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

Definitions

• the present invention refers to a culture medium for the differential detection and enumeration of food and beverage contaminant yeasts of the Dekkera and Brettanomyces genera, containing ethanol and -cumaric acid in its preparation. It is also an object of the present invention a method using said culture medium for the differential detection and enumeration of yeasts of the Dekkera and Brettanomyces genera. It is still an object of the present invention the use of said culture medium in a yeast identification gallery.
• the objective of the present invention consists in providing the food and beverage industry with a culture medium and a method using said culture medium, able to isolate, differentially detect and number yeasts of the Dekkera and Brettanomyces genera, by means of the color changing of the culture medium and of the colonies therein developed, and production of a characteristic phenol-like aroma.
• the study and characterization of the yeast microflora present in many different habitats generally involves a first strain isolation and purifying stage in generic yeast culture media, which is followed by a second identification stage by classical taxonomic methods or by molecular biology based techniques.
• the slow development of the yeasts of the Dekkera and Brettanomyces genera makes its isolation in the commonly used media extremely difficult, since these yeasts are overtaken by faster developing ones that coexist with them.
• a culture medium was developed, which is partially selective and totally differential for yeasts of the Dekkera and Brettanomyces genera, which is based on 4-ethylphenol detection, by its characteristic aroma, and acetic acid, by means of an adequate acid-base color change, produced by those yeasts when developed in a ethanol and p-cumaric acid containing medium.
• the present invention refers to a culture medium for the differential detection and enumeration of food and beverage contaminant yeasts of the Dekkera and Brettanomyces genera.
• Said medium comprises a nutrient base, ethanol as a non-fermentable energy source and inhibiting other yeasts species, p-cumaric acid as an aromatic compound promoting substract produced by said yeasts species, an acid-base indicator whose turning points lie within the acid range (particularly bromocresol green), an antibiotic that inhibits several species of yeasts (particularly cycloheximide), and agar-agar when the medium is intended for use in a solid form.
• the nutrient base is "Yeast Nitrogen Base” and the acid-base indicator is bromocresol green.
• the culture medium according to the invention further contains a bacterial growth inhibitor, particularly chloramphenicol and/or oxytetracycline, which is specially useful for the detection of yeasts of the
• the medium object of the invention when in the liquid form, is prepared by sterilizing filtration, then being dispensed into adequate test tubes.
• agar-agar is dissolved in demineralized water and then sterilized in an autoclave; following the sterilization and cooling to about 50°C, the other components are added under aseptic conditions, having previously been filtration sterilized.
• the pH adjustment for both media is done before the sterilization, to a pH value that depends on the acid-base indicator used.
• the mixture is homogenized and poured into Petri dishes, the medium being ready for use after solidifying.
• the present invention also refers to a method for the detection and enumeration of yeasts of the Dekkera and Brettanomyces genera, using a partially selective and totally differential culture medium, characterized as above.
• the method according to the invention allows the detection and enumeration of yeasts of the Dekkera and Brettanomyces genera in a simple way using the culture medium according to the present invention.
• the culture medium is used by direct contact with a sample under analysis, adequate dilutions being used if the enumeration of the contaminant yeasts is intended .
• the thus inoculated medium is then incubated at an adequate temperature for the growth of the yeasts, during a time period enough, usually 5 to 1 0 days, for the development of clearly visible colonies (in case of the solid medium) or for the clouding of the solution (in case of the liquid medium) .
• the detection of the yeasts of the Dekkera and Brettanomyces genera in the Petri dishes is accomplished by direct observation of cream colored colonies, by the change of medium color according to the type of acid-base indicator that was used, and by the presence of a characteristic phenol-like aroma. If the incubation time is extended, the colonies acquire a darker coloring .
• the detection in the liquid medium is accomplished by the change of the medium color and by the presence of the phenol-like aroma.
• one of the objectives of the present invention consists in providing the food industry with a procedure that allows the isolation, differential detection and enumeration of yeasts of the Dekkera and Brettanomyces genera by the production of characteristic colonies, medium color change and production of a phenol-like aroma, thus avoiding the inconveniences of said yeasts identification after their isolation, object being accomplished using the culture medium and method according to the present invention, as described above.
• the medium according to the invention is useful for inclusion in yeast identification galleries.
• the present invention refers to a culture medium for the differential detection and enumeration of yeasts of the Dekkera and Brettanomyces genera which comprises "Yeast Nitrogen Base” as a nutrient base, ethanol as a non-fermentable energy source, p-cumaric acid as an aromatic compound (4-ethylphenol) promoting substrate, bromocresol green as an acid-base indicator with turning points in the acid range, an antibiotic that inhibits several species of yeasts (cycloheximide), and a bacterial growth inhibitor antibiotic.
• yeast Nitrogen Base as a nutrient base
• ethanol as a non-fermentable energy source
• p-cumaric acid as an aromatic compound (4-ethylphenol) promoting substrate
• bromocresol green as an acid-base indicator with turning points in the acid range
• an antibiotic that inhibits several species of yeasts cycloheximide
• a bacterial growth inhibitor antibiotic a culture medium for the differential detection and enumeration of yeasts of the
• Example 1 Preparation of a culture medium according to the invention
• the culture medium, object of the present invention can be prepared using the following formulation (g/L) : Yeast Nitrogen Base (6.7), as the nutrient base; ethanol (48), as the non-fermentable energy source and as an inhibitor for some of the yeasts; p-cumaric acid (0.1 ), as the phenol-like producing aroma substract; bromocresol green (0.222), previously dissolved in NaOH, as the acid-base indicator; cycloheximide (0.01 ) , as the inhibitor antibiotic for some of the yeast species; chloramphenicol (0.1 ) and/or oxytetracycline (0.1 ), as the bacteria inhibitor antibiotic; and agar-agar (20), as the gelling agent.
• formulation g/L
• the culture medium is sterilized according to the following: the agar-agar is dissolved in 70% of the total needed water, the pH is adjusted to 5.4 with a strong acid, and the resulting solution is sterilized in an autoclave at 1 20°C, for 20 minutes. The other components are dissolved in the remaining of the demineralized water, the pH is adjusted to 5.4 with a strong acid, and the resulting solution is sterilized by filtration through a 0.22 ⁇ m pore diameter membrane. Both of the above solutions are then mixed together when the agar-agar solution reaches 50°C. The medium is then homogenized and dispensed into Petri dishes, allowing it to solidify prior to the inoculation.
• Example 2 two wines suspected of having been altered were analyzed using the culture medium of Example 1 .
• 20 ml samples of each wine were filtered under aseptic conditions, through 0,22 ⁇ m pore diameter cellulose acetate membranes.
• Each membrane was placed on the surface of a Petri dish containing the medium of the invention and incubated at 25°C. After 3 days it was possible to observe colonies in one of the dishes, along with the change of the medium color from blue to yellow; when the dish was opened, the presence of a phenol-like aroma was not detected. In the other dish no colonies were detect after 3 days. After 9 days the dish where the colonies had been observed maintained the same characteristics.
• the culture medium object of the invention for the enumeration of yeasts in wines where the presence of filamentous fungi is suspected
• the Most Probable Number enumeration technique is used, using test tubes with the liquid culture medium. Consequently, agar-agar is not used in the medium formulation, the medium being completely sterilized by filtration.
• the presence of yeasts of the Dekkera or Brettanomyces genera is detected when there is a clouding of the medium, a turning of its color from blue to yellow, and the presence of a phenol-like aroma; therefore, all the test tubes that show these characteristics are considered positive, and all the others negative.

