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Improving alcoholic fermentation by activation of Saccharomyces species during the rehydration stage
LWT - Food Science and Technology 50 (2013) 126e131
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Improving alcoholic fermentation by activation of Saccharomyces species during the rehydration stage P. Díaz- Hellín, J. Úbeda*, A. Briones Tecnología de Alimentos, IRICA, Universidad de Castilla La Mancha, Avda. Camilo José Cela 10, Edifico Marie Curie, 13071 Ciudad Real, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 October 2011 Received in revised form 10 June 2012 Accepted 12 June 2012
The rehydration of selected active dry yeasts is essential for improving control of the wine fermentation process. To help yeast cells withstand the extreme conditions to which they are subjected during rehydration, winemakers often add adjuvants directly to the must. However, few commercial activators are added directly to the yeast rehydration water, even though this might render hydration more efficient due to a favourable osmotic gradient. The present study examined the effect of nine “activators” added directly to the water to rehydrate four commercial wine yeast strains by measuring the vitality, viability and metabolic activity of the process, plus residual sugars, glycerol and ethanol levels at days 2, 8 and 15. The fermentation kinetics were monitored and data were adjusted to a modified Gompertz equation. The activators, some of which had hitherto rarely been tested in combination with Saccharomyces, were selected for their potential activity and ability to enhance processes and/or improve cell structures. Semi-quantitative results revealed that the four wine yeasts responded very differently to the activators, indicating a high degree of strain specificity. There appeared to be no “universal activator”, indicating that a “tailor-made” activator will likely need to be formulated for each commercial yeast strain. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Active dry yeasts Activators Vitality Viability Fermentation kinetics Saccharomyces sp.
1. Introduction Twenty years ago, wine fermentation was a spontaneous process triggered by the native yeast occurring naturally in vineyards and wineries. Today, very few winemakers use traditional fermentation methods; instead, most wineries rely on the inoculation of commercial active dry yeasts. Although industrially produced Saccharomyces biomass is mostly used in fresh or extruded form for the manufacture of bread and beer, requiring an unbroken cold chain, a small proportion of commercial yeast is dried for use in wine fermentation. Since, unlike bread yeasts, wine yeasts are required on a seasonal basis rather than throughout the year, manufacturers find that drying enables major savings in transport and storage (Rodríguez-Porrata et al., 2011). The manufacture of active dry yeasts involves fed-batch biomass production and subsequent dehydration. Both are highly stressful processes, prompting the yeast cell to accumulate protective molecules such as trehalose and glycogen, which play a key role in cell growth (Parrou et al., 1999). In the course of dehydration, the cell also loses essential vital material, including nucleotides, ions * Corresponding author. Tel.: þ34 926295300x3424; fax: þ34 926295318. E-mail addresses: [email protected] (P.D.- Hellín), [email protected] (J. Úbeda), [email protected] (A. Briones). 0023-6438/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.lwt.2012.06.011
and other soluble cell components (Rapoport, Khrustaleva, Chamanis, & Beker, 1995; Suarez, 2004), some of which are biosynthesized de novo during rehydration, allowing most cells to resume growth. Dry cells are rehydrated in water between 34 C and 40 C for around 30 min. Rehydration prompts interactions between cytosol structural macromolecules and the plasma membrane, giving rise to biochemical reactions that are fundamental for cell division (Beker, Blumbergs, Ventina, & Rapoport, 1984). Although it is essential to the success of subsequent fermentation, wineries tend to pay insufficient attention to the rehydration process. Initial airing tends to be neglected, and little effort is made to optimize the timeetemperature combination. In combination with other synergically adverse variables, including the use of musts with an imbalance of sugar/assimilable nitrogen, the excretion of toxic metabolites by yeasts, low environmental or process temperatures, excess sulphite addition and/or overclarification of white wine musts, this can lead to stuck or sluggish fermentations, resulting in wines with sugar concentrations up to 2 g/L (Berthels, Otero, Bauer, Thevelein, & Pretorius, 2004). Many yeast-manufacturing companies also market nitrogencontaining products to be added directly to the must, including amino acids, DAP, inactive yeasts and hulls, and vitamins such as B1, a deficit of which can give rise to stuck or sluggish fermentations (Bataillon, Rico, Sablayrolles, Salmon, & Barre, 1996). Fermentation
P.D.- Hellín et al. / LWT - Food Science and Technology 50 (2013) 126e131
activators consist of two major sub-types: growth factors affecting cell proliferation and activity, and survival factors, such as membrane sterols, that help yeast cells to tolerate toxic compounds, such as ethanol, acetic acid and medium-chain fatty acids. The addition of sterols is essential since they cannot be synthesized by yeasts during fermentation (Bisson, 1999; Redón, Guillamón, Mas, & Rozès, 2009). There are currently very few commercial fermentation activators available; of these, inactive dry yeasts are widely used because they have a favourable effect on the fermentation rate and the duration of the process (Soubeyrand et al., 2005). However, other compounds added in low amounts to small volumes of rehydration water might prove more effective than supplements added to the must, due to a favourable osmotic gradient. Although such water supplements are too expensive to use on an industrial scale, compound products containing varying proportions of these supplements are available, and ensure ease of use. The fermentation profile can be analysed by monitoring kinetic parameters based on carbon dioxide (CO2) release: the area under the curve at a fixed time, temperature, specific growth rate and asymptotic value (Tofalo et al., 2009). This information, together with data on the major wine fermentation compounds, ethanol, glycerol and residual sugars, enables selection of the most appropriate variables. One measure that provides an accurate picture of cell physiology is vitality, defined as CO2 production per unit of time (RodríguezPorrata et al., 2008). Other key cell parameters include viability or capacity for cell division and cell metabolic activity. The present study sought to determine the effect of nine “activators” added directly to the water to rehydrate four commercial wine yeast strains, and to evaluate their subsequent adaptation to a synthetic must by measuring biochemical and physiological parameters. Activators, some of which had hitherto rarely been tested in combination with Saccharomyces, were selected for their potential activity and their ability to enhance processes and/or improve cell structures. Some are known to enhance cellmembrane plasticity under anaerobic conditions (RodríguezPorrata et al., 2011), while others are involved in specific metabolic cycles, have antioxidant properties, or are components of essential enzymes required for cellular metabolism. Their impact was assessed by analysing the vitality, viability and metabolic activity of the yeasts throughout fermentation. Fermentation kinetics and levels of residual sugars, ethanol and glycerol were also analysed. 2. Material and methods 2.1. Yeast strains and metabolic activators tested Four commercial Saccharomyces sp. yeasts (L1, L2, L3 and L4) were rehydrated in a sterile physiological solution at 35 C, with gentle mixing by inversion for 30 min. L1 gives the aromatic characters of varieties rich in aroma precursors. It does not produce volatile phenols and is a discreet producer of acetaldehyde and pyruvic acid, with a high fermentative rate and alcoholic strength at low temperatures. L2 results in rapid fermentation on clarified musts and is not recommended for use at concentrations above 13%. It has a high nitrogen requirement, a fermentation temperature of 12e35 C, a high sugar/alcohol yield, and leads to high levels of glycerol production. L3 produces high levels of manno-proteins due to its b-glycosidase activity, and ferments well at low temperatures. It develops in purulent form and forms sediments that can be re-suspended in cases of maturation over lees. It produces good levels of ethanol forms small amounts of acetic acid and some succinic acid, and does not form hydrogen sulphide. L4 is
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a rapid starter leading to constant and complete fermentation between 15 and 30 C. It has the ability to metabolize high amounts of malic acid, producing significant amounts of esters and high levels of alcohol. 2.2. Metabolic activators tested The following compounds were added individually to the rehydration water, yielding a total of nine different rehydration water trials: glycerol (126.1 g/L); Tween 80 (0.3 g/L); þ ergosterol (4.5$102 g/L); ascorbic acid (1.76$102 g/L); glutathione (2$103 g/ L); nicotinic acid (5$103 g/L); oleic acid (2.82$102 g/L); manganese (1.51 g/L); tetrahydrofolic acid (FH4) (0.56 g/L) and pyruvate (9$102 g/L) (SigmaÒ). Activators were selected for their involvement in different metabolic cycles and included cell-membrane components (glycerol, Tween 80 þ ergosterol, Tween 80 þ oleic acid) that enhance membrane plasticity and thus favour nutrient transport; vitamins (nicotinic acid, tetrahydrofolic acid), which play a role in energy-generating reactions through the biochemical oxidation of carbohydrates, fats and proteins, antioxidants (ascorbic acid, glutathione); manganese, a component of certain ATPdependent coenzymes and reactions; and pyruvic acid, a key compound in cellular metabolism. Strains rehydrated with water alone were used as controls. 2.3. Microfermentations Rehydrated yeast cells were inoculated at a concentration of 106 cells/mL into 200 mL of sterile synthetic must (120 g/L glucose, 80 g/L fructose, 3 g/L YNB, plus 3 g/L each of malic, citric and tartaric acid) at pH 3.7. The substrate was not aired to ensure that the yeast cells would be operating in the worst possible conditions. The initial must contained an excess of yeast assimilable nitrogen (0.36 g/L) and amine nitrogen (0.04 g/L) to ensure that nitrogen deficiency was not a limiting factor for fermentation. Duplicate fermentations were carried out at 28 C for 15 days, without shaking, in 250-mL Erlenmeyer flasks fitted with Müller valves. 2.4. Cell vitality Cell vitality was measured after 2, 8 and 15 days of fermentation using a mTrac 4200 Microbiological Analyser (SY-LAB instruments, Austria). This apparatus measures variations in impedance in a 0.2% KOH solution caused by absorption of the CO2 released by 5 mL of YPD broth (10 g/L yeast extract, 20 g/L glucose and 20 g/L peptone) inoculated with 3.3 106 cells/mL. The decline in impedance is reflected in negative exponential growth curves. The point of inflection between cell adaptation and the first third of the exponential phase corresponding to an impedance value (M) ¼ 15 (25 C) was selected as the detection time (DT). Measurements were performed in triplicate, and the kinetics were monitored for up to 24 h. Absolute values for detection time (DT) displayed excessive bias, so comparisons were made using relative measurements (dM). For this purpose, each yeast was rehydrated in physiological solution both alone and with the selected activator; the DT of each was measured at M ¼ 15, and the following formula was applied: dM ¼ (MH2O Mm)/MH2O, where MH2O is the DT with water alone and Mm is the DT for rehydrated water supplemented with the activator. 2.5. Cell viability and metabolic activity The cell viability of yeasts at 2, 8 and 15 days was measured by spreading appropriate dilutions onto YM agar plates, which were then incubated at 28 C for at least 48 h. Cell metabolic activity was
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analysed by fluorescence microscopy, using the FUNÒ1 cell stain LIVE/DEADÒ Yeast Viability Kit (Probes). The percentages of metabolically active (orange-staining) and inactive (yellow-green staining) cells were counted in a Thoma counting chamber. To determine vitality comparisons were made relative to the control: Rf ¼ (%live/dead H2O control) (%live/dead H2O activator). 2.6. Fermentation kinetics The fermentation kinetics were monitored by gravimetric measurement of CO2 production, weighing flasks at least once a day until the 15th day of fermentation. Kinetic parameters were measured for all the yeasteactivator combinations tested. Experimental data were fitted to a modified Gompertz equation (Zwietering, Jongenberger, Rombouts, & Van’t Riet, 1990):
