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 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 pl , between 4.5 and 4.8, particularly bromocresol green, and optionally agar and an antibiotic inhibitor of bacterial growth, such as cloramphenicol.
• 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.
• 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 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.
• 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 3 shows the morphology of Zygosaccharomyces bailii 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.
• 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.
• Zygosaccharomyces bailii 1 5 blue Zygosaccharomyces bisporus 5 blue Zygosaccharomyces rouxii 3 blue * 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 membranaefacie ⁇ s 1 3 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
• 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 (OD 640 ) lies within the range of 0.7 to 1 .0. 1 0 ⁇ l 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 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 2 The same procedure as Example 2 was used, but using the culture medium in its liquid form. 25 ⁇ l of the cell suspension are transferred to 225 ⁇ l 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 ⁇ l 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.
• the culture is homogenized by mechanical mixing at 1 60 rpm.
• 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.
• Example 3 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 suspensions.
• 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 ⁇ m), the filters are then placed 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.
• a reference culture medium corresponding to a recovery ratio of 1 00%) a generic yeast culture medium is used (yeast extract medium, 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. cerevisiae, P. membranaefaciens and D. anomala was significantly reduced, lower the 0.01 %.
• the culture medium according to the invention will be useful and appropriate for the identification of Z. bailii in contaminated wines samples.
• non-typical colonies ca. 2-3%) with a light blue coloring or with an intense blue coloring can be present.
• the first of these (Fig. 4), with a morphology similar to that of S. cerevisiae, were judged as belonging to this species. The light coloring of these colonies is due to the incorporation of the indicator after the color change induced by the presence of Z. bailii.
• the second kind of colonies (Fig. 5), with a similar morphology to that of P. membranaefaciens were judged as belonging to this species, the intense coloring being due to the high affinity of these cells for the indicator after the color changing induced by the presence of Z. bailii. This characteristic was equally observed for the pure cultures of P.
• membranaefaciens that showed a very intense green coloring in contrast with those of S. cerevisiae, that under these conditions, showed a green-cream coloring.
• the discrimination between those colonies is clear as can be seen in the appended Figures 4 and 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).
• CFU colony forming units
• Other commercial culture media presently used for the detection 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 medium allows the detection of lactic and acetic bacteria as well as yeasts belonging to the non-fermenting flora.
• the culture medium described in Example 1 is an ideal culture medium for the 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.
• 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. 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. bailii 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.
• 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. bailii in a medium with 0,5% formic acid concentration. Both concentrations are suitable to guarantee a negative response from the other tested species.
• the medium with 0.5% (v/v) of formic acid is not the best suited one for the detection of Z. bailii 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.
• 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.
• 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 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.
• 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.

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