YEAST DIVERSITY IN NEW , STILL FERMENTING WINE " FEDERWEISSER "

The aim of this study was to isolate and identify yeasts in different new wine “federweisser” samples. We collected the samples at the end of the August 2015 and in the middle of the September 2015. Used 15 new wine samples in this study (5 white and 10 red) were from the local Slovak winemakers. Irsai Oliver (3), Moravian Muscat (2), Agria/Turan (1), Dornfelder (3), Blue Frankish (3), Pinot Noir (1) and Saint Laurent (2). Three cultivation media were used for detection of yeasts in “federweisser” samples. Malt extract agar base (MEA), Wort agar (WA) and Wild yeast medium (WYM) were used for the cultivation of yeasts. Cultivation was performed by spread plate method. Ethanol/formic acid extraction procedure was used for preparation of samples. MALDI-TOF Mass Spectrometer (Microflex LT/SH) (Bruker Daltonics, Germany) was used for the identification of yeasts. We identified seven different strains of Saccharomyces cerevisiae (23; 70%), two strains of Kloeckera apiculata [teleomorph Hanseniaspora uvarum] (7; 21%), and one strain of Pichia kluyveri (1; 3%), Pichia occidentalis [anamorph Candida sorbosa] (1; 3%) and Metschnikowia pulcherrima (1; 3%) in 15 new wine “federweisser” samples. Saccharomyces cerevisiae was dominant species in each new wine sample, and formed creamy convex colonies with circular edge. Metschnikowia pulcherrima formed convex to pulvinate, circular white-pink colored colonies, Kloeckera apiculata formed flat, circular smooth colonies with turquoise center with gray edge, Pichia occidentalis formed irregular pulvinate light-cream colored colonies, and Pichia kluyveri formed turquoise, convex, undulate and smooth colonies on Malt extract agar base with bromocresol green.


INTRODUCTION
Federweisser wine is grape must which is just undergoing the process of fermentation.Grape must is the juice of the wine grapes which is gained after the pressing of grapes.After corresponding treatment and storing, the must would become wine after finishing the process of fermentation.Because of this, Federweisser is not specially produced as some kind of drink but as an early product of wine production.The fermentation causes the splitting of the fructose of the grapes in alcohol and carbon dioxide.Because of the yeasts and bacteria in the must, the fermentation goes on very quickly.That is why Federweisser is drinkable only a couple of days.But the cool storage can lengthen the process of fermentation.In the refrigerator, Federweisser can be kept about 10 days.The grape must is considered "Federweisser" wine as soon as the alcohol concentration is about 4 to 6%.At the beginning, it tastes quite sweet.During the process of fermentation, the sweetness subsides.Due to the concentration of carbon dioxide, Federweisser tastes very prickly and tangy.Because of the high carbon dioxide concentration, a corking or air tight closure of the Federweisser is not possible.Especially in the past, this caused a transportation problem.Federweisser could only be offered regionally and was limited.Grape must is inoculated with a pure culture of yeasts (S. cerevisiae), usually 10-20 g.100 L -1 of must.Federweisser is very good for cold or warm drinking (Malík et al., 2012).
Yeasts are found throughout nature.However, they do The growth of these species, known collectively as non-Saccharomyces yeasts (or wild yeasts), is limited to the first 2 or 3 days of fermentation, after which they die as a result of ethanol toxicity.As these yeasts disappear, highly fermentative strains of the species Saccharomyces cerevisiae and Saccharomyces bayanus begin to multiply until they become solely responsible for alcoholic fermentation.The yeasts present in the must during the first few hours after filling the tanks belong to the same genera as those found on the grapes, predominantly Kloeckera (Hanseniaspora).In these spontaneous vinification conditions, Saccharomyces yeasts begin to develop after around 20h and are present alongside the grape-derived yeast flora.After 3 rd or 4 th day of fermentation, Saccharomyces yeasts predominate and are ultimately responsible for alcoholic fermentation (Ribéreau- Gayon et al., 2006).This change in the yeast population is linked to the increasing presence of ethanol, the anaerobic conditions, and the use of sulfites during harvesting and in the must, the concentration of sugar, and the greater tolerance of high temperatures shown by S. cerevisiae compared with other yeasts (Fleet and Heard, 1993;Fleet, 2007)

. S. cerevisiae comprises numerous strains with varying biotechnological properties (Ribéreau-Gayon et al., 2006).
The aim of this study was to isolate and identify yeasts in different Slovak new wine "federweisser" samples.