y ¼ A$expf exp½ðmmax $e=AÞ$ðl tÞ þ 1g where y ¼ growth at time t (h), A ¼ asymptote value, when t: N, mmax ¼ maximum growth rate (h1) and l ¼ lag time (h). 2.7. Measurement of glucose/fructose, glycerol and ethanol Glucose, fructose, glycerol and ethanol were measured at 2, 8, and 15 days of fermentation by HPLC using a BioRad HPX-87H 300 7.8 mm column and a refractive index detector. Sulphuric acid (0.245 g/L) was used as the mobile phase and the oven temperature was 25 C. 3. Results and discussion 3.1. Cell vitality, viability and metabolic activity at different fermentation times using different activators The vitality, viability and metabolic activity of the four selected yeast strains, rehydrated and supplemented with each of the nine different activators, were evaluated at 2, 8 and 15 days’ fermentation. Nevertheless, the analysis was focused on the last two sampling points, as no significant differences had developed by the 2nd day. Vitality is a measure of CO2 production per unit of time based on the cell number, thus providing an indication of a yeast’s fermentation activity at any given moment. Fig. 1 shows the dM values for each activatoreyeast strain combination after 8 (A) and 15 days of fermentation (B): the higher the dM value, the more vital the cell. The response to the rehydration-water supplements tested varied considerably. Differences were more extreme at 8 days than at 15 days. On day 8, a total of 13 yeasteactivator combinations displayed dM values equal to or greater than 0.10, in comparison with 10 by 15 days. A similar pattern was observed for negative vitality, with seven dM combinations displaying values of less than 0.10 at 8 days, in comparison with 5 by 15 days fermentation. There would thus appear to be no “universal” activator, with the exception of ascorbic acid, which yielded positive results for all four yeast strains at both measurements. The supplements tested behaved differently depending on the yeast strain and the fermentation time. Oleic acid showed dM values of up to 0.50, almost double those obtained for the other supplements. Strain L1 produced negative results in nearly all cases at 8 days of fermentation. Strains L2, L3 and L4 showed different responses to the same supplement. Nicotinic acid, for example, prompted a sharp decline in vitality at 15 days in strains L1eL3, but at 8 days had a negligible effect on all except strain L2. Ascorbic acid yielded positive results for all four yeast strains at both fermentation times (Fig. 1).
Fig. 1. Vitality as dM, relative measurements, at the 8th (a) and 15th (b) of the fermentation process for L1 to L4 strains and the nine activators tested (a e pyruvate; b e FH4; c e manganese; d e oleic acid; e e nicotinic acid; f e glutathione; g e ascorbic acid; h e ergosterol; i e glycerol) dM of control ¼ 0. L1 L2 L3 and L4 -.
Since the best sources of cellular energy aside from air are assimilable sugars, sugar consumption was plotted against vitality at 8 and 15 days of fermentation (Fig. 2). As Fig. 2A shows, Detection Time (DT) of strains rehydrated with supplements and with water alone was approximately 5 h at 8 days, while varied much more after 15 days. After 8 days, residual sugar varied between 4 and 100 g/L, or 20 g/L and less than 2 g/L by 15 days. Yeast strains L1 and L2 displayed sluggish sugar metabolism at both 8 and 15 days, with as much as 25% of the sugar remaining unconsumed. Strain L3 exhibited an intermediate metabolic rate, while L4 was much faster. After 15 days of fermentation, strains L1 and L2 rehydrated with water alone displayed similar detection times as their counterparts rehydrated with activator supplements (range: 4.6e6.8 h) (Fig. 2B). However, sugar consumption varied widely, with residual sugar levels ranging from 1 to roughly 50 g/L. In addition, controls displayed an intermediate DT, suggesting that some activators had a positive effect and others a negative one. The vitality of strain L4 varied markedly as a function of the activator used. The control strain was sluggish, with a detection time of 24 h at 15 days of fermentation. All activators led to a drop in detection time, with the steepest decline observed following the addition of oleic acid. Residual sugar levels were below 2 g/L in all cases. L3 showed intermediate values. With Nicotinic acid, vitality decreased while residual sugar levels remained steady, whereas both ergosterol and ascorbic acid increased vitality and reduced residual sugar levels.
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therefore of immense importance. The use of certain adjuvants in difficult winemaking conditions would greatly increase residual vitality: oleic acid, for example, increased L4 vitality by 40% with respect to its control. Given that fermentation problems tend to arise in the latter third of the process, it is important that the yeast cells completely exhaust the sugar and retain as much vitality as possible in order to avoid additional complications. The data on viability and metabolic activity (Fig. 3), like those for vitality, were expressed in absolute terms with reference to those recorded in control (i.e. water-only) strains. The data refer to the percentage of cells growing on YM agar or displaying positive vital fluorochrome staining. Plate counts (not shown) gave similar results for all “activated” cultures and controls (107 cells/mL) after 15 days of fermentation. The exception was L4, the viability of which was reduced by an order of a Log magnitude (to around 106 cells/mL) in the presence of pyruvate and nicotinic acid, and even further reduced (<106 cells/ mL) in the presence of the other activator supplements tested. It should be noted that this strain exhibited the lowest vitality after 15 days’ fermentation, and completely exhausted the sugars. The presence of certain supplements in the rehydration water e including the antioxidants glutathione and ascorbic acid, vitamins such as tetrahydrofolic acid, and pyruvic acid and ergosterol e improved the metabolic activity of some yeast strains with the best results both first ones. A clear relationship was observed with regard to vitality in the case of nicotinic acid and ascorbic acid. No relationship was observed in the case of glycerol, whilst for other activators there was a negative relationship: a strain may therefore be metabolically active but display low vitality, or vice versa; in some cases, the two properties may exhibit similar trends. 3.2. Fermentation kinetics of commercial yeast strains rehydrated with different activator supplements Fig. 2. Residual sugars (g/L) vs vitality as detection time (DT, h1) at 8th (a) and 15th (b) days of the fermentation process for L1 (A), L2 (-), L3 (:), L4 (C),control for each strain () and the nine activators tested. (aC) L4 þ oleic acid, (b:) L3 þ nicotinic acid, (c:) L3 þ ergosterol, (d:) L3 þ ascorbic acid. Standard deviation was less than 10% in all cases.
