that it is able to identify other yeast species that are not winemaking ( noble ) yeasts , which ( only ) causes a problem in case of sweet wines with a higher residue of sugar . we have also run the experiments using different azure and eosin dyes . all of the stains &# 39 ; colours changed with the increase of ph . yet , these stains did not provide the previously experienced colour reaction and were not appropriate to distinguish the species of brettanomyces / dekkera from other , examined yeasts . according to the invention , for getting the desired results , we need substituted or unsubstituted bis - 3 , 7 - diamino - phenothiazines together with the substituted derivatives of fluorescein . there was a medium stain that was known before with the same components , eosin and methylene blue , but it was not used for identifying yeast contamination . for example , the united kingdom publication no . gb1248197 [ abbott lab ( us ), “ diagnostic method and apparatus for the detection of bacteria ”] discloses an eosin methylene blue agar medium that contains lactose , however , identifying the yeasts in the reference are not based on eosin methylene blue medium . for our understanding , previously they used the azure - ii - eosin for other purposes , mainly for colouring tissue samples . according to the japanese disclosure document no . jp56106588a they used eosin - y ( 0 . 5 g , 7 . 23 × 10 − 4 mol ) and methylene blue ( 0 . 065 g , 2 . 03 × 10 − 4 mol ) in one medium only , however , the document does not provide any information about the different discolouration of the medium and / or the colonies as a result of growing different types of yeasts . the chromogenic stain to be used according to the invention is therefore the combination of at least one type of substituted or unsubstituted bis - 3 , 7 diaminophenothiazine stain and at least one type of substituted fluorescein stain . preferably , the combination of the substituted or unsubstituted bis - 3 , 7 diaminophenothiazine stain of formula i herebelow and the substituted fluorescein stain of formula ii herebelow . the chemical structure of the bis - 3 , 7 diaminophenothiazine stain of formula i is r1 , r2 , r3 and r4 are independently h , methyl or ethyl , preferably h or methyl , q1 , q2 , q3 and q4 are independently h , c1 - 4 alkyl , halogen , pseudohalogen , — no or — no 2 , preferably h , methyl or ethyl , preferably h or methyl , most preferably h . preferably , the bis - 3 , 7 diaminophenothiazine stain of formula i is present in a cationic form , such as a salt formed with an anion . the anion is preferably a halide ion , most preferably chloride ion . according to a variation , the anion is formed by the substituted fluorescent stain of formula ii . the chemical structure of the substituted fluorescent stain of formula ii is wherein r1 , r2 , r3 és r4 are independently halogen , pseudohalogen , — no or — no 2 , q1 and q2 are h , c1 - 4 alkyl , c1 - 4 alkoxy , halogen , pseudohalogen , — no or — no 2 , 5 - or 6 - member heterocycle or q1 and q2 together form a 5 - or 6 - member heterocycle , in which case q1 and q2 are situated on adjacent c atoms . preferably , r1 and r4 are halogen , more preferably br or — no 2 . preferably , the substituted fluorescein stain of formula ii is used in an anionic form , preferably in the form of a salt formed with a cation . preferably , the cation is sodium ion , potassium ion or ammonium ion . according to a further preferred variation , the cation is formed by the bis - 3 , 7 diaminophenothiazine stain of formula i . preferably , the chromogenic medium of the invention comprises the at least one type of substituted or unsubstituted bis - 3 , 7 diaminophenothiazine stain and the at least one type of substituted fluorescein stain in essentially identical molar amounts , i . e . the amount of the at least one type of substituted or unsubstituted bis - 3 , 7 diaminophenothiazine stain and the amount of the at least one type of substituted fluorescein stain in the culture medium are at most 50 % or 30 %, preferably at most 20 % or 10 % different relative to the component that is present in smaller amount , that is , the ratio of the molar amounts is from 1 . 5 : 1 to 1 : 1 or from 1 . 3 : 1 to 1 : 1 , preferably from 1 . 2 : 1 to 1 : 1 or from 1 . 