WO2000073494A1 - Milieu de culture pour la détection des zygosaccharomyces - Google Patents

Milieu de culture pour la détection des zygosaccharomyces Download PDF

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Publication number
WO2000073494A1
WO2000073494A1 PCT/PT2000/000004 PT0000004W WO0073494A1 WO 2000073494 A1 WO2000073494 A1 WO 2000073494A1 PT 0000004 W PT0000004 W PT 0000004W WO 0073494 A1 WO0073494 A1 WO 0073494A1
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WO
WIPO (PCT)
Prior art keywords
culture medium
zygosaccharomyces
yeasts
green
medium
Prior art date
Application number
PCT/PT2000/000004
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English (en)
Other versions
WO2000073494B1 (fr
Inventor
Cecília LEÃO
Manuela CÔRTE-REAL
Dorit Schuller
Original Assignee
Universidade Do Minho
Stab Vida - Investigacão E Servicos Em Ciências Biológicas, Lda.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Universidade Do Minho, Stab Vida - Investigacão E Servicos Em Ciências Biológicas, Lda. filed Critical Universidade Do Minho
Priority to BR0011107-4A priority Critical patent/BR0011107A/pt
Priority to NZ515657A priority patent/NZ515657A/xx
Priority to EP00935748A priority patent/EP1185685A1/fr
Priority to JP2001500804A priority patent/JP2003501047A/ja
Priority to AU51163/00A priority patent/AU5116300A/en
Priority to CA002375111A priority patent/CA2375111A1/fr
Publication of WO2000073494A1 publication Critical patent/WO2000073494A1/fr
Publication of WO2000073494B1 publication Critical patent/WO2000073494B1/fr

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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 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.
  • 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.
  • 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.
  • the study of the yeast microflora present in the most diverse habitats comprises a first strain isolation stage, using the general selective yeast culture media, and a second identification stage of the 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 large range of physiologic 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.
  • the medium according to the invention is differential for the Zygosaccharomyces bailii and Zygosaccharomyces bisporus yeasts, 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.
  • a new differential and selective culture medium was developed, which permits the identification of Zygosaccharomyces bailii 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 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.
  • 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.
  • 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 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.
  • 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.
  • 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. 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 1 265 and Z. bailii IGC 3806: positive response; T. delbrueckii ISA 1 229 and /. Orientalis 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. bailii strains induced the medium to change color to blue, while all the others maintained the green 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.
  • Figure 5 shows the morphology of P.
  • the Z. bailii colonies are totally distinguishable by its morphology and blue color.
  • the differential and selective culture medium for identification of Zygosaccharomyces bailii and Zygosaccharomyces bisporus yeasts in a sample, after 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.
  • 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.
  • 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.
  • 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 Z. bailii and Z. bisporus yeasts.
  • a culture medium comprising the following ingredients:
  • Table 1 Culture medium composition for the detection of the Zygosaccharomyces bailii and Zygosaccharomyces bisporus yeasts
  • Vitamin Solution Composition according to Table 2 - 0.05 (v/v)
  • 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 HCI 1 M.
  • the sterilization is accomplished by filtration. This solution and the base medium are annealed at 50 ⁇ 5°C before being mixed together. The whole medium is homogenized and dispensed into Petri dishes.
  • 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.
  • the inoculation may be done with a cotton smear containing an equivalent biomass amount.
  • 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.
  • 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.
  • 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.
  • 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 period of 48 hours. The same is valid for the detection of Z. bailii in a medium with 0, 5 % acid formic concentration.
  • 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 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.

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  • 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)

Abstract

L'invention porte sur un milieu de culture différentiel et sélectif pour la détection de levures des types Zygosaccharomyces bailii et Zygosaccharomyces bisporus, réduisant considérablement le temps et le travail usuellement nécessaire à cette détection. L'invention permet de détecter les Zygosaccharomyces bailii et Zygosaccharomyces bisporus en un seul essai ne nécessitant que la préparation et l'inoculation d'un milieu de culture liquide ou solide. Ledit milieu consiste en un milieu minéral de base additionné d'oligo-éléments et de vitamines, de glucose et d'acide formique, seules sources d'énergie et de carbone, et d'un indicateur de pH, du vert de bromocrésol colorant le milieu en vert et virant au bleu sous l'action des susdites levures. La coloration en bleu, spécifique de ces colonies s'observe dans le milieu après 48 à 96 heures d'incubation, selon la méthode d'inoculation utilisée. L'invention peut s'utiliser avec des souches de levures préalablement isolées et purifiées, ou avec des suspensions de populations mixtes de levures comportant d'autres levures que les Zygosaccharomyces bailii et Zygosaccharomyces bisporus, en vue de leur détection dans l'industrie alimentaire, notamment dans les vins et autres boissons. Le milieu peut également être intégré à des galeries d'essais d'identification de levures.
PCT/PT2000/000004 1999-05-31 2000-05-31 Milieu de culture pour la détection des zygosaccharomyces WO2000073494A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
BR0011107-4A BR0011107A (pt) 1999-05-31 2000-05-31 Meio de cultura diferencial e seletivo para as leveduras zygosaccharomyces bailii e zygosaccharomyces bisporus, utilização do mesmo, e, processo de detecção de leveduras zygosaccharomyces bailii e zygosaccharomyces bisporus
NZ515657A NZ515657A (en) 1999-05-31 2000-05-31 Culture medium for the detection of Zygosaccharomyces
EP00935748A EP1185685A1 (fr) 1999-05-31 2000-05-31 Milieu de culture pour la detection des zygosaccharomyces
JP2001500804A JP2003501047A (ja) 1999-05-31 2000-05-31 ザイゴサッカロミセスの検出用培地
AU51163/00A AU5116300A (en) 1999-05-31 2000-05-31 Culture medium for the detection of zygosaccharomyces
CA002375111A CA2375111A1 (fr) 1999-05-31 2000-05-31 Milieu de culture pour la detection des zygosaccharomyces