An inverse correlation was observed between sugar concentrations and strain vitality. The strains displaying the greatest vitality after 15 days of fermentation (L1 and L2) failed to exhaust the sugar in the medium, whereas the least vital strains (L3 and L4) consumed almost all of the sugar. The inherent ability of a yeast strain to metabolize sugar is a fundamental consideration in winemaking; selecting the most suitable strain in this respect is
Fig. 3. Rf, relative metabolic activity assessed by fluorescence at the 15th day of the fermentation process for L1eL4 strains with the nine activators tested (a e pyruvate; b e FH4; c e manganese; d e oleic acid; e e nicotinic acid; f e glutathione; g e ascorbic acid; h e ergosterol; i e glycerol) L1 L2 L3 and L4 -. Positive values of Rf indicate low % of fluorescence activity compared to controls, whilst negative ones indicated greater activity than controls.
The most effective way of analysing fermentation behaviour is by measuring kinetic parameters such as CO2 production, the area under the curve (AUC) at a pre-established time, specific growth rate, lag phase and asymptotic value during the stationary phase, and by measuring levels of major fermentation compounds such as ethanol, glycerol and residual sugars. Experimental data displayed a good fit to Zwietering’s modification of the Gompertz model, based on the equation outlined in the materials and methods. The fitting of CO2 production curves adapted to this model is shown in Fig. 4, where the profile of each strain rehydrated with water alone is shown, together with the activators that yielded the best and worst results with that strain. The fermentation kinetics of strain L1 (Fig. 4A) were improved by the addition of nicotinic acid, whereas for strains L2 (Fig. 4B) and L3 (Fig. 4C) the best activators were glycerol and ergosterol, respectively. The fermentation kinetics for strains L1, L2 and L3 were all worsened by the addition of oleic acid. The addition of glycerol in the rehydration of strain L4 (Fig. 4D) had no effect on fermentation kinetics, whereas manganese increased the latent period and significantly reduced the area under the curve. The four yeast strains displayed considerable differences in fermentation behaviour. After 15 days of fermentation, residual sugar levels in the growth medium of strains L1 and L2 were unacceptably high. The addition of ergosterol and FH4 resulted in a marked increase in sugar consumption and conversion to glycerol, and a consequent decrease in alcohol production. However, none of the activators tested succeeded in reducing residual sugar content to acceptable levels (<2 g/L) (data not shown). Table 1 shows kinetic parameters such as maximum growth rate (mmax), asymptotic value (A) and area under the curve (AUC) together with data on chemical composition (residual sugars,
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P.D.- Hellín et al. / LWT - Food Science and Technology 50 (2013) 126e131
Fig. 4. Fitting of CO2 production curves to the Gompertz model for strains L1 (a), L2 (b), L3 (c) and L4 (d). The figure shows the profile of each strain rehydrated with water alone (-), together with the activators that yielded the best (C) and worst ( ) results with that strain. L1: control (-) nicotinic acid (C) oleic acid ( ); L2: control (-) ergosterol (C) oleic acid ( ); L3: control (-) glycerol (C) oleic acid ( ); L4: control (-) glycerol (C) manganese ( ). Standard deviation was less than 10% in all cases.
glycerol and ethanol) for two of the strains rehydrated in the presence of all the activators tested (L3 and L4). In strain L3, mmax ranged from 0.066 to 0.105 h1. Cells rehydrated in the presence of ergosterol, glycerol and the antioxidants ascorbic acid and glutathione displayed the greatest maximum growth rates. In particular, ergosterol contributed to a larger AUC and a higher asymptotic value compared to the other additives. In contrast, the addition of oleic acid resulted in the lowest maximum growth rates, AUC, and asymptotic values. A similar trend towards higher mmax (ranging from 0.090 to 0.175 h1) in the presence of ergosterol, glycerol and glutathione was observed in L4. After 15 days of fermentation, strain L4 rehydrated in water alone had a sugar content of 1.1 g/L. This was not improved by activator supplementation, although a decline in alcohol content was observed. The sugar content following supplementation with glycerol, nicotinic acid and oleic acid was similar to the control, but manganese content increased, and ascorbic acid, pyruvic acid, glutathione and alcohol content decreased. The use of additives may help improve sugar consumption when rehydrating strains associated with high levels of sugar consumption (Table 1). Ascorbic acid and ergosterol improved sugar metabolism, yielding values of 2.2 and 1.7 g/L, respectively, but also modified glycerol and ethanol values with respect to the control. In summary, these data show that kinetic and major chemical parameters are useful for evaluating activator supplements. If the
Table 1 Kinetics parameters and residual sugar concentration, glycerol and ethanol of L3 (a) and L4 (b) strains at the 15th day of fermentation. Maximum specific rate (mmax); asymptote value (A); under curve value (AUC); glucose þ fructose (Residual Sugars, R.S.); glycerol (Glyc.) and alcoholic grade ( Alcoholic). n ¼ 2 replicates. SD in all values was 15%.