1 : 1 to 1 : 1 , most preferably the rate of the molar amounts is 1 : 1 or vice versa . most preferably , the culture medium of the invention contains multiple types of substituted or unsubstituted bis - 3 , 7 aminophenothiazine stains , in the general formula i of which r1 , r2 , r3 and r4 are h , methyl or ethyl so as that the substituents r1 , r2 , r3 and r4 are different in the different stains ( r1 , r2 , r3 and r4 may not be identical ). most preferably , r1 , r2 , r3 and r4 are h or methyl and the bis - 3 , 7 diaminophenothiazine component stains are different in the degrees of methylation . accordingly and preferably , methylene blue and the demethylated intermediers thereof or the mixture thereof may be used in the stain , selected from a n ′, n ′- dimethylphenothiazin - 5 - ium - 3 , 7 - diamine salt , preferably acetate or chloride ( azure a : cas 531 533 ) most preferably the mixture of azure b and methylene blue is present in the stain . similarly , the substituted fluoresceins may be of one or more types . most preferably , an eosin stain or a mixture of eosin stains is used , which may preferably comprise for example eosin b or eosin y . the eosin stain is preferably eosin b or eosin y , the formulas of which are , respectively most preferably , the stain used according to the invention is azure ii eosinate . azure ii eosinate ( cas 53092 - 85 - 6 ) is a mixture of methylene blue and azure b in the ratio of 1 : 1 and of eosin y . azure ii eosinate is available from various manufacturers ( such as fluka , sinopharm , cn és nile chemicals , ind .). it is apparent for the skilled artisan that further substituted variants or salts of the stains of the inventions may be used , provided they are chromatic and the colour changes in the presence of yeast . considering growth media , any growth medium being suitable for culturing yeasts and containing at least an agent which inhibits the growth of saccharomyces strains can be used , e . g . growth media disclosed in the background of the invention . based on the above , the invention concerns a method for selective culturing of brettanomyces / dekkera yeasts and differential staining of their colonies , where a growth medium suitable for culturing yeast cells is prepared , which is made selective by the addition of an appropriate chemotherapeutic agent or antibiotic inhibiting the growth of saccharomyces strains and by the addition of chromogenic stain of the invention . the culture medium may be of varied composition . theoretically , any growth medium suitable for culturing yeasts is appropriate and known by a person skilled in the art . the growth medium preferably contains ingredients selected from the following group : sugar , e . g . glucose ; aminoacid - or peptid - containing extract or hydrolizate , such as pepton , yeast extract , “ yeast nitrogen base ” or “ yeast carbon base ; geling agent , e . g . agar ; and optionally salt . highly preferably , the growth medium comprises glucose , yeast nitrogen base and agar . additionally , the growth medium also contains substances inhibiting the reproduction of microbes having a role in the normal or healthy fermentation of foodstuff . provided the foodstuff in which the detection method is performed is a foodstuff prepared by fermentation , the growth - inhibiting substance feasibly prevents the growing of microorganisms performing the natural fermentation of the foodstuff , e . g . it blocks the growing of noble yeast . it is obvious for a person skilled in the art that the growth inhibitor should be applied at least in such a concentration which is already sufficient enough to block the growth of such microorganisms . at the same time the inhibitor concentration may have an upper threshold not to inhibit the growth of yeasts , the presence of which is desired to be tested . preferably , the foodstuff is a foodstuff fermented by saccharomyces species , such as beer , wine or other yeast containing product or intermediate , and the agent inhibiting the growth of saccharomyces strains is an appropriate chemotherapeutic agent or antibiotic , e . g . cycloheximide applied in a concentration , e . g . of 0 . 5 - 50 μg / ml , preferably 1 - 20 μg / ml , particularly preferably 2 - 10 μg / ml , highly preferably something like 5 μg / ml , thereby the selectivity of the growth medium is enhanced or it is made selective . the sample may be any sample used in the production of such foodstuff , e . g . a sample taken from devices used in the process or a sample drawn from the liquid used for cleaning the devices . the prepared culture medium is brought to a form suitable for sample application . according to a certain variation a gel is prepared and a plate is poured into , e . g ., a petri dish . alternatively , any other solid ( e . g ., in a form of gel ) culture medium can be applied where the sample can be plated and the progeny ( e . g . colonies ) of a single cell can be separated . in addition to petri dishes any other culturing container having large surface can be preferably used , where the sample can be spread on the surface of medium formed in it , and it can be closed ( e . g ., has a lid ) and in which the microorganism colonies can be detected and feasibly visualized . it is preferable for the culturing container to be made of glass or plastic , more preferably plastic , and preferably it has a transparent lid . kolle dishes or roux flaks may also be used , they also have large surfaces but the sample should be introduced into the dish through a small opening and performing uniform plating also presents difficulties . then the opening should be closed in a way that allows some aeration but the sample does not get uncontaminated . consequently , according to the invention , sterilisable culturing dishes with lid may be used , in which samples to be tested can be plated on a large surface . from the samples ( e . g ., water used for washing barrels or other surfaces ) or from their suitable dilutions a predetermined amount is plated on the surface of culture media then they are incubated at 10 - 37 ° c ., preferably at 20 - 30 ° c ., particularly preferably at room temperature for about 5 - 20 days , preferably for 8 - 16 days , and highly preferably for 10 - 14 days . it is obvious for a person skilled in the art that the incubation time is necessarily longer at lower temperatures . on culture medium prepared according to the invention , saccharomyces yeasts stop growing , and the colour of brettanomyces / dekkera colonies become pink and they can be discriminated from microorganisms which are not harmful or just slightly harmful to the wine . the results are evaluated visually . in the event of sample application , the result can be made quantitative by giving the number of cultivable yeast cells per 1 ml . detection sensitivity of brettanomyces / dekkera yeasts may be enhanced by filtering a higher amount of wine through membranes with 0 . 45 μm or 0 . 22 μm pore size , then by placing the membrane on the surface of the culture medium . in this case it should be ensured that no air bubbles are present between the membrane and the agar surface . furthermore , the invention relates to culture media for performing the above method where the medium is in a powder or in a ready - to - use form , and also to the kits containing them and other components necessary for performing the examination ( e . g . sample application devices ) and the user instructions as well . preferably , the reagent kit of the invention comprises the culture medium necessary for performing the method of the invention in the form and amount pre - weighed for each test and in a form poured into plastic petri dishes in advance . the method developed by us is cheap and it does not require special instrumentation and easy to perform by anyone . the procedure requires no sterile laboratory conditions and only little attention is to be paid to ensure that the right sample is placed on the surface of the culture medium . the culture medium contains components easily available . in addition to components used for growth , the medium contains antibiotics inhibiting the growth of yeasts , e . g .— other culture media similar to selective brettanomyces — cycloheximide as well . this antibiotic is used for the identification of different species in yeast diagnostics . in the concentration used by the inventors , it prevents the growth of most yeasts playing a role in wine - making ( e . g . saccharomyces ) while this concentration is still tolerated by the species causing the degradation of wine . the present invention is further illustrated , but not limited by the following examples . in the following examples , unless indicated otherwise , the following concentrations and compositions were applied . composition of medium that was suitable for growing yeast cells was the following : 1 % glucose , 0 . 67 % “ yeast nitrogen base ”, 2 % agar , which was made selectively by using 5 μg / ml cycloheximide as an antibiotics for blocking the growth of saccharomyces strains . azure - ii - eosinate was used in a 30 μg / ml concentration . we make a 10 scale dilution sequence in 5 steps from destilled water that we gained from must . from each dilution we streak 50 μl onto the surface of the selective , chromogen medium in the petri dishes . we make the grafting in three parallel running measurements . we incubate the petri dishes between 20 - 25 ° c . for 10 - 14 days . the pink colonies that appear on the surface of the medium after the incubation time is over imply the brettanomyces / dekkera infection . we choose the dishes in which we can easily identify the number of colonies . if we multiply the number of colonies by twenty , plus the value of the dilution we get the plate count of the brettanomyces / dekkera of the must applied to 1 ml . a positive dish can be examined under uv light as well . the fluorescence of the colonies confirms the obtained results . we shake up the wine before taking a sample , then we filtrate 500 ml of it through a membrane filter with 0 . 45 μm pore diameter . we place the membrane filter on the surface of the selective chromogenic medium in the petri dish , in a way that it fits properly ( there should be no air bubble between them ). the petri dishes are incubated on 20 - 25 ° c . for 10 - 14 days . example 3 : identifying brettanomyces / dekkera species from red wine stored in barrels we centrifugate 50 ml from the red wine in the barrel ( 3000 rpm , 10 min , hereus multifuge 3s ). we suspend the pellet in 1 ml destilled water . from the suspension we streak 100 μm on the surface of the selective chromogenic medium in the petri dish . the petri dishes are incubated on 20 - 25 ° c . for 10 - 14 days . after washing the barrels , we filtrate 500 ml from the wash water through a membrane filter with 0 . 