Isolation and Identification of Spoilage Yeasts in Wine Samples by ...

Kantor A. et al./Scientific Papers: Animal Science and Biotechnologies, 2015, 48 (1)

Isolation and Identification of Spoilage Yeasts in Wine Samples by MALDI-TOF MS Biotyper Attila Kántor*, Miroslava Kačániová Slovak University of Agriculture in Nitra, 949 76, Tr. A. Hlinku 2, Nitra

Abstract Many genera and species of microorganisms can be found in grape musts and wines at various times during the winemaking process. For instance, Saccharomyces, Brettanomyces, and Pediococcus can be found together in wine. There are many species of yeast involved in wine spoilage during storage. Aim of this study was to isolate the spoilage yeasts from wine samples with using special selective agar media and identified on species level by MatrixAssisted Laser Desorption/Ionization-Time of Fly Mass Spectrometry (MALDI-TOF MS).Six red wines used in this study. We identified 10 yeast species from 152 isolates. The most common species in wine samples was Saccharomyces cerevisiae. We also identified four Candida species, two Zygosaccharomyces species and one species from genus Rhodotorula, Saccharomycodes and Dekkera. Keywords: wine; yeasts; MALDI-TOF MS Biotyper.

fermented alcoholic beverages, the concept of spoilage yeast has a more complex meaning than in non-fermented foods, where any yeast able to change food sensorial characteristics can be regarded as a ‘‘spoilage yeast.’’ In fermented drinks or foods, yeast activity is essential during the fermenting process. Detrimental and beneficial activity must therefore be distinguished. In the wine industry where alcoholic fermentation occurs in the presence of many yeast species and bacteria (mainly lactic and acetic acid), it isvery difficult to draw a line between beneficial fermenting activity and spoilage activity. For this reason, spoilage yeasts are rarely sought during wine fermentation, but during storage or aging and during the bottling process.Microbial spoilage of wines may also be due to the activity of lactic and acetic bacteria. In fact, most traditional wine ‘‘diseases’’ are bacterial in origin [2, 4, 7].The distribution of yeast species in cellar-aging wine includes Dekkera(teleomorphBrettanomyces), film yeasts for example Candida valida, Candida krusei,Candida stellata,Candida vini, etc.), Saccharomycodes, and Zygosaccharomyces, all of

1. Introduction The most important microorganisms in wine production are yeasts. They influence fermentation speed, wine flavour and other wine qualities[1-3]. Grapes are a primary source of natural yeasts in wine production.The grape yeast flora can be either beneficial or detrimental to the quality of wine products.It has been found that freshly crushed grape juice harbors a diversity of yeast species, principally within the genera Kloeckera(teleomorphHanseniaspora), Pichia, Candida, Metschnikowia,and Kluyveromyces. Occasionally, species in other genera such as Cryptococcus, Rhodotorula, Debaryomyces, Issatchenkia, Zygosaccharomyces, Torulaspora, Dekkera, Schizosaccharomyces,Saccharomycodesand Saccharomyces have also been isolated from wine grapes of several wine producing areas [1, 3-6].In * Corresponding author: Mgr. +421376415812, [email protected]

Attila

Kántor,

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which can result in serious wine spoilage [8].Spoilage species usually grow slower than other microorganisms in general purpose culture media and, therefore, they tend to be underestimated in samples heavily contaminated [9].Fermentative yeasts include those responsible for wine fermentation, where Saccharomyces cerevisiaeis the most important, but other species (S. bayanus, S. pastorianus and S. paradoxus) may also conduct or participate in the process [10, 11]. These species may also be seen as wine spoilers if their activity persists beyond the fermentative steps of wine or sparkling wine production. The exception is that of Saccharomycesspp. forming desirable films on the surface of particular oxidative ageing processes of Sherry-like wines [12]. Aim of this study was to isolate variousyeast species from six unfiltered red wines and identify them by MALDI-TOF MS Biotyper.

