Comparative Analysis of Fatty Acid Composition in Some Saccharomyces cerevisiae Strains

Aims: Comparative study of fatty acid composition of biomass in alcohol and wine S. cerevisiae strains, yeast. Methodology: Yeast strains were identified biochemically and by ITS rDNA gene sequencing. Capillary electrophoresis and gas-liquid chromatography were used for analyzing composition and content of carboxylic acids in the yeast biomass, Champagne wine and ethanol produced by yeast. Results: Comparative analysis of fatty acid composition in biochemically active yeasts S. с erevisiae Y-503, S. cerevisiae Y-3980 and S. cerevisiae Litto – Levure С HA showed that the distinctive feature of alcohol Y-503 strain from wine strains was the lack of 16 fatty acids, the predominance of eicosapolyene acids (up to 58.1% in biomass and 56.6% in ethanol) and almost half the content of saturated fatty acids. All strains were found to be predominated with palmitic (16:0) and linoleic (18:2 ω -6) acids that is typical for yeast Saccharomyces . This pattern was observed in Champagne wine and ethanol. Conclusion: The results of comparative analysis of fatty acid composition of new and traditionally used commercial yeast strains may be useful in the selection of strains in order to manufacture high quality products in winemaking and alcohol industry.


INTRODUCTION
Today microbial lipid production has received applied interest [1][2][3][4]. Due to high growth rate and biomass yield, fast lipid accumulation, yeasts exhibit advantages over bacteria and another microorganisms [5,6] for subsequent lipid isolation and processing in biofuel production [1]. All types of microorganisms produce lipids but they differ from each another by the capacity to produce and accumulate lipids. Many oleaginous microorganisms are capable of producing lipids even at a level greater than 20% of their dry cell weight [7] Moreover a systematic lipid biosynthesis engineering approach centered on diacylglycerol acyltransferase gene overexpression in Yarrowia lipolytica for increased lipid yield has been developed [8]. S. cerevesiae as nonoleaginous microorganism do not accumulate lipids in high quantity, but being traditional organism exploitable in food industry it is also of current interest [9]. The ability of yeast to produce or accumulate fatty acids is dependent on nutrient medium composition, carbon source, pH and many other factors (cultivation time, temperature, etc.) [10]. Numerous studies have reported fatty acid composition of yeasts [1,11,12], the interrelation between the lipid composition and the ability of S. cerevisiae strains to adapt to cultivation conditions [13], the possibility of utilizing various yeast species as potential producers of dietetically important major fatty acids [11]. Fatty acids along with carbonyl and sulphur compounds are known to play a role in the sensory quality of wine [14,15]. A number of commercial S. cerevisiae strains are used around the world in winemaking and alcohol industry. It is of applied interest to investigate new native and selected yeast strains that are responsible for the production of wines with peculiar flavours [9]. The aim of this study was evaluation of fatty acid composition of biomass in newly selected in our laboratory alcohol and wine S. cerevisiae strains versus traditionally used commercial wine strain, Champagne wine and ethanol, produced by yeast. New S. cerevisiae strains have successfully passed laboratory and industrial testing and were recommended for industrial use.

Molecular and Genetic Studies
The Y-503 strain was shown to belong to the S. cerevisiae taxon by UP-PCR method in the group of S. Bulat (Laboratory of Eucaryote Genetics, Department of Molecular and Radiation Biophysics, Petersburg Nuclear Physics Institute, Russian Academy of Sciences). The Y-503 strain is heterozygous tetraploid. Species identification of Y-3980 strain was carried out by analyzing the nucleotide sequences of ITSI-5.8S-ITS2 region of rDNA. The amplified DNA fragment was sequenced in the scientific and manufacturing company «Syntol» (Moscow). Species identification was carried out by comparing the nucleotide sequences obtained with the data of genetic bank of NCBI (www.ncbi.nlm.nih.gov) and CBS (www.cbs.knaw.nl).

Cultivation
To cultivate S. cerevisiae Y-503, culture media with the following composition were used (g / l): In the development media molasses was diluted with geothermal water of different composition and in the control medium molasses was diluted with water till hydrocarbon content amounted to 20.0 g/100 cm 3 (in each variant). To optimize S. cerevisiae cultivation conditions the selection of media composition was made. The yeast was cultivated in 3000 ml flasks (working volume 1500 ml) for 120 h at 30ºС, рН 4.5 in the laboratory using the depth method in a batch mode under anaerobic conditions. Cell cultures grown in nutrient media that were identical to control or development media were used as inoculum. To obtain inoculum (with hydrocarbon content 20 g/100 cm 3 ) the cells were sequentially adapted to the media containing hydrocarbon: 7.9 -10.8 -12.4 -20.0 g/100 cm 3 . The inoculum was cultivated for 5 days at temperature 30±1ºС in a batch mode under anaerobic conditions. The content of inoculum was 10% of culture medium volume. 80% struktol -oily substance -(0.1 ml/1.5 l of medium) was used as defoamer. After 120 hours of cultivation and separation of the yeast by centrifugation (5000 g, 15 min) in a stationary laboratory centrifuge CLS -344.2, the nutrient medium was named «fermented substrate».
The yeasts S. cerevisiae Y-3980 and S. cerevisiae Litto -Levure СHA were cultivated in the medium of the following wine materials (%): Pinot -40, Riesling -20, Aligote -20, Chardonnay -20. Fermentation mixture contained 2.5% of carbohydrate, 11.2% of alcohol, 0.3 g/dm 3 of phenolic substances, 17.8 g/dm 3 of reductones as well as 9.1 g/dm 3 of titratable and 0.17 g/dm 3 of volatile acidity. The inoculum (cultivated for 48 hours in deep culture of the grape must) was 3% of the medium volume. Champagnization of wine materials was carried out in a batch mode in the acratophore (Champagne -fermenting tank) at temperature 9 -10ºС, pressure 0.4 -0.5 MPa, рН 3.3-3.5 for 20 days. The concentration of ethanol in alcohol stripper obtained by distilling the fermented substrate was determined using standard procedure.