a Control Glycerol Ergosterol Ascorbic Ac. Glutathione Nicotinic Ac. Oleic Acid Manganese FH4 Pyruvate b Control Glycerol Ergosterol Ascorbic Ac. Glutathione Nicotinic Ac. Oleic Acid Manganese FH4 Pyruvate
mmax (h1) A (g) AUC (g*h) R.S. (g/L) Glyc. (g/L)
0.083 0.103 0.105 0.092 0.098 0.097 0.066 0.082 0.080 0.079
17.4 17.5 19.4 17.6 17.2 17.2 16.4 15.6 17.4 17.9
2714.6 3856.8 4326.0 3599.2 2901.4 3512.4 2471.0 2658.4 3280.8 3280.8
3.0 4.3 1.7 2.2 3.4 2.7 2.3 4.8 5.7 8.9
5.5 4.7 4.1 6.6 5.8 5.9 6.4 6.3 5.4 5.3
13.4 15.4 11.4 13.4 12.6 14.9 13.3 14.2 12.6 13.6
0.149 0.175 0.170 0.126 0.162 0.124 0.090 0.099 0.137 0.137
18.7 18.0 18.8 17.4 18.8 17.2 18.5 12.5 17.0 18.3
4690.3 4832.6 4955.0 4149.0 4100.7 4018.5 3373.2 2545.6 4268.7 4723.5
1.1 1.4 1.6 1.4 1.4 1.7 2.0 1.2 1.4 1.7
7.6 6.0 6.6 6.7 7.2 5.8 9.5 6.9 7.1 6.0
14.6 14.4 12.8 12.6 12.5 14.5 14.5 14.8 12.8 12.3
Alcoholic
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aim is to achieve less aggressive kinetics e as measured by the area under the curve e together with acceptable vitality (Fig. 1B) and appropriate residual sugar, ethanol and glycerol levels, ascorbic acid may be the activator of choice in combination with yeast strain L4. In contrast, nicotinic acid had an adverse effect on the vitality of L3 and L4 after 15 days, although the kinetic and chemical data were acceptable. 4. Conclusions The inherent ability of a yeast strain to metabolize sugar is a fundamental consideration in winemaking. Given that fermentation problems tend to arise in the latter third of the process, it is important that the yeast cells completely exhaust the sugar and retain as much vitality as possible in order to avoid additional complications. Choosing the most suitable strain is therefore of immense importance. The use of certain adjuvants in difficult winemaking conditions has the potential to greatly increase residual vitality, as demonstrated in this study by the 40% increase in L4 vitality following the addition of oleic acid. However, the results obtained for vitality, kinetics, major compounds, viability and sugar metabolism using the various fermentation activators tested varied considerably, indicating a high degree of strain-specificity. These results suggest that there is no “universal” activator apart from ascorbic acid and that formulation of a “tailor-made” activator is likely to be required for each commercial yeast strain. Acknowledgements This research was supported by the Regional Project Programme (PII-2109-0178-4509) from the government of Castilla La Mancha.
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References Bataillon, M., Rico, A., Sablayrolles, J. M., Salmon, J. M., & Barre, P. (1996). Early thiamin assimilation by yeasts under enological conditions: impact on alcoholic fermentation kinetics. Journal of Fermentation and Bioengineering, 82, 145e150. Beker, M. J., Blumbergs, J. E., Ventina, E. J., & Rapoport, A. I. (1984). Characteristics of cellular membranes at rehydration of dehydrated yeast Saccharomyces cerevisiae. European Journal of Applied Microbiology and Biotechnology, 19, 347e352. Berthels, N. J., Otero, R. R. C., Bauer, F. F., Thevelein, J. M., & Pretorius, I. S. (2004). Discrepancy in glucose and fructose utilisation during fermentation by Saccharomyces cerevisiae wine yeast strains. FEMS Yeast Research, 4, 683e689. Bisson, L. (1999). Stuck and sluggish fermentations. American Journal of Enology and Viticulture, 50, 107e111. Parrou, J. L., Enjalbert, B., Plourde, L., Bauche, A., Gonzalez, B., & Francois, J. (1999). Dynamic responses of reserve carbohydrate metabolism under carbon and nitrogen limitations in Saccharomyces cerevisiae. Yeast, 15, 191e203. Rapoport, A. I., Khrustaleva, G. M., Chamanis, G. Y., & Beker, M. E. (1995). Yeast anhydrobiosis permeability of the plasma-membrane. Microbiology, 64, 229e232. Redón, M., Guillamón, J. M., Mas, A., & Rozès, N. (2009). Effect of lipid supplementation upon Saccharomyces cerevisiae lipid composition and fermentation performance at low temperature. European Food Research and Technology, 228, 833e840. Rodríguez-Porrata, B., López-Martínez, G., Redón, M., Sancho, M., Mas, A., Rozès, N., et al. (2011). Enhancing yeast cell viability after dehydration by modification of the lipid profile. World Journal of Microbiology & Biotechnology, 27, 75e83. Rodríguez-Porrata, B., Novo, M., Guillamón, J., Rozès, N., Mas, A., & Cordero Otero, R. (2008). Vitality enhancement of the rehydrated active dry wine yeast. International Journal of Food Microbiology, 126, 116e122. Soubeyrand, V., Luparia, V., Williams, P., Doco, T., Vernhet, A., Ortiz-Julien, A., et al. (2005). Formation of micella containing solubilized sterols during rehydration of active dry yeasts improves their fermenting capacity. Journal of Agricultural and Food Chemistry, 53, 8025e8032. Suarez, J. A. (2004). Levaduras vínicas. Funcionalidad y su uso en bodegas (1st ed.). Madrid: AMV Ediciones Mundi-Prensa. Tofalo, R., Chaves-López, C., Di Fabio, F., Schirone, M., Felis, G. E., Torriani, S., et al. (2009). Molecular identification and osmotolerant profile of wine yeasts that ferment a high sugar grape must. International Journal of Food Microbiology, 130, 179e187. Zwietering, M. H., Jongenberger, I., Rombouts, F. M., & Van’t Riet, K. (1990). Modeling of the bacterial growth curve. Applied and Environmental Microbiology, 56, 1875e1881.
