ZA200109748B - Culture medium for the detection of zygosaccharomyces. - Google Patents
Culture medium for the detection of zygosaccharomyces. Download PDFInfo
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- ZA200109748B ZA200109748B ZA200109748A ZA200109748A ZA200109748B ZA 200109748 B ZA200109748 B ZA 200109748B ZA 200109748 A ZA200109748 A ZA 200109748A ZA 200109748 A ZA200109748 A ZA 200109748A ZA 200109748 B ZA200109748 B ZA 200109748B
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- South Africa
- Prior art keywords
- culture medium
- zygosaccharomyces
- green
- yeasts
- medium
- Prior art date
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- 239000001963 growth medium Substances 0.000 title claims description 89
- 238000001514 detection method Methods 0.000 title claims description 26
- 241000235017 Zygosaccharomyces Species 0.000 title claims description 13
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 95
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 86
- 239000002609 medium Substances 0.000 claims description 63
- 241000235029 Zygosaccharomyces bailii Species 0.000 claims description 57
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 47
- 235000019253 formic acid Nutrition 0.000 claims description 47
- 241000235034 Zygosaccharomyces bisporus Species 0.000 claims description 40
- 238000000034 method Methods 0.000 claims description 33
- 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 claims description 26
- 239000008103 glucose Substances 0.000 claims description 26
- 230000008859 change Effects 0.000 claims description 15
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 13
- 235000014101 wine Nutrition 0.000 claims description 13
- 230000008569 process Effects 0.000 claims description 11
- 229940088594 vitamin Drugs 0.000 claims description 11
- 235000013343 vitamin Nutrition 0.000 claims description 11
- 239000011782 vitamin Substances 0.000 claims description 11
- 229930003231 vitamin Natural products 0.000 claims description 11
- 229920001817 Agar Polymers 0.000 claims description 10
- 239000002696 acid base indicator Substances 0.000 claims description 10
- 239000008272 agar Substances 0.000 claims description 10
- 235000013305 food Nutrition 0.000 claims description 9
- 230000012010 growth Effects 0.000 claims description 9
- FRPHFZCDPYBUAU-UHFFFAOYSA-N Bromocresolgreen Chemical compound CC1=C(Br)C(O)=C(Br)C=C1C1(C=2C(=C(Br)C(O)=C(Br)C=2)C)C2=CC=CC=C2S(=O)(=O)O1 FRPHFZCDPYBUAU-UHFFFAOYSA-N 0.000 claims description 8
- 230000001580 bacterial effect Effects 0.000 claims description 8
- 150000001875 compounds Chemical class 0.000 claims description 8
- 239000003112 inhibitor Substances 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 6
- 239000007003 mineral medium Substances 0.000 claims description 6
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 241000894006 Bacteria Species 0.000 claims description 5
- 230000001954 sterilising effect Effects 0.000 claims description 5
- 238000004659 sterilization and disinfection Methods 0.000 claims description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 4
- 239000003242 anti bacterial agent Substances 0.000 claims description 4
- 235000013361 beverage Nutrition 0.000 claims description 4
- 230000003115 biocidal effect Effects 0.000 claims description 4
- 239000011575 calcium Substances 0.000 claims description 4
- 229910052799 carbon Inorganic materials 0.000 claims description 4
- 239000008367 deionised water Substances 0.000 claims description 4
- 229910021641 deionized water Inorganic materials 0.000 claims description 4
- 239000011734 sodium Substances 0.000 claims description 4
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 3
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- 239000001166 ammonium sulphate Substances 0.000 claims description 3
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 3
- 229960002685 biotin Drugs 0.000 claims description 3
- 235000020958 biotin Nutrition 0.000 claims description 3
- 239000011616 biotin Substances 0.000 claims description 3
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 3
- 239000004327 boric acid Substances 0.000 claims description 3
- 229960005069 calcium Drugs 0.000 claims description 3
- 229910052791 calcium Inorganic materials 0.000 claims description 3
- LLSDKQJKOVVTOJ-UHFFFAOYSA-L calcium chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Ca+2] LLSDKQJKOVVTOJ-UHFFFAOYSA-L 0.000 claims description 3
- 229940052299 calcium chloride dihydrate Drugs 0.000 claims description 3
- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 claims description 3
- 150000004683 dihydrates Chemical class 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- WOSISLOTWLGNKT-UHFFFAOYSA-L iron(2+);dichloride;hexahydrate Chemical compound O.O.O.O.O.O.Cl[Fe]Cl WOSISLOTWLGNKT-UHFFFAOYSA-L 0.000 claims description 3
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 3
- CDUFCUKTJFSWPL-UHFFFAOYSA-L manganese(II) sulfate tetrahydrate Chemical compound O.O.O.O.[Mn+2].[O-]S([O-])(=O)=O CDUFCUKTJFSWPL-UHFFFAOYSA-L 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- 229960003512 nicotinic acid Drugs 0.000 claims description 3
- 235000001968 nicotinic acid Nutrition 0.000 claims description 3
- 239000011664 nicotinic acid Substances 0.000 claims description 3
- -1 oligoelements Natural products 0.000 claims description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 3
- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 claims description 3
- 229960004172 pyridoxine hydrochloride Drugs 0.000 claims description 3
- 235000019171 pyridoxine hydrochloride Nutrition 0.000 claims description 3
- 239000011764 pyridoxine hydrochloride Substances 0.000 claims description 3
- 229910052708 sodium Inorganic materials 0.000 claims description 3
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 claims description 3
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 claims description 3
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 2
- 238000011161 development Methods 0.000 claims description 2
- 239000004615 ingredient Substances 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 239000011591 potassium Substances 0.000 claims description 2
- 229910052700 potassium Inorganic materials 0.000 claims description 2
- 238000003908 quality control method Methods 0.000 claims description 2
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 claims 3
- 230000001419 dependent effect Effects 0.000 claims 1
- 244000005700 microbiome Species 0.000 claims 1
- 238000004886 process control Methods 0.000 claims 1
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 70
- 238000011534 incubation Methods 0.000 description 30
- 241000894007 species Species 0.000 description 18
- 241000235062 Pichia membranifaciens Species 0.000 description 14
- 230000004044 response Effects 0.000 description 14
- 239000006285 cell suspension Substances 0.000 description 12
- 238000011081 inoculation Methods 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 10
- 241001522017 Brettanomyces anomalus Species 0.000 description 9
- 238000011084 recovery Methods 0.000 description 9
- 239000007787 solid Substances 0.000 description 9