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• General Health & Medical Sciences (AREA)
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• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Apparatus Associated With Microorganisms And Enzymes (AREA)

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• 1999
  • 1999-05-31 PT PT102306A patent/PT102306B/pt not_active IP Right Cessation
• 2000
  • 2000-05-31 JP JP2001500805A patent/JP2003501048A/ja not_active Abandoned
  • 2000-05-31 DE DE60022035T patent/DE60022035D1/de not_active Expired - Lifetime
  • 2000-05-31 BR BR0011117-1A patent/BR0011117A/pt not_active IP Right Cessation
  • 2000-05-31 NZ NZ515656A patent/NZ515656A/xx unknown
  • 2000-05-31 AT AT00935749T patent/ATE302284T1/de not_active IP Right Cessation
  • 2000-05-31 CA CA002377144A patent/CA2377144A1/en not_active Abandoned
  • 2000-05-31 AU AU51164/00A patent/AU777883B2/en not_active Ceased
  • 2000-05-31 EP EP00935749A patent/EP1185686B1/en not_active Expired - Lifetime
  • 2000-05-31 WO PCT/PT2000/000005 patent/WO2000073495A1/en not_active Ceased
  • 2000-05-31 CN CN00810102.7A patent/CN1367843A/zh active Pending
• 2001
  • 2001-11-27 ZA ZA200109750A patent/ZA200109750B/xx unknown

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