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PT10230599A PT102305B (pt) 1999-05-31 1999-05-31 Meio de cultura para a deteccao das leveduras zygosaccharomyces bailii e zygosaccharomyces bisporus
PT102305 1999-05-31

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WO2000073494A1 true WO2000073494A1 (fr) 2000-12-07
WO2000073494B1 WO2000073494B1 (fr) 2001-02-08

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PCT/PT2000/000004 WO2000073494A1 (fr) 1999-05-31 2000-05-31 Milieu de culture pour la détection des zygosaccharomyces

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EP (1) EP1185685A1 (fr)
JP (1) JP2003501047A (fr)
CN (1) CN1367842A (fr)
AU (1) AU5116300A (fr)
BR (1) BR0011107A (fr)
CA (1) CA2375111A1 (fr)
NZ (1) NZ515657A (fr)
PT (1) PT102305B (fr)
WO (1) WO2000073494A1 (fr)
ZA (1) ZA200109748B (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014155369A2 (fr) 2013-03-29 2014-10-02 Szegedi Tudományegyetem Milieu chromogène sélectif

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5850741B2 (ja) * 2011-12-27 2016-02-03 株式会社明治 酵母及びカビの検出具
WO2014030774A1 (fr) 2012-08-24 2014-02-27 国立大学法人山口大学 Milieu de culture pour levure

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998026270A2 (fr) * 1996-12-09 1998-06-18 Biolog, Inc. Matrice de gel a violet de redox

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998026270A2 (fr) * 1996-12-09 1998-06-18 Biolog, Inc. Matrice de gel a violet de redox

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Title
ARCH. MICROBIOL., vol. 136, no. 3, 1983, pages 203 - 208 *
ARCH. MICROBIOL., vol. 142, no. 3, 1985, pages 302 - 306 *
BOKIN BOBAI, vol. 15, no. 8, 1987, pages 389 - 396 *
CHEMICAL ABSTRACTS, vol. 100, no. 9, 27 February 1984, Columbus, Ohio, US; abstract no. 66580, W. BABEL ET AL.: "Improvement of growth yield of yeast on glucose to the maximum by using an additional energy source" page 468; column 1; XP002148563 *
CHEMICAL ABSTRACTS, vol. 103, no. 15, 14 October 1985, Columbus, Ohio, US; abstract no. 119605, P. M. BRUINENBERG ET AL.: "Utilization of formate as an additional energy source by glucose-limited chemostat cultures of Candida utilis CBS 621 and Saccharomyces cerevisiae CBS 8066. Evidence for the absence of transhydrogenase activity in yeasts." page 392; column 2; XP002148562 *
CHEMICAL ABSTRACTS, vol. 107, no. 23, 7 December 1987, Columbus, Ohio, US; abstract no. 216107, S. KOMEMUSHI ET AL.: "Development of selective media for yeasts related to winemaking" page 458; column 1; XP002148561 *
CHEMICAL ABSTRACTS, vol. 125, no. 9, 26 August 1996, Columbus, Ohio, US; abstract no. 113126, P. CERRUTTI ET AL.: "Inhibitory effects of vanillin on some food spoilage yeasts in laboratory media and fruit purees" page 1030; column 1; XP002148560 *
CHEMICAL ABSTRACTS, vol. 133, no. 3, 17 July 2000, Columbus, Ohio, US; abstract no. 29819, M.-I. DE SILONIZ ET AL.: "Advances in the development of a methodology to identify common yeast contaminants of high sugar food products" page 449; column 1; XP002148559 *
FOOD TECHNOL. BIOTECHNOL., vol. 37, no. 4, 1999, pages 277 - 280 *
INT. J. FOOD MICROBIOL., vol. 29, no. 2,3, 1996, pages 379 - 386 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014155369A2 (fr) 2013-03-29 2014-10-02 Szegedi Tudományegyetem Milieu chromogène sélectif
US9845487B2 (en) 2013-03-29 2017-12-19 Szegedi Tudományegyetem Selective chromogenic medium

Also Published As

Publication number Publication date
EP1185685A1 (fr) 2002-03-13
CA2375111A1 (fr) 2000-12-07
WO2000073494B1 (fr) 2001-02-08
JP2003501047A (ja) 2003-01-14
PT102305B (pt) 2002-01-30
ZA200109748B (en) 2003-02-27
NZ515657A (en) 2004-01-30
BR0011107A (pt) 2002-03-05
AU5116300A (en) 2000-12-18
CN1367842A (zh) 2002-09-04
PT102305A (pt) 2000-11-30

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