- 239000000725 suspension Substances 0.000 description 9
- 238000004040 coloring Methods 0.000 description 8
- 238000005374 membrane filtration Methods 0.000 description 8
- 239000002253 acid Substances 0.000 description 7
- 239000000203 mixture Substances 0.000 description 6
- 241000235036 Debaryomyces hansenii Species 0.000 description 5
- 241000235072 Saccharomyces bayanus Species 0.000 description 5
- 241001123227 Saccharomyces pastorianus Species 0.000 description 5
- 241000229116 Zygotorulaspora florentina Species 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 241001149671 Hanseniaspora uvarum Species 0.000 description 3
- 235000014663 Kluyveromyces fragilis Nutrition 0.000 description 3
- 241001508814 Lodderomyces elongisporus Species 0.000 description 3
- 241000235645 Pichia kudriavzevii Species 0.000 description 3
- 235000018370 Saccharomyces delbrueckii Nutrition 0.000 description 3
- 244000253911 Saccharomyces fragilis Species 0.000 description 3
- 235000018368 Saccharomyces fragilis Nutrition 0.000 description 3
- 241000235347 Schizosaccharomyces pombe Species 0.000 description 3
- 244000288561 Torulaspora delbrueckii Species 0.000 description 3
- 235000014681 Torulaspora delbrueckii Nutrition 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000011109 contamination Methods 0.000 description 3
- 229940031154 kluyveromyces marxianus Drugs 0.000 description 3
- 244000027711 Brettanomyces bruxellensis Species 0.000 description 2
- 235000000287 Brettanomyces bruxellensis Nutrition 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 241000235648 Pichia Species 0.000 description 2
- 241000223254 Rhodotorula mucilaginosa Species 0.000 description 2
- 241001489222 Saccharomycodes ludwigii Species 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 239000006160 differential media Substances 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 1
- 241000722885 Brettanomyces Species 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 244000286779 Hansenula anomala Species 0.000 description 1
- 235000014683 Hansenula anomala Nutrition 0.000 description 1
- 241000223252 Rhodotorula Species 0.000 description 1
- 241001489223 Saccharomycodes Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010876 biochemical test Methods 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- 230000001332 colony forming effect Effects 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 230000033629 detection of yeast Effects 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000013095 identification testing Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 238000012543 microbiological analysis Methods 0.000 description 1
- 244000005706 microflora Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000014639 sexual reproduction Effects 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000009417 vegetative reproduction Effects 0.000 description 1
- 210000005253 yeast cell Anatomy 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
- C12Q1/045—Culture media therefor
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Toxicology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
15-12-2000 'PCT/PT00/00004 | DESCPAMD © gag10-PCT So :
Culture medium for Jehel detection of Zygosaccharomyces baiti—and [2rgosaccharomyces bisporns-Yeasty'
Object of the Invention
The present invention refers to a differential and selective culture medium _ containing glucose, formic acid and an acid-base indicator, for the detection in a sample, after 48 hours, of Zygosaccharomyces bailii and
Zygosaccharomyces bisporus yeasts, two of the most dangerous species when considering food deterioration, and to a process for the detection of
Zygosaccharomyces bailii and Zygosaccharomyces bisporus yeasts using the referred culture medium. ein further object of the present invention the use of the referred culture medium in a gallery of yeasts identification tests. : State of the prior art
Yeasts are a growing problem in the food industry. The use of milder preservation processes in order to maintain the organoleptic properties of the product, of packages with modified atmospheres, and of new formulations, designed to avoid bacterial contamination are, nevertheless, favorable to yeast contamination. Although some pathogenic yeast species have been detected in food and the opportunistic strains may be dangerous to a fraction of the population, the fundamental risk of contamination that arises is not one of sanitary nature, but it consists in the spoilage effects that certain yeasts, such as Zygosaccharomyces bailii and Zygosaccharomyces bisporus have in food products, with the consequent economic losses involved. -
Heretofore, the study of the yeast microflora present in the most diverse habitats (e.g. food, nature), comprises a first strain isolation stage, using the general selective yeast culture media, and a second identification stage of the Fal isolated strains, through the use of conventional and/or molecular biology based methods. The classical yeast identification methods are based in a series of vegetative and sexual reproduction characteristics, and comprise a h ! LEE Com
Printed:10-04-2001 1
] PCT/PT00/00004 a large range of physiological and biochemical tests. It is a demanding work that only produces results after at least one to two weeks, and requires a great deal of experience for the correct interpretation of the results. The molecular biology based methods are, generally, faster than the classical ones, but they also require a good amount of operator experience and involve expensive equipment and reactants. ’
There are some culture media commercially available for the detection of yeasts in wines, namely the Wallerstein Laboratory Nutrient Medium, WLN, used for detecting fermenting yeasts, and the Wallerstein Laboratory
Differential Medium, WLD, which allow the detection of lactic and acetic bacteria as well as of yeasts belonging to the non-fermenting flora (both from
Difco). However, these prior art media are not capable to differentiate the yeasts, particularly the Zygosaccharomyces bailii species.
There is therefore the necessity for a culture medium and a process for the detection and identification of Zygosaccharomyces baifii and
Zygosaccharomyces bisporus, rapid and efficient, and which is thus an alternative means to the conventional techniques for the rapid detection of these species.
Description of the invention it was surprisingly found that Zygosaccharomyces bailii and
Zygosaccharomyces bisporus yeasts, when grown in a medium containing glucose, formic acid and an appropriated acid-base indicator, lead to a rapid change in the medium color and to the formation of colored colonies after at least 48 hours, these changes being characteristic and exclusive of the referred yeasts in the referred culture medium.
It was also found that the medium according to the invention is differential for the Zygosaccharomyces bailii and Zygosaccharomyces bisporus yeasts, 2
AMENDED SHEET
¥ through the inclusion of an appropriate acid-base indicator, and can be selective for the growth of the referred yeasts, depending on the formic acid concentration present in the medium.