with 100 µL of each wine sample on the surface of solidified medium and evenly spread with sterile glass spreader. Petri dishes were incubated at 25°C for 5 days. Isolated yeasts after incubation were identified by colony morphology (colour, surface, edge, elevation) and inoculated separately on the surface of fresh cultivation media. After that we identified the yeast species by MALDIFOF MS Biotyper. MALDI-TOF MS Biotyper: We used MALDITOF MS biotyper (Bruker Daltonics, Germany) for identification of yeasts isolated from wine samples. After incubation, isolated colonies were picked and suspended in 300 µL of sterile distilled water and mixed thoroughly. 900 µL of absolute ethanol was added. The mixture was centrifuged at 12 000 × g for 2 min. After the supernatant was discarded, the pellet was centrifuged again. Residual ethanol was completely removed by pipetting and the pellet was allowed to dry at room temperature. Subsequently 10 µL of formic acid (70%) was added and mixed with the pellet with a sterile toothpick. Next, 10 µL of acetonitrile (100%) was added and mixed thoroughly. The solution was centrifuged at maximum speed for 2 minutes again, and 1 µL of the supernatant was spotted on a polished MALDI target plate (Bruker Daltonics, Germany). Immediately after drying 1 µL of the matrix solution was added to each spot and allowed to air dry. The matrix used was a saturated solution of α-cyano-4-hydroxycinnamic acid (HCCA) (Bruker Daltonics, Germany) dissolved in 50% acetonitrile with 0.025% trifluoroacetic acid (TFA). The matrix solution preparation (2.5 mg of HCCA) contains 500 µL of acetonitrile, 475 µL of ultra-pure water and 25 µL of trifluoroacetic acid. Next added 250 µL of this solution to the 2.5 mg of HCCA. Samples were then processed in the MALDI-TOF MS (Microflex LT/SH, Bruker Daltonics, Germany) with flex Control software and results obtained with Realtime Classification software (RTC) (Bruker Daltonics, Germany).

2 Materials and Methods Wine samples: Six red wine samples were used in this study. Alibernet 2012, Cabernet Sauvignon 2012, Merlot 2013, Rimava 2013, Pinot Noir 2013 and Blue Frankish 2014 red wines were used for the isolation and identification of yeasts. We collected 200 mL of each unfiltered wines (before microfiltration) and immediately after that the samples were storage at 6-8°C in refrigerator for next analysis. Samples were incubated in laboratory at room temperature (25 ± 2°C) for one week. After that we prepared wine samples for microbiological analysis. Cultivation media: We used four different cultivation media for detection of various yeast species in wines,namely: Wort agar (WA) (HiMedia, India); Yeast extract Peptone Dextrose agar (YPD) (Conda, Spain), Malt Extract agar (MEA) (Biomark, India) and Sabouraud Dextrose agar (SDA) (Conda, Spain). All media were supplemented with chloramphenicol (100mg/L) to inhibit bacterial grow. Chloramphenicol was added into cultivation media before sterilization by autoclaving at 115-121°C for 15 minutes. Also acid base indicator bromocresol green (BG)(20 mg/L) (pH range: 3.8-5.4) was added into the MEA andWA cultivation media before sterilization. Cultivation media were inoculated

3. Results and discussion We isolated total 152 isolates of yeasts from six wine samples. The most common identified yeast species was Saccharomyces cerevisiae (83 isolates), Rhodotorula glutinis (27 isolates) and

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Candida valida (18 isolates). Saccharomyces cerevisiae isolated from all wine samples, Rhodotorula glutinis from four and Candida valida from three wine samples. We identified total 10 yeast species, but these three were the most common in wine samples. We also identified less than 10 isolates of Candida sorbosa (9 isolates), Candida krusei (4 isolates), Candida zemplinina (1 isolate), Dekkera bruxellensis (1 isolate), Saccharomycodes ludwigii (4 isolates), Zygosaccharomyces bailii (2 isolates) and Zygosaccharomyces florentinus (3 isolates).Table 1 shows the results from the identified species of

yeasts. Candida sorbosa [Pichia occidentalis] is most often associated with fruit and natural fermentations of food products, as seen from strain isolation histories, above. Hierro et al. [13]reported C. sorbosato be relatively common in wine fermentations. In Loureiro and MalfeitoFerreira [2] review shows that some yeast species are not dangerous for wine production and storage (Rhodotorulasp.), but for example Dekkera is a dangerous species which not necessarily frequent contaminant in wines. They are slowly growing species.

Table 1. Identified yeast species and number of isolates Yeast species Anamorph [Teleomorph]

No. of isolates

Cultivation media

Candida sorbosa [Pichia occidentalis]

9

WA, MEA

Candida krusei [Issatchenkia orientalis]

4

WA, MEA

Candida valida [Pichia membranifaciens]

18

WA, MEA, SDA

Candida zemplinina

1

WA

Dekkera bruxellensis [Brettanomyces bruxellensis]

1

YPD

Rhodotorula glutinis

27

YPD, MEA, SDA

Saccharomyces cerevisiae

83

WA, MEA, SDA, YPD

Saccharomycodes ludwigii

4

WA

Zygosaccharomyces bailii

2

WA, SDA

Zygosaccharomyces florentinus

3

WA, MEA

Total

152 WA: Wort agar, MEA: Malt Extract agar,

YPD: Yeast extract peptone dextrose agar, SDA: Sabouraud dextrose agar.