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Improving alcoholic fermentation by activation of Saccharomyces species during the rehydration stage P. Díaz- Hellín, J. Úbeda*, A. Briones Tecnología de Alimentos, IRICA, Universidad de Castilla La Mancha, Avda. Camilo José Cela 10, Edifico Marie Curie, 13071 Ciudad Real, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 October 2011 Received in revised form 10 June 2012 Accepted 12 June 2012
The rehydration of selected active dry yeasts is essential for improving control of the wine fermentation process. To help yeast cells withstand the extreme conditions to which they are subjected during rehydration, winemakers often add adjuvants directly to the must. However, few commercial activators are added directly to the yeast rehydration water, even though this might render hydration more efficient due to a favourable osmotic gradient. The present study examined the effect of nine “activators” added directly to the water to rehydrate four commercial wine yeast strains by measuring the vitality, viability and metabolic activity of the process, plus residual sugars, glycerol and ethanol levels at days 2, 8 and 15. The fermentation kinetics were monitored and data were adjusted to a modified Gompertz equation. The activators, some of which had hitherto rarely been tested in combination with Saccharomyces, were selected for their potential activity and ability to enhance processes and/or improve cell structures. Semi-quantitative results revealed that the four wine yeasts responded very differently to the activators, indicating a high degree of strain specificity. There appeared to be no “universal activator”, indicating that a “tailor-made” activator will likely need to be formulated for each commercial yeast strain. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Active dry yeasts Activators Vitality Viability Fermentation kinetics Saccharomyces sp.
1. Introduction Twenty years ago, wine fermentation was a spontaneous process triggered by the native yeast occurring naturally in vineyards and wineries. Today, very few winemakers use traditional fermentation methods; instead, most wineries rely on the inoculation of commercial active dry yeasts. Although industrially produced Saccharomyces biomass is mostly used in fresh or extruded form for the manufacture of bread and beer, requiring an unbroken cold chain, a small proportion of commercial yeast is dried for use in wine fermentation. Since, unlike bread yeasts, wine yeasts are required on a seasonal basis rather than throughout the year, manufacturers find that drying enables major savings in transport and storage (Rodríguez-Porrata et al., 2011). The manufacture of active dry yeasts involves fed-batch biomass production and subsequent dehydration. Both are highly stressful processes, prompting the yeast cell to accumulate protective molecules such as trehalose and glycogen, which play a key role in cell growth (Parrou et al., 1999). In the course of dehydration, the cell also loses essential vital material, including nucleotides, ions * Corresponding author. Tel.: þ34 926295300x3424; fax: þ34 926295318. E-mail addresses: [email protected] (P.D.- Hellín), [email protected] (J. Úbeda), [email protected] (A. Briones). 0023-6438/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.lwt.2012.06.011
and other soluble cell components (Rapoport, Khrustaleva, Chamanis, & Beker, 1995; Suarez, 2004), some of which are biosynthesized de novo during rehydration, allowing most cells to resume growth. Dry cells are rehydrated in water between 34 C and 40 C for around 30 min. Rehydration prompts interactions between cytosol structural macromolecules and the plasma membrane, giving rise to biochemical reactions that are fundamental for cell division (Beker, Blumbergs, Ventina, & Rapoport, 1984). Although it is essential to the success of subsequent fermentation, wineries tend to pay insufficient attention to the rehydration process. Initial airing tends to be neglected, and little effort is made to optimize the timeetemperature combination. In combination with other synergically adverse variables, including the use of musts with an imbalance of sugar/assimilable nitrogen, the excretion of toxic metabolites by yeasts, low environmental or process temperatures, excess sulphite addition and/or overclarification of white wine musts, this can lead to stuck or sluggish fermentations, resulting in wines with sugar concentrations up to 2 g/L (Berthels, Otero, Bauer, Thevelein, & Pretorius, 2004). Many yeast-manufacturing companies also market nitrogencontaining products to be added directly to the must, including amino acids, DAP, inactive yeasts and hulls, and vitamins such as B1, a deficit of which can give rise to stuck or sluggish fermentations (Bataillon, Rico, Sablayrolles, Salmon, & Barre, 1996). Fermentation
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activators consist of two major sub-types: growth factors affecting cell proliferation and activity, and survival factors, such as membrane sterols, that help yeast cells to tolerate toxic compounds, such as ethanol, acetic acid and medium-chain fatty acids. The addition of sterols is essential since they cannot be synthesized by yeasts during fermentation (Bisson, 1999; Redón, Guillamón, Mas, & Rozès, 2009). There are currently very few commercial fermentation activators available; of these, inactive dry yeasts are widely used because they have a favourable effect on the fermentation rate and the duration of the process (Soubeyrand et al., 2005). However, other compounds added in low amounts to small volumes of rehydration water might prove more effective than supplements added to the must, due to a favourable osmotic gradient. Although such water supplements are too expensive to use on an industrial scale, compound products containing varying proportions of these supplements are available, and ensure ease of use. The fermentation profile can be analysed by monitoring kinetic parameters based on carbon dioxide (CO2) release: the area under the curve at a fixed time, temperature, specific growth rate and asymptotic value (Tofalo et al., 2009). This information, together with data on the major wine fermentation compounds, ethanol, glycerol and residual sugars, enables selection of the most appropriate variables. One measure that provides an accurate picture of cell physiology is vitality, defined as CO2 production per unit of