Identification of yeasts
We used MALDI-TOF Mass Spectrometer (Bruker Daltonics, Germany) for identification of yeasts isolated from federweisser samples.After incubation of yeasts at 25 °C for 5 days, 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 13 000 × 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 Real-time Classification software (RTC) by used database "Taxonomy" (Bruker Daltonics, Germany).

RESULTS AND DISCUSSION
After cultivation time, we obtained results from number of CFU (colony forming unit) in 100 µL of new wine sample of each used decimal dilutions.For better interpretation of results logarithmic conversion was applied on numerical results.Natural logarithm (Log e ) was used in Microsoft ® Office Excel program by function LN.The highest number of yeasts cultivated on malt extract agar (MEA) was found in sample number thirteen Pinot Noir 6.43 log CFU.100 µL -1 and the lowest number of yeasts cultivated on MEA was present in the third sample Moravian Muscat 4.62 log CFU.100 µL -1 .The highest number of yeasts cultivated on Wort agar (WA) was found also in sample number 13 Pinot Noir 6.39 log CFU.100 µL -1 , but the lowest number of yeasts cultivated on WA was present in the fourth sample Irsai Oliver 5.38 log CFU.100 µL -1 .The highest number of yeasts cultivated on Wild yeast medium (WYM) was found in sample number seven Blue Frankish 6.33 log CFU.100 µL -1 and the lowest number of yeasts cultivated on WYM was present in the fifteenth sample Dornfelder 4.20 log CFU.100 µL -1 .Table 1 contains results from microbiology of new wines obtained by spread plate method with used specific decimal dilutions 10 -4 and 10 -5 .Yeasts were countable at these two used dilutions.In this study we identified seven different strains of Saccharomyces cerevisiae (23), two strains of  Kloeckera apiculata (7), and one strain of Pichia kluyveri (1), Pichia occidentalis (1) and Metschnikowia pulcherrima (1) in fifteen federweisser samples.Pichia kluyveri was identified in Blue Frankish sample number five and Pichia occidentalis (anamorph Candida sorbosa) in sample number six (Irsai Oliver).We also identified one strain of Metschnikowia pulcherrima in sample number five (Blue Frankish).
The most common species in new wine samples was Saccharomyces cerevisiae and we identified seven different strains namely: DSM 1334, DSM 3798, DSM 70868, DTY3, CBS 1171, WS LLH and strain 991400574.Second most common species in new wine samples was Kloeckera apiculata (Hanseniaspora uvarum).K. apiculata was found in 7 new wine samples, two different strains (DSM 2768 and DSM 70788).Seven different strains of Saccharomyces cerevisiae was found in 15 new wine samples, what can be seen in Table 2.
S. cerevisiae is the most important yeast for wine production and is responsible for the metabolism of grape sugar to alcohol and CO 2 .For these reasons S. cerevisiae is often simply referred to as "the wine yeast" (Fleet, 1993; Pretorius et al., 1999;Swiegers and Pretorius, 2005).From all of identified yeasts, Saccharomyces cerevisiae was the dominant species, and we identified this species in all 15 new wine samples (70%).Grapes contain different species of yeast belongs to non-Saccharomyces yeasts such as Kloeckera (dominant genera), Metschnikowia, Candida, Pichia, Rhodorotula, Aureobasidium etc. Saccharomyces yeasts are not present in grape surface, or present in very low levels (less than 50 CFU.When alcoholic fermentation starts non-Saccharomyces yeast population decrease.After the start of alcoholic fermentation when the ethanol concentration reaches 5 to 6% these yeast will be die (Fugelsang et al., 2007).As fermentation progresses, the levels of these yeasts decrease, while that of Saccharomyces increases (Fleet and Heard 1993).By the end of fermentation, Saccharomyces is the majority of the yeasts found, and often the only yeast isolated.New, still fermenting wine contains 4 to 6% ethanol and mostly contains only Saccharomyces cerevisiae, which is always predominant in new wines.But yeasts as Kloeckera, Metschnikowia, Candida, Pichia etc. can be identified in new wine samples in low populations.Some winemakers use commercial pure cultures and the others prefer to encourage the growth of some non-Saccharomyces yeasts early in alcoholic fermentation but eventually inoculate with Saccharomyces (Fugelsang et al., 2007).We identified except S. cerevisiae also Kloeckera apiculata in 7 new wine samples (21%) in lower population.Very interesting was that we isolated and identified only 3 another species of yeasts: Metschnikowia pulcherrima (3%), Pichia kluyveri (3%) and Pichia occidentalis (3%).In study Kántor et al., (2015) bromocresol green was used as a supplement also in Malt extract agar base (MEA) from BioMark ™ (India).But in that study, cultivation media was not supplemented with yeast extract and glucose, only bromocresol green was added.After sterilization by autoclaving, that medium had a dark blue color.However in this study we supplemented Malt extract agar base (BioMark ™ , India) with yeast extract and glucose, and after sterilization by autoclaving had medium olive-green color.Malt extract agar base (BioMark ™ , India) contains only malt extract (30 g.L -1 ), mycological peptone (5 g.L -1 ) and agar (15 g.L -1 ).Supplementation was desired in this case with glucose, yeast extract and bromocresol green.Yeasts grow very well in this modified medium, and bromocresol green is very helpful in differentiating of yeasts.Figure 1 shows the different colony morphology of 4 yeast species grown on MEA in sample number 5 (Blue Frankish).As you can see, the number of Saccharomyces cerevisiae was the highest, then Kloeckera apiculata and after that Pichia kluyveri and Figure 1 Yeast species isolated from new wine "Federweisser" (Sample no. 5, dilution 10 -4 ).
only one colony on this petri dish belonged to Metschnikowia pulcherrima.M. pulcherrima produced maroon colored pigment called pulcherrimin, which was visible from the bottom of the petri dish.Metschnikowia pulcherrima formed convex to pulvinate, circular whitepink colonies.Kloeckera apiculata formed flat, circular smooth colonies with turquoise center with gray edge.Pichia kluyveri formed turquoise, convex, undulate and smooth colonies and Pichia occidentalis formed irregular pulvinate light-cream colonies.

CONCLUSION
In this study we isolated and identified yeast species in 15 Slovak new wine "Federweisser" samples.We identified the yeast isolates by MALDI-TOF mass spectrometry biotyper (Bruker Daltonics, Germany).The most dominant species was Saccharomyces cerevisiae which was isolated from all 15 new wine samples, which was a very good result.By mass spectrometry we identified 7 different strains of S. cerevisiae.The second most common species was Kloeckera apiculata (Hanseniaspora uvarum) found in 7 new wine samples (2 strains).We also identified other non-Saccharomyces yeasts such as Metschnikowia pulcherrima (1 strain), Pichia occidentalis (1 strain) and Pichia kluyveri (1 strain).

Benito et al., 2011; Ciani et al., 2006; Comitini et al., 2011; Cordero Otero et al., 2003; Di Maio et al., 2012;
(Polsinelli et al., 1996)re found in specific habitats where different species form communities (Lachance and Starmer, 1998).Within the winemaking environment (habitat), the vineyard (grape surfaces) and cellar (equipment surfaces and must) can be considered specialized niches where the wine related yeasts can form communities(Polsinelli et al., 1996).The yeast species found in different niches associated with grape growth (vineyards) and wine production (wineries, grape must, fermentation and wine) can be arbitrarily divided into two groups, i.e. the Saccharomyces group and the non-Saccharomyces group.The Saccharomyces group with its primary representative, Saccharomyces cerevisiae, is present on grape skins in low numbers (Rankine, 1972; Török et al., 1996, König et al., 2009), and application in co-inoculation or sequential inoculation with S. cerevisiae is increasingly popular (Ciani et al., 2006; Ciani and Maccarelli, 1998; Comitini et al., 2011; Jolly et al., 2006; Soden et al., 2000), particularly for their effects on wine composition, flavour and aroma (

(Fleet, 2007; Fugelsang and Edwards, 2007). Yeasts
, which play a central role in the winemaking process, are unicellular fungi that reproduce by budding (

Table 2
Yeast species in new wine "federweisser" samples.