Thus, according to the present invention, a new differential and selective culture medium was developed, which permits the identification of
Zygosaccharomyces baili and Zygosaccharomyces bisporus yeasts, assuring results after 48 hours of incubation, and which is therefore an alternative means to the conventional techniques for the rapid detection of these species, allowing a drastic reduction in the time and work involved in their identification.
The culture medium according to the present invention comprises a base mineral medium, supplemented with vitamins, oligoelements, glucose and formic acid as the only carbon and energy sources, an appropriated acid-base indicator, namely one having a pK; between 4.5 and 4.8, particularly bromocresol green, and optionally agar and an antibiotic inhibitor of bacterial : growth, such as cloramphenicol.
According to the present invention, the formic acid is present in the culture medium in a concentration from 0.1% to 0.5% (v/v), the concentration being selected depending if the culture medium is to be selective or only differential.
When the concentration of formic acid is increased in the culture medium according to the present invention, the selectivity of the medium for Z. bailii and Z bisporus yeasts increases, although at expenses of some differentiability, as shown in examples 6 and 7 below.
According to the present invention, the glucose is present in a concentration from 0.05% and 0.1% (p/v), preferably 0,1% (p/v).
The culture medium according to the present invention further allows, through the choice of appropriated conditions, in particular the inoculation methodology, the enumeration of Z. bailii and Z. bisporus yeasts in a sample, regardless the presence of other yeasts, since it is selective as shown in examples 4 and 8.
B in an embodiment of the present invention, the acid-base indicator is bromocresol green which provides the medium with a green color, that is converted to blue by the Zygosaccharomyces bailii and Zygosaccharomyces bisporus yeasts. Additionally, the colonies of the referred yeasts present, in the medium of the invention, a blue coloring.
In another embodiment, the culture medium according to the invention may contain additionally an inhibitor of bacterial growth, being particularly useful for application in samples of mixed populations including bacteria.
The culture medium object of the invention is prepared by autoclave sterilization of the base mineral medium in deionized water. The medium is then allowed to cool, and before solidifying, the glucose, formic acid, oligoelements and vitamins, prepared as adequate solutions and previously
FJ sterilized, are added under aseptic conditions. The whole medium is homogenized and aseptically dispensed into Petri dishes.
The present invention also refers to a process of detection of
Zygosaccharomyces bailii and Zygosaccharomyces bisporus yeasts present in a sample, using a culture medium according to the present invention, as characterized above.
According to this feature of the present invention, a process was developed which comprises: (i) preparing a medium according to the present invention; (ii) inoculating it, by spreading, streaking or by deposition of a drop of cell
X suspension, with a sample to be analyzed for Zygosaccharomyces bailii and/or
Zygosaccharomyces bisporus yeasts; (ii) incubating it in a incubator at a suitable temperature and for a time enough for the yeast development (minimum 48 hours); and (iv) observing the color changing in the medium and the colonies formation, to conclude the presence of the referred yeasts when occurs a changing of the medium color and formation of colored colonies in agreement with the acid-base indicator used.
The present invention can be used with previously isolated and purified strains, there being no kind of limitation concerning the type of inoculation that is used. However, the time needed to observe the turning of the indicator depends on the cell concentration of the inoculum and on the method of inoculation.
The present invention can also be used with cell suspensions of mixed yeast : populations, containing yeasts other than Zygosaccharomyces bailii and : Zygosaccharomyces bisporus, providing information about the presence of these species, every time that blue colonies are detected in conjunction with a change in the medium color.
One of the objects of the present invention is to provide the food industry, particularly the wine and beverages industry, with a procedure for the detection of Zygosaccharomyces bailii and Zygosaccharomyces bisporus yeasts. The procedure is simple and easily reproducible by any microbiological analysis laboratory. Additionally, the production of the culture medium doesn’t require new technologies. Once prepared, the culture medium finds immediate use in any industrial facility or quality control laboratory, since there is no need for highly skilled personnel other than the one in charge of the routine microbiological analyses.
15122000 PCT/PTO0/0N004 © DEscPAMD « 84810 - PCT
Further, the culture medium according to the present invention can be used to integrate galleries of identification of yeasts.
Figure 1 is a photograph showing the response of several yeasts (Z. bailii ISA oo 1265 and Z. bailii IGC 3858. positive response; 7. delbrueckii ISA 1229 and /.
Drientalis IGC 3806: negative response) in a solid medium according to the present invention containing glucose (0.1% w/v) and formic acid (0.3% v/v) at the end of 96 hours of incubation at 30°C. The Z. bailii yeasts shown a positive response revealed by a blue coloring of the culture medium in the dish, while the negative responses are shown as a green coloring which did not change during the incubation. . : Figure 2 is a photograph showing the response of several yeasts in a liquid medium according to the present invention containing glucose (0.1% w/v) and formic acid (0.3% w/v) at the end of 48 hours of incubation at 30°C. All the
Z. baflii strains induced the medium to change color to blue, while all the , others maintained the green color.
Figure 3 shows the morphology of Zygosaccharomyces baijlii yeast colonies in a culture medium according to the present invention containing 0.3% (v/v) of formic acid and 0.1% (w/v) of glucose, obtained by the use of the method of membrane filtration, after 96 hours of incubation at the temperature of 30°C.
The colonies can be observed well defined with a blue color, -
Figure 4 shows the morphology of S. cerevisiae and Zygosaccharomyces bailii yeast colonies in a culture medium according to the present invention . containing 0.2% (v/v) of formic acid and 0.1% (w/v) of glucose, obtained by the use of the method of membrane filtration, after 96 hours of incubation at the temperature of 30°C. The Z bailii colonies shown a blue coloring, perfectly distinct from the creme coloring of the other colonies. 6 Mmended sheed
Printed:10-04-2001 A
] PCT/PT00/00004
S.A
Figure 5 ‘shows the morphology of P. membranifaciens and
Zygosaccharomyces baijlii yeast colonies in a culture medium according to the present invention containing 0.2% (v/v) of formic acid and 0.1% (w/v) of glucose, obtained by the use of the method of membrane filtration, after 96 —_-- hours of incubation at the temperature of 30°C. The Z. bailii colonies are totally distinguishable by its morphology and blue color.
In a preferred embodiment of the present invention the differential and selective culture medium, for identification of Zygosaccharomyces baifii and
Zygosaccharomyces bisporus yeasts in a sample, after atleast 48 hours of incubation, comprises a base mineral medium, including bromocresol green as the acid- : base indicator, supplemented with oligoelements and vitamins, 0.05% to 0.1% (w/v) of glucose and 0.1% to 0.5% (v/v) of formic acid as the only energy and carbon sources, and optionally agar and an inhibitor of bacterial growth.
In this embodiment of the invention, the bromocresol green provides the medium with a green coloring that will be converted into blue through the action of the Zygosaccharomyces bailii and Zygosaccharomyces bisporus yeasts during incubation under appropriate conditions. Additionally, the colonies of these yeasts will also present a blue color. The change of color of the culture medium is characteristic of these yeast species, as illustrated in examples 1 and 2, thus allowing the detection of the presence thereof in a sample only by the color changing.
The process according to the present invention will now, be illustrated by means of the non limitative examples below: 7
AMENDED SHEET
. PCT/PT00/00004
Example 1
This example illustrates the preparation of a solid culture medium according to the present invention and shows that it is effective in the identification of
To Z. bailii and Z. bisporus yeasts.
A culture medium is prepared comprising the following ingredients:
Table 1 Culture medium composition for the detection of the Zygosaccharomyces bailii and Zygosaccharomyces bisporus yeasts i - ___——
Compound Concentration (%)
Base Medium Ammonium sulphate {NH,4},S0O, 0.5 (w/v)
Potassium dihydrogen phosphate KH,PO, 0.5 (wiv) -
Magnesium sulphate heptahydrate MgSO, -7H,0 0.05 (wiv)
Calcium chloride dihydrate CaCl,-2 H,0 0.013 (w/v) :
Bromocresol green C,.H,,Br,0:S 0.005 (wiv) :
Agar - 2.0 {w/v) .
Glucose - CeH,.04 0.1 (w/v)
Formic acid - . } CH,0, 0.4 (v/v)
Oligoelements Solution A (Composition according to Table 2) - 0.05 (v/v)
Oligoelements Solution B (Composition according to Table 2) -- 0.05 (viv)
Vitamin Solution (Composition according to Table 2) - 0.05 (viv)
Table 2 Oligoelements and vitamin solutions composition
Compound . Concentration {%)
Oligoelements Solution A Boric acid H,BO, 0.1 (wiv) : . Potassium lodide Ki 0.02 {w/iv)
Sodium molibdate dihydrate Na,MoQ,-2H,0 0.04 (wiv)
Oligoelements Solution B Copper sulphate pentahydrate LuS0,-5H,0 0.008 {w/iv)
Iron chloride hexahydrate FeCl, - 6 H,0 0.02 {wiv)
Manganese sulphate tetrahydrate MnSO,-4H,0 0.08 (w/v)
Zinc sulphate heptahydrate ZnSO, -7H,0 0.08 {wiv}
Hydrochloric acid HCI 10°N 0.8 viv)
Vitamin Solution Biotin’ C,oH1sN,0,5 0.001 (w/v)
Calcium panthotenate C,H,(NO, 1/2 Ca 0.08 {w/v}
Mioinositol CeH,204 4.0 (wiv)
Niacin CHsNO, 0.16 (w/v)
Pyridoxine hydrochloride CH,,NO,HC! C.16 (wiv)
Thiamin hydrochloride C,;H,,CIN,OS-HCI 0.16 {w/iv) -YYYYYYYYYYY tr mm YY WW
AMENDED SHEET
) PCT/PT00/00004
I
The base medium compounds are dissolved in 4/5 of the estimated deionized water volume, and the sterilization is accomplished in autoclave at 121°C, for 20 minutes. The pH must be adjusted to 4.5.
The other medium compounds (glucose, formic acid, oligoelements solution A, — oligoelements solution B, and vitamin solution) are dissolved in the remaining water volume so that the final concentration of these compounds equals the values mentioned in Table 1. The pH must be adjusted to 4.5 with NaOH 10M. The sterilization is accompanied by filtration. This solution and the base medium are brought to 50 + 5°C before being mixed together. The whole medium is homogenized and dispensed into Petri dishes.
The yeast strains to be identified, previously purified and inoculated in agar slants with a generic yeast culture medium (yeast extract medium, peptone, and glucose), are incubated for 48 hours at 28°C. An loopful is transferred to : the culture medium with glucose and formic. acid, prepared above. The inoculation is made by streaking and the plates are incubated at 30°C, for a minimum time of 48 hours. Alternatively, the inoculation may be done with a cotton smear containing an equivalent biomass amount.
The results obtained are presented in Table 3. Typical responses of the below mentioned yeasts are shown in Figure 1. 9
AMENDED SHEET i PCT/PT00/00004
Table 3 Inoculation by streaking - response of several ‘yeasts in the culture medium containing glucose and formic acid (0.4% v/v) after 48 hours of incubation at 30°C. ——————————————————————————————————————————————————————————————————————————————————————————————
Species N. of tested strains Culture medium color —_—
Zygosaccharomyces bailii 15 blue
Zygosaccharomyces bisporus 5 blue
Zygosaccharomyces bisporus 3 blue*
Zygosaccharomyces nouxii 6 green - Zygosaccharomyces florentinus 1 green
Saccharomyces bayanus’ 2 green
Saccharomyces cerevisiae 21 green
Saccharomyces pastorianus 2 green
Saccharomycodes ludwigii 3 green . Schizosaccharomyces pombe 4 green
Pichia membranifaciens 13 green
Pichia anomala 7 green
Dekkera anomala 3 green :
Dekkera bruxellensis 4 green
Debaryomyces hansenii 2 green
Issatchenkia orientalis 6 green
Kluyveromyces marxianus 5 green
Kloeckera apiculata 1 green
Lodderomyces elongisporus 2 green
Rhodotorula mucilaginosa 2 - green
Torulaspora delbrueckii 7 green . -* the change in the medium color was observed after an additional incubation period of 24-48 hours
A change in the culture medium color from green to blue was observed in all of the tested Z. bailii strains. However, it was observed that 3 of the 8 tested
Z.- bisporus strains converted the ‘medium color only after an additional incubation period of 24 to 48 hours. For all the strains of the other species tested a negative result was observed, since the medium color did not change.
The results that were obtained show that the culture medium according to the present invention is suitable and effective for the detection of Z. bailii and Z. bisporus directly inoculated from cultures in solid medium after a minimum cubation period of 48 hours. 10
AMENDED SHEET
- PCT/PT00/00004
Example 2
The same procedure as Example 1 was used, differing only in that the inoculation was made with single strain cell suspensions instead of cells originated in solid medium. The cells are also originated from agar slants as disclosed in Example 1. The cell suspensions are prepared in deionized water in such a way that the optical density (ODg,,) lies within the range of 0.7 to ee 1.0. 10 pl drops of these suspensions are placed on the surface of Petri dishes containing the medium disclosed in Examplel. The plates were incubated at 30°C for 48 hours.
The results obtained are presented in Table 4. These results are identical to the ones presented for Example 1.
Table 4 Application of cell suspensions on the surface of the solid medium - response of several yeasts in ithe culture medium containing glucose and formic acid {0.4% v/v} after 48 hours of incubation at 30°C.
Species N. of tested strains Culture medium color nt
Zygosaccharomyces bailii 15 blue
Zygosaccharomyces bisporus 5 blue
Zygosaccharomyces bisporus 3 blue*
Zygosaccharomyces nouxii 6 green
Zygosaccharomyces florentinus 1 green
Saccharomyces bayanus 2 green
Saccharomyces cerevisiae 21 green
Saccharomyces pastorianus 2 green
Saccharomycodes ludwigii 3 greep
Schizosaccharomyces pombe 4 green
Pichia membranifaciens “13 green
Pichia enomala 7 green
Dekkera anomala 3 green
Dekkera bruxeliensis 4 green
Debaryomyces hansenii 2 green
Issatchenkia orientalis 6 green
Kluyveromyces marxianus 5 green
Kloeckera apiculata 1 green
Lodderomyces elongisporus 2 green
Rhodotorula mucilaginosa 2 green
Torulaspora delbrueckii 7 green - : * the change in the mediurn color was observed after an additional incubation period of 72-86 hours 11 :
AMENDED SHEET
The culture medium according to the present invention is suitable and effective for the detection of Z. bailii and Z. bisporus from pure culture suspensions after a minimum incubation period of 48 hours.
Example 3
The same procedure as Example 2 was used, but using the culture medium in its liquid form. 25 pl of the cell suspension are transferred to 225 pl of the medium disclosed in Example 1 but without the agar (contained in the wells of a microplate), and in a concentration such that after the 25 pl addition of the cell suspension the final medium components concentration equals the ones disclosed in Example 1. The incubation conditions are similar to those described in Example 2. In addition the culture is homogenized by mechanical mixing at 160 rpm.
The results obtained are presented in Table 5. These results are similar to the ones obtained in the above examples.
i PCT/PT00/00004 - ? Table 5 Inoculation of cell suspensions in liquid medium - response of several yeasts in the culture medium containing glucose and formic acid {0.4% v/v) after 48 hours of incubation at 30°C.
Species N. of tested strains Culture medium color
Zygosaccharomyces bailii 15 blue
Zygosaccharomyces bisporus 5 blue
Zygosaccharomyces bisporus 3 blue”
Zygosaccharomyces nouxii 6 green
Zygosaccharomyces florentinus 1 green . Saccharomyces bayanus 2 green oo Saccharomyces cerevisiae 21 green
Saccharomyces pastorianus 2 green
Saccharomycodes fudwigii 3 green
Schizosaccharomyces pombe 4 green
Pichia membranifaciens 13 green - Pichia anormala 7 green :
Dekkera anomala 3 green
Dekkera bruxellensis 4 green
Debaryomyces hansenii 2 green
Issatchenkia orientalis 6 green
Kluyveromyces marxianus 5 green
Kloeckera apiculata 1 green
Lodderomyces elongisporus 2 : green
Rhodotorula mucifaginosa 2 green
Torulaspora delbrueckii 7 green eS EEE eee * the change in the medium color was observed after an additional incubation period of 48-72 hours
The culture medium according to the present invention, in the liquid form, is equally suitable and effective for the detection of Z. bailii and Z. bisporus from pure culture suspensions after.a minimum incubation period of 48 hours.
Example 4
This Example shows that the culture medium according to -the present : invention is selective for yeasts of the Z. bailii and Z. bisporus species in samples of mixed yeasts populations.
A similar procedure as in Example 3 is used, differing only in that the cell suspensions used are pure or mixed (in equal ratios) yeast cell suspensions, and in that the method of membrane filtration is used. The cell suspension is prepared as in Example 2. The mixed cultures are prepared from pure culture 13
AMENDED SHEET
PCT/PT00/00004 suspensions. In this case, the inoculations are accomplished using an aliquot of the suitably diluted suspension that is filtered under vacuum through a sterilized filtration membrane (pores of 0.45 pm), the filters are then placed on Petri dishes, and the dishes containing the filters on the surface of the medium disclosed in Example 1, are incubated at 30°C for 96 hours. As a reference culture medium (corresponding to a recovery ratio of 100%) a generic yeast culture medium is used (medium containing yeast extract, peptone, and glucose).
The results obtained are presented in Table 6. The recovery ratio of Z. bailii - cells in the medium disclosed in Example 1 is about 60 to 70 %, regardless of the presence of other yeast species. The culture medium was shown to be highly selective since the recovery ratio of S. cerevisssiae, P. membranifaciens and
D. anomala was significantly reduced, lower the 0.01%.
S. cerevisiae, P. membranifaciens and D. anomala being representative examples of contaminant species in wines, the culture medium according to : the invention will be useful and appropriate for the identification of 2. ba/lii in contaminated wines samples.
Table 6 Recovery ratio {%) obtained by the method of membrane filtration after 96 hours of incubation at 30°C. -———,,,,—,—,—_,_—_,—_,ee
Species Z. bailii recovery ratio 0 ee TS
Zygosaccharomyces bailii 65
Zygosaccharomyces bailii 57 "Saccharomyces cerevisiae nd
Zygosaccharomyces bailii 67
Pichia membranifaciens n.d.