time (RodríguezPorrata et al., 2008). Other key cell parameters include viability or capacity for cell division and cell metabolic activity. The present study sought to determine the effect of nine “activators” added directly to the water to rehydrate four commercial wine yeast strains, and to evaluate their subsequent adaptation to a synthetic must by measuring biochemical and physiological parameters. Activators, some of which had hitherto rarely been tested in combination with Saccharomyces, were selected for their potential activity and their ability to enhance processes and/or improve cell structures. Some are known to enhance cellmembrane plasticity under anaerobic conditions (RodríguezPorrata et al., 2011), while others are involved in specific metabolic cycles, have antioxidant properties, or are components of essential enzymes required for cellular metabolism. Their impact was assessed by analysing the vitality, viability and metabolic activity of the yeasts throughout fermentation. Fermentation kinetics and levels of residual sugars, ethanol and glycerol were also analysed. 2. Material and methods 2.1. Yeast strains and metabolic activators tested Four commercial Saccharomyces sp. yeasts (L1, L2, L3 and L4) were rehydrated in a sterile physiological solution at 35 C, with gentle mixing by inversion for 30 min. L1 gives the aromatic characters of varieties rich in aroma precursors. It does not produce volatile phenols and is a discreet producer of acetaldehyde and pyruvic acid, with a high fermentative rate and alcoholic strength at low temperatures. L2 results in rapid fermentation on clarified musts and is not recommended for use at concentrations above 13%. It has a high nitrogen requirement, a fermentation temperature of 12e35 C, a high sugar/alcohol yield, and leads to high levels of glycerol production. L3 produces high levels of manno-proteins due to its b-glycosidase activity, and ferments well at low temperatures. It develops in purulent form and forms sediments that can be re-suspended in cases of maturation over lees. It produces good levels of ethanol forms small amounts of acetic acid and some succinic acid, and does not form hydrogen sulphide. L4 is
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a rapid starter leading to constant and complete fermentation between 15 and 30 C. It has the ability to metabolize high amounts of malic acid, producing significant amounts of esters and high levels of alcohol. 2.2. Metabolic activators tested The following compounds were added individually to the rehydration water, yielding a total of nine different rehydration water trials: glycerol (126.1 g/L); Tween 80 (0.3 g/L); þ ergosterol (4.5$102 g/L); ascorbic acid (1.76$102 g/L); glutathione (2$103 g/ L); nicotinic acid (5$103 g/L); oleic acid (2.82$102 g/L); manganese (1.51 g/L); tetrahydrofolic acid (FH4) (0.56 g/L) and pyruvate (9$102 g/L) (SigmaÒ). Activators were selected for their involvement in different metabolic cycles and included cell-membrane components (glycerol, Tween 80 þ ergosterol, Tween 80 þ oleic acid) that enhance membrane plasticity and thus favour nutrient transport; vitamins (nicotinic acid, tetrahydrofolic acid), which play a role in energy-generating reactions through the biochemical oxidation of carbohydrates, fats and proteins, antioxidants (ascorbic acid, glutathione); manganese, a component of certain ATPdependent coenzymes and reactions; and pyruvic acid, a key compound in cellular metabolism. Strains rehydrated with water alone were used as controls. 2.3. Microfermentations Rehydrated yeast cells were inoculated at a concentration of 106 cells/mL into 200 mL of sterile synthetic must (120 g/L glucose, 80 g/L fructose, 3 g/L YNB, plus 3 g/L each of malic, citric and tartaric acid) at pH 3.7. The substrate was not aired to ensure that the yeast cells would be operating in the worst possible conditions. The initial must contained an excess of yeast assimilable nitrogen (0.36 g/L) and amine nitrogen (0.04 g/L) to ensure that nitrogen deficiency was not a limiting factor for fermentation. Duplicate fermentations were carried out at 28 C for 15 days, without shaking, in 250-mL Erlenmeyer flasks fitted with Müller valves. 2.4. Cell vitality Cell vitality was measured after 2, 8 and 15 days of fermentation using a mTrac 4200 Microbiological Analyser (SY-LAB instruments, Austria). This apparatus measures variations in impedance in a 0.2% KOH solution caused by absorption of the CO2 released by 5 mL of YPD broth (10 g/L yeast extract, 20 g/L glucose and 20 g/L peptone) inoculated with 3.3 106 cells/mL. The decline in impedance is reflected in negative exponential growth curves. The point of inflection between cell adaptation and the first third of the exponential phase corresponding to an impedance value (M) ¼ 15 (25 C) was selected as the detection time (DT). Measurements were performed in triplicate, and the kinetics were monitored for up to 24 h. Absolute values for detection time (DT) displayed excessive bias, so comparisons were made using relative measurements (dM). For this purpose, each yeast was rehydrated in physiological solution both alone and with the selected activator; the DT of each was measured at M ¼ 15, and the following formula was applied: dM ¼ (MH2O Mm)/MH2O, where MH2O is the DT with water alone and Mm is the DT for rehydrated water supplemented with the activator. 2.5. Cell viability and metabolic activity The cell viability of yeasts at 2, 8 and 15 days was measured by spreading appropriate dilutions onto YM agar plates, which were then incubated at 28 C for at least 48 h. Cell metabolic activity was
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analysed by fluorescence microscopy, using the FUNÒ1 cell stain LIVE/DEADÒ Yeast Viability Kit (Probes). The percentages of metabolically active (orange-staining) and inactive (yellow-green staining) cells were counted in a Thoma counting chamber. To determine vitality comparisons were made relative to the control: Rf ¼ (%live/dead H2O control) (%live/dead H2O activator). 2.6. Fermentation kinetics The fermentation kinetics were monitored by gravimetric measurement of CO2 production, weighing flasks at least once a day until the 15th day of fermentation. Kinetic parameters were measured for all the yeasteactivator combinations tested. Experimental data were fitted to a modified Gompertz equation (Zwietering, Jongenberger, Rombouts, & Van’t Riet, 1990):