Analysis of Low-molecular Organic Compounds
The content study of free low-molecular organic acids in biomass, aqueous-alcoholic solutions and Champagne wine was carried out by the capillary electrophoresis device "Capel-105" (Russia). The method of direct interesterification with methanol solution of sodium methoxide was used to obtain methyl esters of fatty acids from triglycerides.

Fractionation of Methyl Esters of Fatty Acids
Fractionation of methyl esters of fatty acids (biomass, aqueous-alcoholic solutions, Champagne wine) and determination of impurities in ethanol were carried out with the gas chromatography using TRACE GC 2000 gas chromatograph (CE Instruments, Italy) with a flame ionization detector (PID) on a capillary gas column HP-FFAP (SGE Analytical Science, Australia) (50 m x 0.32 mm x 0.52 mm) with 10% diethylene glycol-succinate (Chromlab, Russia). The column temperature was 185ºC; the evaporator temperature -230ºС. Nitrogen was used as the carrier gas (1.8-2.7 dm 3 /h). Acids were identified according to the period of retention of individual fractions using standard solutions of certain fatty acids. The content of methyl esters of fatty acids was obtained by the methods of statistical analysis and presented as mean values with standard deviation.

The Study of Yeast Biomass
It is well known that volatile fatty acids being components of various aromatic compounds produced by S. cerevisiae can affect the oenological characteristics of sparkling wine [16]. The data in the Table 1  It is shown that the content of palmitic (16:0) and linoleic (18:2ω-6) acids is predominated in the biomass of all strains that is typical for the yeast Saccharomyces. The study of the biomass of wine strains revealed the increased content of saturated acids (1.64 and 1.78 times) as compared to the alcohol S. сerevisiae Y-503 strain (see Table 1). However, the saturated capric (10:0), and lauric (12:0) fatty acids (that are contained in all species of yeasts and important for the quality of Champagne wine) in wine yeast were higher 1.55/2.61 and 1.57/2.34 times, respectively, on average than in alcohol Y-503 strain.
The total content of unsaturated fatty acids in the biomass of S. cerevisiae Y-3980 and S. cerevisiae Litto Levure CHA, on the contrary, was 2.3 times less than in S. сerevisiae Y-503. All studied strains are able to synthesize eicosapolyene acids, and their total content in the biomass of S. сerevisiae Y-503 reaches 58.10% and in wine strains -26.27% on average of the total acid amount. Accordingly, the maximum values of linoleic (18:2 ω-6), arachidonic (20:4ω-6), and dihomo-γ-linolenic (20:3ω-6) acids (well known as precursors of the extensive group of physiologically and pharmacologically active compounds) were observed in the alcohol strain. Some fatty acids in the biomass of all studied strains were found in a small amount, less than 1% (see Table 1).

The Study of Champagne Wine and Ethanol
Analyzing the data of the fatty acid pool of Champagne wine and ethanol we identified specific characteristics in the composition of the fatty acids depending on the strains used. Many researchers demonstrated the value of the fatty acids in the biosynthesis of aroma-producing complex in sparkling wine and the formation of sensory properties including fruit and herbal aroma, sunflower tones, foamy and antioxidant properties [9,16,17].

CONCLUSION
According to our studies, the amount of fatty acids in Champagne wine and ethanol corresponds to the standard physiological and biochemical indicators of product quality [17,20]. Comparative analysis of fatty acid composition of biochemically active S. сerevisiae Y-503, S. cerevisiae Y-3980 and S. cerevisiae Litto Levure CHA strains showed that the distinctive characteristic of alcohol Y-503 strain was the absence of 16 fatty acids compared to wine strains. Biomass of Y-503 strain was characterized by the predominance of eicosapolyene acids and almost half the content of saturated fatty acids compared to wine strains. Accordingly, this pattern was detected in Champagne wine and ethanol. Comparing the new S. cerevisiae Y-3980 wine strain with commercial S. cerevisiae Litto Levure CHA have led to the conclusion that the new strain may be recommended to wine industry. Future more extensive studies are required in particular to select new alcohol and wine strains with proper characteristics. The results of the study may be useful for selection of natural strains in order to obtain high quality products of alcohol industry and winemaking with peculiar characteristics, highlighting regional specificities.