Dekkera anomala n.d _— ee, ne
Seccharomyces cerevisiae < 0,002
Pichia membranifaciens 0,011 : _— eer
Dekkera anomala <0,004 _— n.d. not determined } 14
AMENDED SHEET
15-12-2000 PCT/ PT00/00004 DESCPAMD 84810 - PCT
The Figures 3, 4, and 5 show the colony morphology of different species in pure and mixed cultures, being remarkable the easy discrimination between the 3 species using only the colonies color and morphology. ’ 5 ( imeet page 45a >
Example 5
This example shows the differential ability of the culture medium according to the present invention and the enumeration of Z. bailii cells in wine samples.
The enumeration of Z. bailii cells in wine samples is made using membrane filtration {according to the method disclosed in Example 4). For the determination of the number of colony forming units (CFU)/ml of wine is done after 96 hours of incubation at the temperature of 30°C. Other commercial culture_media presently used for the detegtion of yeasts in wines (Wallerstein
Laboratory Differential Medium, WLD, and Wallerstein Laboratory Nutrient
Medium, WLN, both marketed by Difco) are tested in parallel. The WLN medium is used for the detection of fermenting yeasts, while the WLD 15 Able: Coli
Printed:10-04-2001 1
. PCT/PT00/00004 oN : in mixed cultures of S. cersvisiae and Z bailli, a few colonies (ca. 2-3%) with a light blue coloring and with a morphology similar to that of S. cerevisiae can be present, and that were judged as belonging to this species. The light colofing is due to the affinity of these cells for the indicator after the color changing induced by the presence of Z bailii.
In mixed cultures of P. membranifaciens and Z bailii, an intense blue coloring of the
TT colonies formed by P.membranifaciens was observed. This characteristic is equally due to the high affinity of these cells for the indicator after the color changing induced by the presence of Z. baili. However, the discrimination between those colonies is clear as can be seen in the appended Figure 5. 15a
AMENDED SHEET
CY medium allows the detection of lactic and acetic bacteria as well as yeasts belonging to the non-fermenting flora.
The results are summarized in Table 7.
Table 7 Number of CFU/mi in 2 contaminated wines, obtained by the method of membrane filtration after 96 hours of incubation in culture medium containing glucose and formic acid (0.4% v/v) at 30°C © Culture medium Wine 1 Wine 2
Medium disclosed in Example 1 75 90M + 170 @
WLN 685 620
WLD 10 200 ™ cream-yellowish colored colonies 2 plue colored colonies, typical of Z. bailii
The identification of biue colored colonies and white-yellowish colonies as belonging or not to the Z. bailii species was confirmed by molecular methods.
The culture medium described in Example 1 is an ideal culture medium for the ) 15 isolation of yeasts of the Zygosaccharomyces bailii species, allowing the discrimination between this yeast and other yeasts species, just by the color.
The WLN and WLD media do not show this differentiation ability that is a ’ characteristic of the medium of the present invention. This property makes this medium superior to those presently commercially available.
Example 6
This example shows the effect of formic acid concentration in the solid culture medium according to the present invention.
A culture medium was prepared as in Example 1, but using different concentrations of formic acid, and inoculation was done with various yeast strains following the same procedure as described in Example 2, as presented in Table 8.
PCT/PT00/00004
The results obtained are presented in Table 8. For the lower formic acid concentration the basification of the solid culture medium is observed for all the strains belonging to the species Z. bailii and Z. bisporus. The increase in concentration resulted for 3 Z. ba/lii strains in a slower change in the culture medium color. All the strains of the other tested species induced no color change in the culture medium. ~ Table B Application of cell suspension drops on the surface of solid medium - response of several yeasts in the culture medium containing glucose and formic acid at different concentrations after 48 hours of incubation at 30°C. _— : Species N. of tested Culture medium color strains -_ } . formic acid 0.3% (v/v) formic acid 0,5% (v/v) _—_— er Srv lormicacid Uo (Viv
Zygosaccharomyces bailii 12 blue blue . Zygosaccharomyces bailii 3 blue. blue*
Zygosaccharomyces bisporus 8 blue ’ n.d.
Zygosaccharomyces florentinus 1 green green
Saccharomyces bayanus 2 green green
Saccharomyces cerevisiae 21 green green
Saccharomyces pastorianus 2 green green
Pichia membranifaciens 13 green green .
Debaryomyces hansenii 2 green green -_— een green * the change in the medium color was observed atter an additional incubation period of 48-72 hours n.d. not determined
The present culture medium is therefor suitable and effective for the detection of Z. bailii and Z. bisporus from pure culture suspensions, applied as a drop on the surface of the solid medium, for all the tested concentrations of formic acid, after a minimum incubation period of 48 hours. The results obtained show that for 0,3% acid formic concentration, the culture medium according to the invention is appropriate and efficient for the detection of Z. bailii and Z. bisporus in pure culture suspensions, inoculated in liquid culture medium, after a minimum incubation period of 48 hours. The same is valid for the detection of Z. bali in a medium with 0,5% formic acid concentration. Both concentrations are suitable to guarantee a negative response from the other tested species. However, the medium with 0.5% (v/v) of formic acid is not 17
AMENDED SHEET
. PCT/PT00/00004 the best suited one for the detection of Z. bali strains that show lower tolerance to acid conditions.
Example 7
This Example shows the effect of formic acid concentration in a culture medium according to the present invention.
A culture medium was prepared as in Example 3, but using different : concentrations of acid formic and inoculation was done with various yeast strains following the procedure of Example 3, as presented in Table 9.
These results obtained are similar to the ones in Example 6 and are presented in Table 9. In Figure 2 are shown typical responses of yeast strains belonging and not belonging to the Z. bailii species. :
Table 9 Inoculation of cell suspensions in liquid medium - response of several yeasts in the culture medium containing glucose and formic acid at different . concentrations after 48 hours of incubation at 30°C.