y ¼ A$expf exp½ðmmax $e=AÞ$ðl tÞ þ 1g where y ¼ growth at time t (h), A ¼ asymptote value, when t: N, mmax ¼ maximum growth rate (h1) and l ¼ lag time (h). 2.7. Measurement of glucose/fructose, glycerol and ethanol Glucose, fructose, glycerol and ethanol were measured at 2, 8, and 15 days of fermentation by HPLC using a BioRad HPX-87H 300 7.8 mm column and a refractive index detector. Sulphuric acid (0.245 g/L) was used as the mobile phase and the oven temperature was 25 C. 3. Results and discussion 3.1. Cell vitality, viability and metabolic activity at different fermentation times using different activators The vitality, viability and metabolic activity of the four selected yeast strains, rehydrated and supplemented with each of the nine different activators, were evaluated at 2, 8 and 15 days’ fermentation. Nevertheless, the analysis was focused on the last two sampling points, as no significant differences had developed by the 2nd day. Vitality is a measure of CO2 production per unit of time based on the cell number, thus providing an indication of a yeast’s fermentation activity at any given moment. Fig. 1 shows the dM values for each activatoreyeast strain combination after 8 (A) and 15 days of fermentation (B): the higher the dM value, the more vital the cell. The response to the rehydration-water supplements tested varied considerably. Differences were more extreme at 8 days than at 15 days. On day 8, a total of 13 yeasteactivator combinations displayed dM values equal to or greater than 0.10, in comparison with 10 by 15 days. A similar pattern was observed for negative vitality, with seven dM combinations displaying values of less than 0.10 at 8 days, in comparison with 5 by 15 days fermentation. There would thus appear to be no “universal” activator, with the exception of ascorbic acid, which yielded positive results for all four yeast strains at both measurements. The supplements tested behaved differently depending on the yeast strain and the fermentation time. Oleic acid showed dM values of up to 0.50, almost double those obtained for the other supplements. Strain L1 produced negative results in nearly all cases at 8 days of fermentation. Strains L2, L3 and L4 showed different responses to the same supplement. Nicotinic acid, for example, prompted a sharp decline in vitality at 15 days in strains L1eL3, but at 8 days had a negligible effect on all except strain L2. Ascorbic acid yielded positive results for all four yeast strains at both fermentation times (Fig. 1).
Fig. 1. Vitality as dM, relative measurements, at the 8th (a) and 15th (b) of the fermentation process for L1 to L4 strains and the nine activators tested (a e pyruvate; b e FH4; c e manganese; d e oleic acid; e e nicotinic acid; f e glutathione; g e ascorbic acid; h e ergosterol; i e glycerol) dM of control ¼ 0. L1 L2 L3 and L4 -.
Since the best sources of cellular energy aside from air are assimilable sugars, sugar consumption was plotted against vitality at 8 and 15 days of fermentation (Fig. 2). As Fig. 2A shows, Detection Time (DT) of strains rehydrated with supplements and with water alone was approximately 5 h at 8 days, while varied much more after 15 days. After 8 days, residual sugar varied between 4 and 100 g/L, or 20 g/L and less than 2 g/L by 15 days. Yeast strains L1 and L2 displayed sluggish sugar metabolism at both 8 and 15 days, with as much as 25% of the sugar remaining unconsumed. Strain L3 exhibited an intermediate metabolic rate, while L4 was much faster. After 15 days of fermentation, strains L1 and L2 rehydrated with water alone displayed similar detection times as their counterparts rehydrated with activator supplements (range: 4.6e6.8 h) (Fig. 2B). However, sugar consumption varied widely, with residual sugar levels ranging from 1 to roughly 50 g/L. In addition, controls displayed an intermediate DT, suggesting that some activators had a positive effect and others a negative one. The vitality of strain L4 varied markedly as a function of the activator used. The control strain was sluggish, with a detection time of 24 h at 15 days of fermentation. All activators led to a drop in detection time, with the steepest decline observed following the addition of oleic acid. Residual sugar levels were below 2 g/L in all cases. L3 showed intermediate values. With Nicotinic acid, vitality decreased while residual sugar levels remained steady, whereas both ergosterol and ascorbic acid increased vitality and reduced residual sugar levels.
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therefore of immense importance. The use of certain adjuvants in difficult winemaking conditions would greatly increase residual vitality: oleic acid, for example, increased L4 vitality by 40% with respect to its control. Given that fermentation problems tend to arise in the latter third of the process, it is important that the yeast cells completely exhaust the sugar and retain as much vitality as possible in order to avoid additional complications. The data on viability and metabolic activity (Fig. 3), like those for vitality, were expressed in absolute terms with reference to those recorded in control (i.e. water-only) strains. The data refer to the percentage of cells growing on YM agar or displaying positive vital fluorochrome staining. Plate counts (not shown) gave similar results for all “activated” cultures and controls (107 cells/mL) after 15 days of fermentation. The exception was L4, the viability of which was reduced by an order of a Log magnitude (to around 106 cells/mL) in the presence of pyruvate and nicotinic acid, and even further reduced (<106 cells/ mL) in the presence of the other activator supplements tested. It should be noted that this strain exhibited the lowest vitality after 15 days’ fermentation, and completely exhausted the sugars. The presence of certain supplements in the rehydration water e including the antioxidants glutathione and ascorbic acid, vitamins such as tetrahydrofolic acid, and pyruvic acid and ergosterol e improved the metabolic activity of some yeast strains with the best results both first ones. A clear relationship was observed with regard to vitality in the case of nicotinic acid and ascorbic acid. No relationship was observed in the case of glycerol, whilst for other activators there was a negative relationship: a strain may therefore be metabolically active but display low vitality, or vice versa; in some cases, the two properties may exhibit similar trends. 3.2. