Journal of Agricultural Chemistry and Environment, 2014, 3, 48-52
Published Online April 2014 in SciRes. http://www.scirp.org/journal/jacen
How to cite this paper: Csutorás, Cs., et al. (2014) Technological Experiments for the Enhancement of Glycerol Content in
High Quality Wines. Journal of Agricultural Chemistry and Environment, 3, 48-52.
Technological Experiments for the
Enhancement of Glycerol Content in High
Cs. Csutorás1*, O. Hudák1, K. Rácz2, L. Rácz1
1Institute of Food Science, Egerfood Regional Knowledge Center, Eszterhazy Karoly University, Eger, Hungary
2Eger Crown Winehouse Ltd., Kerecsend, Hungary
Received December 2013
The glycerol content of grape juices and wines has been determined by gas-chromatographic me-
thods using silyl derivatives. The effect of different storage conditions and yeast cultures on gly-
cerol content has been investigated. Grape juice samples with different starting D-Glucose con-
centrations have been treated with several additives (HPO42−, S2O52−, NADH and HSO3−). Significant
glycerol concentration enhancement has been detected in the case of the addition of NADH (5.14
g/L), however the presence of HSO3− prevented the excessive glycerol formation (1.21 g/L).
Glycerol; Wine Quality; Wine-Making Technology; Gas Chromatography
Glycerol (propane-1,2,3-triol) belongs to a family of non-volatile polyalcohols and represents an important
component of natural saponifiable lipids (e.g. esters formed with fatty acids). Pure glycerol is an odorless, co-
lorless, high viscosity liquid. Several glycerol derivatives (e.g. dihydroxyacetone) play important role in essen-
tial biochemical metabolic processes, one of them is the glycolysis, where the six-carbon atom containing
D-glucose is cleaved to dihydroxyacetone and glycerol aldehyde and finally to pyruvate with three carbon atoms.
Since glycerol molecule is a closely related to these important decomposition products, it has important biologi-
cal significance and appears as side product in several biological processes (alcoholic fermentation, aging and
ripening of wines). Glycerol is involved in several microbiological or enzymatic transformation routes, the na-
turally bound glycerol is w idesp read in natural bio logical sou rc es. Glycero l is widely u sed in th e indu str y for the
production of dye, paper, textile, fuel, cosmetics, medicine and food [1-5]. The chemical analysis of glycerol is
important in clinical diagnostics as well, since the triacyl glyceride level in blood gives vital information con-
cerning diseases of the cardiovascular system.
The biosynthesis of glycerol in cells is closely associated with osmotic cell regulation . The anaerobic
*Corresponding a uthor.
Cs. Csutorás et al.
conversion of glucose into ethanol by Saccharomyces cerevisiae is redox neutral. NAD+ is consumed initially in
the Embden-Meyerhof-Parnas (EMP) pathway that is regenerated during the production of ethanol. However,
when intermediates in the EMP pathway are consumed as precursors for the synthesis of the cellular material,
this balance will be disturbed, since the produced NADH is not converted back into NAD+. The shift in redox
balance stops the metabolism of yeast cells, unless other processes are employed for the regeneration of NAD+.
During the anaerobic growth of S. cerevisiae NADH cannot be oxidized by oxygen, but the conversion of
NADH to NAD+ can be achieved in the cell by the formation of reduced by-products such as glycerol. The pro-
duction of glycerol in yeast cells is caused by the need to maintain a favorable redox balance [6-11] by the con-
version of NADH that is formed during the biomass production, to NAD+. Glycerol formation requires the re-
duction of dihydroxyacetone phosphate to glycerol-3-phosphate (G-3-P) catalyzed by dihydroxyacetone phos-
phate reductase. In the process one molecule NAD+ will be produced from NADH that was formed previously in
the oxidation of glyceraldehyde-3-phosphate. The reaction is catalyzed by G-3-P dehydrogenase (GPDH) fol-
lowed by the dephosphorylation of G-3-P to glycerol by glycerol-3-phosphatase . Glycerol formation may
serve in the cells as a route for the transformation of NADH to NAD+.
Summarizing the biochemical process, glycerol is formed in wines as a by-product of glycolysis of wine
yeasts like S. cerevisiae during wine fermentation. The glycerol concentration in grape juice is low but during
fermentation 4% - 10% of the sugar content is converted into glycerol . Glycerol concentration in wines va-
ries between 1 and 10 g/L [13,14], however red wines generally contain higher levels of glycerol than white
wines. Glycerol does not have direct impact on wine flavor however it enhances the quality by improving phys-
ical properties like viscosity and smoothness. Nowadays there is a high demand on the production of wines with
higher glycerol levels, since there is a close connection between quality and glycerol concentration . The
viscous liquid glycerol has sweet taste thus high concentrations of glycerol favorably influence the organoleptic
evaluation and the viscosity of wine . Several experiments have been carried out for the investigation of
glycerol production during the fermentation of sugar-containing liquids. Enhanced glycerol production can be
achieved by the application of several inorganic salts  or by adjusting the pH to 7 or above resulting in the
reduction of dihydroxyacetone phosphate to glycerol-3-phosphate by NADH .
In EU countries direct addition of glycerol to enhance the quality of wine is prohibited. In our work we aimed
at the search for chemical compounds that influence the redox equilibrium of the fermentation process to en-
hance the glycerol content in wine. We focused in our experiments on the application of additives that are au-
thorized in EU countries.
2. Materials and Methods
All chemicals and reagents (glycerol, MSTFA, pyridine, NADH) were purchased from Sigma Aldrich, St. Louis,
USA. Yeast strains were purchased from a local winery trader (Interker-Wine Ltd). The derivatization process
was performed under water free conditions, 2 mL of wine or grape juice samples were freeze d ried for 12 hours
and the residue was dissolved in 2 mL of pyridine. 100 µL of pyridine solution and 100 µL of N-Methyl-
N-(trimethylsilyl) trifluoroacetamide (MSTFA) were added and stirred for 15 minutes at room temperature to
yield the trimethylsilyl derivative. The reaction was terminated and the excess derivatizing agent was decom-
posed by the addition of 5 mL water. 2 mL heptane was used to extract the derivatized glycerol, the heptane so-
lution was used in gas-chromatographic analysis. GC analysis was performed on a Shimadzu GC 2010 instru-
ment using a HP-5 MS column (30m, 0.25 µm x 0.25 µm). 1 µL of heptane solution was injected, helium gas as
carrier (1 mL/min) was applied.
The injector temperature was 200˚C and the initial temperature of 110˚C was held for 4 minutes, then it was
increased to 150˚C with a speed of 4˚C /min, the temperature was further increased to 250˚C with 20˚C/min rate
and it was held for 11 minutes at this temperature. The detector temperature was 250˚C and the total measuring
time was 30 minutes. The retention time of glycerol under these conditions was 8.07 minutes.
3. Results and Discussion
The effect of the addition of different additives as potassium metabisulfite, diammonium phosphate, ammonium
bisulfite and NADH to grape juices with different sugar concentrations has been examined. Experiments were
carried out at different temperatures, glycerol levels of grape juices and wines were measured by GC methods.
Glycerol is a highly polar compound thus gas chromatographic analysis in free form is not possible due to its
Cs. Csutorás et al.
high boiling point. The sample preparation has been involved silyl derivative formation in order to lower the
boiling point of gl y c e r ol .
The glycerol formation has been studied using several different grape juice and wine samples (Ta b l e 1 and
Table 2). Analysis of samples was performed multiple times during the maturation of wines (5, 10 and 20 days)
and studied the effect of additives at different fermentation temperatures (8˚C - 30˚C). In our experiments vari-
ous yeast strains have been used (Fermol Chardonnay-I, Mycoferm CRU-88-II, Uvaferm Sacharomyces Cerevi-
siae-III, Sacharomyces Bayanus-IV). The initial glycerol content of grape juice was found to be 1.13 g/L.
As maturation progressed, significant concentration increase has been detected for all wine samples and for
all different yeast strains. After 20 days the highest glycerol content (4.12 g/L) was obtained in a sample that
was stored at cellar temperature using Mycoferm CRU-88-II strain.
The results of the experiments with different yeasts are shown in Table 1. Th er e is no significant difference in
glycerol concentrations in the function of the applied yeast strains, the best results were obtained in all cases
with Mycoferm CRU-88 yeast strains. Temperature however seems to be an important factor in glycerol forma-
tion during fermentation. Significant differences were observed in glycerol concentrations during and at the end
of wine fermentation. Higher temperatures are not favorable in the point of view of glycerol formation thus they
should be avoided aiming at the production of high quality wines. By the way at higher temperatures there
would be a failure also in the appropriate aroma composition of wines. The best results in glycerol content of the
produced wine were obtained at lower temperatures, however extremely low temperatures are not favorable,
since fermentation slows down or even stops. The best results were achieved at cellar temperature (13˚C - 14˚C)
where the highest glycerol concentrations were measured.