Species N. of tested Culture medium color . strains ’ formic acid 0,3% (v/v) formic acid 0,5% (v/v)
Zygosaccharomyces bailii 12 biue blue
Zygosaccharomyces bailir 3 blue blue*
Zygosaccharomyces bisporus 8 blue n.d. } Zygosaccharomyces florentinus 1 green green
Saccharomyces bayanus 2. green green
Saccharomyces cerevisiae 21 green green
Saccharomyces pastorianus 2 green green
Pichia membranifaciens 13 green green
Debaryomyces hansenii 2 green green’ * the change in the medium color was observed after an additional incubation period of 48-72 hours n.d. not determined
As in Example 6 the results obtained show that, for 0,3% acid formic concentration, the culture medium according to the present invention is suitable and effective for the detection of Z. bailii and Z. bisporus from pure culture suspensions, inoculated in liquid media after a minimum incubation 18
AMENDED SHEET
. PCT/PT00/00004 period of 48 hours. The same is valid for the detection of Z. bailii in a medium with 0,5% formic acid concentration.
Example 8
This example shows the effect of formic acid concentration in the culture medium according to the present invention on the medium selectivity.
The procedure of Example 4 was used, but using different concentrations of formic acid in the culture medium.
The results obtained are presented in Table 10. These results and the ones from Example 4 show that the recovery ratio of Z. bailii cells in the medium decreases with the increasing of the acid formic concentration, being independent of the presence of other yeast species such those that can be found in contaminated wines. For 2 of these other 3 tested species the recovery ratio also decreases with the increase in the formic acid concentration. i.
Table 10. Recovery ratio {%) obtained by the method of membrane filtration after 96 hours of incubation at 30°C. : _—m—mmm— —-—
Species . Z. bailii recovery ratio formic acid 0.2% formic acid 0.3% formic acid 0.5%
CC ——— lormic acid D.c% formic acid 0.3% formic acid 0.5% * Zygosaccharomyces bailii 82 78 42 ee
Zygosaccharomyces bailii - 82 81 35
Saccharomyces cerevisiae n.d. n.d. n.d. eT > thier, he | eo
Zygosaccharomyces bailii 89 94 34
Pichia membranifaciens n.d. n.d. n.d.
Dekkera anomala n.d. n.d. n.d.
Lee gee ne oo n4@ 00000 0 nd.
Saccharomyces cerevisiae 30 4 <0.002 -———— a _—
Pichia membranifaciens 55 5.9 <0.004 —_— ee °®2 32 <0.004
Dekkera anomala <0.004 <0.004 <0.004 _-_— er Sees SUE n.d. not deterttiined
Thus, it was shown that the culture medium according to the present invention has characteristics of a selective and differential culture medium appropriated and highly effective for the detection, identification and -- 19
AMENDED SHEET enumeration of Zygosaccharomyces bailii and Zygosaccharomyces bisporus yeasts species in samples either containing previously isolated strains of these yeasts or containing mixed yeasts populations. These characteristics of differentiability and selectivity can be optimized. Lower formic acid concentrations provides the medium with remarkable differentiation ability although with lower selectivity. On the other hand, for higher formic acid concentrations the medium is highly selective.
The culture medium can also be supplemented with an inhibitor of bacterial growth, which makes it useful for using with mixed populations samples including also bacteria, as for example food and beverages.
Although the present invention is described based on its preferred embodiments, it should be apparent to any person skilled in the art that variations and modifications within the spirit and scope of the appended : claims are possible.
Claims (19)
1. A differential and selective culture medium for Zygosaccharomyces bailii and Zygosaccharomyces bisporus yeasts, characterized in that it comprises a base mineral medium supplemented with vitamins, oligoelements, glucose and formic acid as the only carbon and energy sources, an appropriated acid-base indicator having a pKi between 4.5 and 4.8 and, optionally an antibiotic inhibitor of bacterial growth and agar.
2. Culture medium according to claim 1 characterized in that glucose is present in a concentration from 0.05% to 0.1% (p/v), preferably 0.1% (p/v).
3. Culture medium according to claim 1 characterized in that formic acid is present in a concentration, dependent of the desired differentiability and selectivity, from 0.1% to 0.5% (v/v), preferably from 0.2% to 0.4% (v/v).
4. Culture medium according to claim 3 characterized in that the formic acid concentration is preferably 0.4% (v/v).
5. Culture medium according to claim 1 characterized in that the base mineral medium comprises ammonium sulphate (0.5% (WV), potassium dihydrogenphosphate (0.5% (w/v)), magnesium sulphate heptahydrate (0.05% (w/v)) and calcium chloride dihydrate (0.013% (w/v)); the oligoelements solution A (0.05% (v/v)) comprises boric acid (0.1% (w/v)), potassium iodide (0.02% (w/v)) and sodium molibdate dihydrate (0.04% (w/v)); the oligoelements solution B (0.05% (v/v)) comprises copper sulphate pentahydrate (0.008% (w/v)), iron chloride hexahydrate
(0.04% (w/v)), manganese sulphate tetrahydrate (0.08% (w/v)), zinc sulphate heptahydrate (0.08% (w/v)) and hydrochloric acid (HCI 10°N, 0.8% (v/v); and the vitamin solution (0.05% (v/v)) comprises biotin (0.001% (w/v)), calcium panthotenate
(0.08% (w/v)), mioinositol (4.0% (w/v)), niacin (0.16% (w/v)), pyridoxine hydrochloride
(0.16% (w/v)) and thiamin hydrochloride (0.16% (w/v)). AMENDED SHEET
A | PCT/PT00/00004
6. Culture medium according to claim 1 characterized in that the acid-base indicator is one having a pK; between 4.5 and 4.8, preferably bromocresol green.
7. Culture medium according to claim 6 characterized in that the pH is adjusted to
4.3-4.8, preferably 4.5. oo 8. Culture medium according to claim 1 characterized in that it further contains an antibiotic inhibitor of bacterial growth, in the usually used concentrations for this purpose, for use with mixed population samples containing bacteria.
9. A culture medium according to any previously claim characterized in that it contains all the ingredients except agar, that is in its liquid form.