Saccharomycodes ludwigii is referred to as the “winemaker’s nightmare” because the yeast is highly resistant to SO2, approximately five times that of Saccharomyces [14]. Ciani and Maccarelli[15]hypothesized that the extraordinary resistance of this yeast could be due to its ability to produce high concentrations of acetaldehyde, which binds SO2. As such, the best known control method is sterile filtration. Fortunately, the yeast has rarely been reported in either cellar aged or bottled wines possibly due to slow growth or poor competition with other yeasts [1].Saccharomycodes spoils wines by formation of cloudiness and sediment as well as off-odors [2, 16]. Another potential post alcoholic fermentation problem is Zygosaccharomyces. This yeast causes

spoilage by forming gas, sediment, or cloudiness in bottled wines [2]. We identified two species of Zygosaccharomyces species. They form small, creamy, greenish-blue, round shaped, raised colonies on WA with bromocresol green. Zygosaccharomyces is osmophilic, resistant to ethanol, SO2, sorbate, andother commonly used preservatives. In study Kraková et al. [17] was isolated Candida zemplinina yeast from Pinot Noir red wine. Candida zemplinina is osmotolerant and psychrotolerant yeast able to grow at high sugar concentrations and at low temperatures [18]. We also identified Candida zemplinina, but in Merlot red wine. One yeast species was isolated from all wines, Saccharomyces cerevisiae. Five different species

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of yeasts were isolated and identified from Cabernet Sauvignon and Pinot Noir wine. From Blue Frankish wine isolated four species and three species from Alibernet and Merlot wines. The best wine sample in this study was Rimava sample,

because from this wine we isolated and identified just Saccharomyces cerevisiae. Table 2 shows the detected or not detected species of identified yeasts from different wines by MALDI-TOF MS biotyper (Bruker Daltonics, Germany).

Table 2. Detected species in red wines Wine sample Yeast species Candida sorbosa

Alibernet

Blue Frankish

C. Sauvignon

Merlot Pinot Noir

Rimava

ND

ND

D

ND

D

ND

Candida krusei

D

ND

ND

ND

ND

ND

Candida valida

D

D

D

ND

ND

ND

Candida zemplinina

ND

ND

ND

D

ND

ND

Dekkera bruxellensis

ND

ND

ND

ND

D

ND

Rhodotorula glutinis

ND

D

D

D

D

ND

Saccharomyces cerevisiae

D

D

D

D

D

D

Saccharomycodes ludwigii

ND

D

ND

ND

ND

ND

Zygosaccharomyces bailii

ND

ND

ND

ND

D

ND

Zygosaccharomyces florentinus

ND

ND

D

ND

ND

ND

D: detected, ND: not detected

Figure 1 shows a pie chart with a percentage representation of different identifiedspecies of yeasts in this study.Saccharomyces, Kloeckera and Metschnikowia are common winery contaminants; their activity is especially dangerous when associated with damaged berries, which encourage their growth, leading to high initial populations at the beginning of fermentation. Surprisingly, the yeast species regarded as the most dangerous to wines, i.e., Dekkera/Brettanomycesspp., Z. bailii, and S. ludwigii, are seldom detected in yeast studies performed in wineries [19, 20].We not identified Kloeckera or Metschnikowia species from wines. In agreement, Heard and Fleet [21] and Clemente-Jimenez et al. [22] reported that inoculation of various species of Kloeckera, Candida, Issatchenkia, Metschnikowia, and Pichia(non-Saccharomyces yeasts)in grape juice

yielded maximum ethanol concentrations of less than 6%.This factor probably contributes to the frequently observed die-off of these yeasts shortly after the start of alcoholic fermentation when the ethanol concentration reaches 5-6% v/v.As populations of non-Saccharomyces yeasts decline, Saccharomyces will dominate and complete alcoholic fermentation [8]. In study Renouf et al [23] reported that common microorganisms present in wine barrels and tanks are Saccharomyces cerevisiae, Oenococcus oeni, but also wood-specific yeast species Cryptococcus. S. cerevisiae was detected only on bottles of recent vintages. Zygosaccharomycesis able to referment sweet wines and detected in bottles of older vintages. Finally dangerous Dekkera was predominant yeast in every bottle, recent or old [23].

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Kantor A. et al./Scientific Scientific Papers: Animal Science and Biotechnologies, 2015, 48 (1)

Figure 1.Percentage Percentage representation of identified yeast species from wine samples

4. Conclusions

References

Ten spoilage yeast species were identified by MALDI-TOF TOF MS biotyper from six unfiltered red wines. Total we determined 152 yeast isolates. The most common spoilage yeast was Saccharomyces cerevisiae, which was isolated from all six wine samples. We also identified dangerous spoilage yeast species Dekkera bruxellensis in Pinot Noir wine, Saccharomycodes ludwigii in Blue Frankish and two Zygosaccharomyces species. Candida film yeasts also identified mostly Candida valida, valida Candida sorbosa and Candida krusei.. We also identified one isolateof Candida zemplinina from Merlot wine. Rhodotorula glutinis also identifiedin ide four wine samples, and this yeast wascommon winery spoilage species.

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Acknowledgements This work has been supported by grant of VEGA 1/0611/14 andby by European Community under project no 26220220180: Building Research Centre „AgroBioTech”.

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