45 μm pore diameter . we place the membrane filter on the surface of the selective chromogenic medium in the petri dish , in a way that it fits properly ( there should be no air bubble between them ). the petri dishes are incubated on 20 - 25 ° c . for 10 - 14 days . we gently shake the grapes that are soaked in destilled water for an hour on room temperature . meanwhile the cells from the grapes are being washed in the water . after this , we pour out the water from the grapes and filtrate it through a membrane filter with 0 . 45 μm pore diameter . we place the membrane filter on the surface of the selective chromogenic medium in the petri dish , in a way that it fits properly ( there should be no air bubble between them ). the petri dishes are incubated on 20 - 25 ° c . for 10 - 14 days . we filtrate 500 ml from the bottled sweet wine through a membrane filter with 0 . 45 μm pore diameter . we place the membrane filter on the surface of the selective chromogenic medium in the petri dish , in a way that it fits properly ( there should be no air bubble between them ). the petri dishes are incubated on 30 ° c . for 10 - 14 days . the appearing blue colonies show a positive result . example 7 : identifying the level of infectivity of collective strains of brettanomyces / dekkera , zygosaccharomyces bailii and lachancea fermentatii we suspend 1 loop from the culture in 5 ml destilled water . from the suspension we streak it on the surface of the differentiating medium with the loop . the petri dishes are incubated on 20 - 25 ° c . for 10 - 14 days . the appearing blue zygosaccharomyces bailii colonies , the pink brettanomyces / dekkera , and the greenish blue lachancea fermentatii with the pink edge indicate infection . example 8 : recovering pure culture of brettanomyces / dekkera strains in culture collection we suspend 1 loop of the infected culture in 5 ml destilled water . with the loop we streak on the differentiating medium from the suspension . the petri dishes are incubated on 20 - 25 ° c . for 10 - 14 days . we make a suspension from the appearing pink colonies ( 1 loop / 5 ml sterile destilled water ), and from the suspension we streak on the differentiating medium with a loop . the petri dishes are incubated on 20 - 25 ° c . for 10 days . if we do not observe other colonies apart from the pink ones , we can be ascertained about the purity of the culture . we conducted the experiment according to example x . with the following stains as well : the colour of the applied stains change in each case with the increase of ph . the stains were not suitable for clearly separating the species of brettanomyces / dekkera from the other , examined yeasts . we conducted the experiment according to example x with the following media : we experienced the most contrasted pink / blue discolouration on ynb medium in case of the azure ii - eosin . the process and the media specified by the invention can be advantageously applied in the first place to monitor cell counts of brettanomyces / dekkera , identify or exclude their proliferation , as well as to detect for a hygienic purpose brettanomyces / dekkera yeasts responsible for the deterioration of wines and provisions in case of utensils used for storage with which they can get directly into contact . the usage of the medium makes the early identification of the growth of brettanomyces / dekkera yeasts — that can trigger the deterioration of food and wine — possible , as well as verifying the effect of the treatments that are aimed at avoiding deterioration . the method can identify other microorganisms , such as yeast species belonging to the zygosaccharomyce genus and lachancea genus . an advantage of this method is that it makes it possible to easily identify the colonies of brettanomyces / dekkera by looking at them , furthermore , colonies of yeast species belonging to the zygosaccharomyces and lachancea genus can be distinguished from the colonies , other species and wild yeasts that are able to grow on a selective medium . the identification that is based on visibility , not only makes the identification process much easier , but it also does not require experience and scent samples , furthermore , it makes scenting samples unnecessary , which bears the possibility of spreading the infection . the reason why it is more specific to brettanomyces / dekkera species than other methods , is that it is able to identify other yeast species that are not winemaking ( noble ) yeasts , which causes a problem in case of sweet wines with a higher residue of sugar . the method that we developed is cheap , it does not require special instruments , anyone can carry it out . circumstances of a sterile laboratory are not necessary , it only requires minimal attention to put the right sample onto the surface of the medium . the medium contains easily accessible components . barata a ., seborro f ., belloch c ., malfeito - 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