10. A differential and selective culture medium for Zygosaccharomyces bailii and Zygosaccharomyces bisporus yeast, characterized in that it is composed of Glucose 0.1% (w/v) Formic acid 0.4% (v/v) Base Medium: Ammonium sulphate 0.5% (w/v) Potassium dihydrogenphosphate 0.5% (w/v) Magnesium sulphate heptahydrate 0.05% (w/v) Calcium chloride dihydrate 0.013% (w/v) Bromocresol green 0.005% (w/v) Agar 2.0% (w/v) Oligoelements Solution A 0.05% (v/v) Boric acid 0.1% (w/v) Potassium lodide 0.02% (w/v) Sodium molibdate dihydrate 0.04% (w/v) Oligoelements Solution B 0.05% (v/v) Copper sulphate pentahydrate 0.008 (w/v) Iron chloride hexahydrate 0.04 (w/v) AMENDED SHEET
PCT/PT00/00004 Manganese sulphate tetrahydrate 0.08 (w/v) Zinc sulphate heptahydrate 0.08 (wiv) Hydrochloric acid, HCI 10°N, 0.8% (viv) Vitamin Solution 0.05% (v/v) Biotin 0.001% (w/v) Calcium panthotenate 0.08% (w/v) CL Mioinositol 4.0% (w/v) Niacin 0.16% (w/v) Pyridoxine hydrochloride 0.16% (w/v) Thiamin hydrochloride 0.16% (w/v) the pH being adjusted to pH 4.5 with NaOH 10M.
11. Culture medium according to any previously claim characterized in that the medium is prepared by dissolving the base medium compounds in 4/5 of the estimated deionized water volume, the sterilization being accomplished in autoclave at 121°C, for 20 minutes, by dissolving the other medium compounds in the remaining water so that the final concentration of these compounds equals the desired values, the sterilization being accomplished by filtration, bringing this solution and the base medium at about 50+5°C, before mixing the same and adjusting the final pH value to the desired value.
12. Process for the detection of Zygosaccharomyces baili and Zygosaccharomyces bisporus yeasts characterized by the use of a differential and selective culture medium for the referred yeast species, comprising a base mineral medium supplemented with vitamins, oligoelements, glucose and formic acid as the only carbon and energy sources, an appropriated acid-base indicator having a pKi between 4.5 and 4.8 and, optionally an antibiotic inhibitor of bacterial growth and agar.
13. Process according to claim 12, characterized in that the acid-base indicator is bromocresol green and in that, after inoculating the referred culture medium with a AMENDED SHEET
PCT/PT00/00004 sample containing Zygosaccharomyces bailii and/or Zygosaccharomyces bisporus yeasts and incubating in conditions appropriated for the growth of the referred yeasts, it is possible to conclude for the presence of said yeasts species, by means of a medium color change from green to blue after about 48 hours, and if desired to number said yeasts species, by development of blue colored colonies after about 96 hours.
14. Process according to claims 12 and 13 characterized in that it is applied to the detection and numbering yeasts of the Zygosaccharomyces baili and Zygosaccharomyces bisporus species in wines, as well as in other beverages or food containing or not mixed yeast populations.
15. Use of a culture medium according to claims 1 to 11, to be included in yeast identification galleries.
16. Use of a culture medium according to claims 1 to 11 in an industry, particularly in the quality and process control in the food and beverage industry.
17. A medium according to claim 1 or claim 10, substantially as herein described and illustrated.
18. A process according to claim 12, substantially as herein described and illustrated.
19. A new medium or a new process for detecting a microorganism, substantially as herein described. AMENDED SHEET
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PT10230599A PT102305B (en) | 1999-05-31 | 1999-05-31 | MEANS OF CULTURE FOR THE DETECTION OF YEAST ZYGOSACCHAROMYCES BAILII AND ZYGOSACCHAROMYCES BISPORUS |
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|---|---|
| ZA200109748B true ZA200109748B (en) | 2003-02-27 |
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| Application Number | Title | Priority Date | Filing Date |
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| ZA200109748A ZA200109748B (en) | 1999-05-31 | 2001-11-27 | Culture medium for the detection of zygosaccharomyces. |
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|---|---|
| EP (1) | EP1185685A1 (en) |
| JP (1) | JP2003501047A (en) |
| CN (1) | CN1367842A (en) |
| AU (1) | AU5116300A (en) |
| BR (1) | BR0011107A (en) |
| CA (1) | CA2375111A1 (en) |
| NZ (1) | NZ515657A (en) |
| PT (1) | PT102305B (en) |
| WO (1) | WO2000073494A1 (en) |
| ZA (1) | ZA200109748B (en) |
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| JP5850741B2 (en) * | 2011-12-27 | 2016-02-03 | 株式会社明治 | Yeast and mold detector |
| EP2889369B1 (en) | 2012-08-24 | 2020-03-25 | Yamaguchi University | Yeast culture medium |
| HUP1300186A2 (en) | 2013-03-29 | 2014-10-28 | Univ Szegedi | Selective chromogen culture-medium |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5882882A (en) * | 1995-04-12 | 1999-03-16 | Biolog, Inc. | Gel matrix with redox purple for testing and characterizing microorganisms |
-
1999
- 1999-05-31 PT PT10230599A patent/PT102305B/en not_active IP Right Cessation
-
2000
- 2000-05-31 WO PCT/PT2000/000004 patent/WO2000073494A1/en not_active Application Discontinuation
- 2000-05-31 CN CN 00808263 patent/CN1367842A/en active Pending
- 2000-05-31 NZ NZ515657A patent/NZ515657A/en unknown
- 2000-05-31 JP JP2001500804A patent/JP2003501047A/en not_active Abandoned
- 2000-05-31 BR BR0011107-4A patent/BR0011107A/en not_active IP Right Cessation
- 2000-05-31 EP EP00935748A patent/EP1185685A1/en not_active Withdrawn
- 2000-05-31 AU AU51163/00A patent/AU5116300A/en not_active Abandoned
- 2000-05-31 CA CA002375111A patent/CA2375111A1/en not_active Abandoned
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2001
- 2001-11-27 ZA ZA200109748A patent/ZA200109748B/en unknown
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|---|---|
| AU5116300A (en) | 2000-12-18 |
| JP2003501047A (en) | 2003-01-14 |
| CN1367842A (en) | 2002-09-04 |
| WO2000073494B1 (en) | 2001-02-08 |
| PT102305A (en) | 2000-11-30 |
| WO2000073494A1 (en) | 2000-12-07 |
| NZ515657A (en) | 2004-01-30 |
| BR0011107A (en) | 2002-03-05 |
| PT102305B (en) | 2002-01-30 |
| EP1185685A1 (en) | 2002-03-13 |
| CA2375111A1 (en) | 2000-12-07 |
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