The effect on glycerol production of different additives used in enology—(NH4)2HPO4, K2S2O5, NADH,
NH4HSO3)—has also been studied. The experiments were carr ied out at different D-Glucose levels (10, 20 and
40 g/L) by applying Mycoferm CRU-88 yeast culture (Table 2). The sugar content of grape juices is crucial in
the production of good quality wines.
The applied high quality white grape juice contained sugar at a concentration of 210 g/L. The addition of ex-
tra amount of glucose to grape juice definitely increased the amount of glycerol in wine during fermentation
which is an experimental proof of the fact that the sugar content of grape juices determines the quality of the
produce d wi ne.
Table 1. Effect of yeasts and temperature on the glycerol content of wines during fermentation.
Wine samp les* Glycerol content (g/L)
5 days 10 days 20 days
R I 2.62 2.83 3.12
R II 2.76 2.92 3.28
R III 2.57 2.72 3.15
R IV 2.53 2.74 3.21
C I 3.12 3.29 4.08
C II 3.18 3.32 4.12
C III 3.15 3.21 4.06
C IV 3.13 3.24 4.05
30˚C I 2.43 3.28 3.43
30˚C II 2.65 3.32 3.52
30˚C III 2.37 3.38 3.48
30˚C IV 2.41 3.35 3.43
8˚C C I 2.13 2.58 3.68
8˚C II 2.18 2.53 3.82
8˚C III 2.11 2.45 3.71
8˚C IV 2.15 2.38 3.75
*R = room temper ature, C = cellar temperature (13˚C - 14˚C), I = Fermol Chardonn ay, II = Mycoferm CRU-88, III = Uvaferm Sach aromyces Ce-
revisiae, IV = Sacharomyces Bayanus.
Cs. Csutorás et al.
Table 2. Effect of glucose and different grape juice additives on the glycerol content of wines during fermentation.
Applied additives Sample Glycerol content (g/L)
8 days 60 days
Grape juice + 10 g/L D-glucose 1.82 3.59
Grape juice + 20 g/L D-Glucose 2.18 3.65
Grape juice + 40 g/L D-Glucose 1.56 3.75
200 mg/L (NH4)2HPO4
Grape juice 1.96 4.04
Grape juice + 10 g/L D-glucose 2.08 4.17
Grape juice + 20 g/L D-Glucose 2.24 4.90
Grape juice + 40 g/L D-Glucose 1.93 4.35
200 mg/L K2S2O5
Grape juice 0.61 3.56
Grape juice + 10 g/L D-glucose 0.44 3.00
Grape juice + 20 g/L D-Glucose 0.21 3.87
Grape juice + 40 g/L D-Glucose 0.16 3.72
500 mg/L NADH
Grape juice 2.23 4.39
Grape juice + 10 g/L D-glucose 2.25 4.63
Grape juice + 20 g/L D-Glucose 2.41 4.56
Grape juice + 40 g/L D-Glucose 2.02 5.84
0.4 mL/L 41% NH4HSO3
Grape juice 0.19 1.52
Grape juice + 10 g/L D-glucose 0.17 1.09
Grape juice + 20 g/L D-Glucose 0.17 1.26
Grape juice + 40 g/L D-Glucose 0.16 1.21
In Table 2, the results of our experiments on the investigation of the effect of different additives on the con-
centration of glycer ol in the produc ed wine are also summarized. Potassium metabisulfite and ammonium bisul-
fite disturb the fermentation process that results in the formation of a lower amount of glycerol compared with
the control experiment. In the case of ammonium bisulfite the hindrance of yeast strains was more significant.
The addition of NADH to grape juice resulted in significant increase of glycerol production of the yeast. The
addition of NADH caused a shift in redox balance th at is an outer stress for yeast cells. Yeast cells answer to the
stress by pr oducing glycerol to de c re a s e the concentrati on of NADH.
Similar effect has been caused by the addition of diammonium phosphate resulting in the formation of higher
levels of glycerol. The presence of ammonium ions is a suitable medium for amino acid production of the yeast
cells. Amino acid biosynthesis results in the formation of NADH that induces yeast cells to produce glycerol.
Glycerol production helps the yeast cells to make the appropriate redox balance of NADH and NAD+.
The fermentation and glycerol content of grape juices and wines has been studied under different conditions
and yeast strains. Gas-chromatographic methods have been applied in the quantitative evaluation of the glycerol
content of wines. The highest glycerol levels have been detected at decreased temperature (8˚C - 14˚C). The
D-Glucose concentration affects the glycerol production during the fermentation. Significant decrease has been
detected, when the grape juice was treated with HSO32− but the addition of NADH or diammonium phosphate
can almost double the glycerol content of wines.
The authors thank the National Development Agency for financial support (TÁMOP-4.2.2.A-11/1/KONV-
2012-0008, TÁMO P-4.2.3-12 /1/KONV-2012-0025, GOP-1.1. 1.-11-2011-0015).
 Novo, L.P., Gurgel, L.V.A., Marabezi, K. and Curvelo, A.A.S. (2011) Delignification of Sugarcane Bagasse Using
Glycerol-Water Mixtures to Produce Pulps for Saccharification. Bioresource Technology, 102, 10040-10046.
Cs. Csutorás et al.
 Melero, J.A., Vicente, G., Morales, G., Paniagua, M., Morales, G. and Muñoz, P. (2012) Etherification of Biodiesel-
Derived Glycerol with Ethanol for Fuel Formulation over Sulfonic Modified Catalysts. Bioresource Technology, 103,
 Gómez-Hens, A. and Fernández-Romero, J.M. (2006) Analytical Methods for the Control of Liposomal Delivery Sys-
tems. Trends in Analytical Chemistry, 25, 167-178. http://dx.doi.org/10.1016/j.trac.2005.07.006
 Salehpour, S. and Dube, M.A. (2012) Towards the Sustainable Production of Higher-Molecular-Weight Polyglycerol.
Macromolecular Chemistry and Physics, 212, 1284-1293. http://dx.doi.org/10.1002/macp.201100064
 Calderon, M., Quadir, M.A., Sharma, S.K. and Haag, R. (2010) Dendritic Polyglycerols for Biomedical Applications.
Advanced Materials, 22, 190-218. http://dx.doi.org/10.1002/adma.200902144
 Zoecklein, W.B., Fugelsang, K.C., Gump, H.B. and Nury, S.F. (1995) Wine Analysis and Production. Chapman &Hall,
New York, 99-100.
 Oura, E. (1977) Reaction Products of Yeast Fermentations. Process Biochemistry, 12, 19-21.
 Gancedo, C. and Serrano, R. (1989) Energy-Yielding Metabolism. In Rose, A.H. and Harrison, J.S., Eds., The Yeasts,
2nd Edition, Vol. 3, Academic Press, London, 205-259.
 Nordstrom, K. (1968) Yeast Growth and Glycerol Formation II. Carbon and Redox Balances. Journal of the Institute of
Brewing, 74, 429-432. http://dx.doi.org/10.1002/j.2050-0416.1968.tb03154.x
 van Dijken, J.P. and Scheffers, W.A. (1986) Redox Balances in the Metabolism of Sugars by Yeast. FEMS Microbiol-
ogy Reviews, 32, 199-224. http://dx.doi.org/10.1111/j.1574-6968.1986.tb01194.x
 Boulton, R.B., Singleton, V.L., Bisson, L.F. and Kunkee, E.R. (1996) Principles and Practices of Winemaking. Chap-
man & Hall, New York, 146-150. http://dx.doi.org/10.1007/978-1-4615-1781-8
 Radler, F. and Schütz, H. (1982) Glycerol Production of Various Strains of Sacharomyces. American Journal of Enol-
ogy and Viticulture, 33, 36-40.
 Rankine, B.C. and Bridson, D.A. (1971) Glycerol in Australian Wines and Factors Influencing Its Formation. Ameri-
can Journal of Enology and Viticulture, 22, 6-12.
 Ough, C.S., Fong, D. and Amerine, M.A. (1972) Glycerol in Wine: Determination and Some Factors Affecting. Ame r-
ican Journal of Enology and Viticulture, 23, 1-5.
 Balli, D., Flari, V., Sakellaraki, E., Schoina, V., Iconomopoulou, M., Bekatorou, A. and Kanellaki, M. (2003) Effect of
Yeast Cell Immobilization and Temperature on Glycerol Content in Alcoholic Fermentation with Respect to Wine
Making. Process Biochemistry, 39, 499-506. http://dx.doi.org/10.1016/S0032-9592(03)00133-X
 Noble, A.C. and Bursick, G.F. (1984) The Contribution of Glycerol to Perceived Viscosity and Sweetness in White
Wine. American Journal of Enology and Viticulture, 35, 110-113.
 Rapin, J.D., Marison, I.W., Von Stockar, U. and Reilly, P.J. (1994) Glycerol Production by Yeast Fermentation of
Whey Pe r meate. Enzyme Microbial Technology, 16, 143-150. http://dx.doi.org/10.1016/0141-0229(94)90077-9
 Wang, Z.X., Zhuge, J., Fang, H. and Prior, B.A. (2011) Glycerol Production by Microbial Fermentation: A Review.
Biotechnology Advances, 19, 201-223. http://dx.doi.org/10.1016/S0734-9750(01)00060-X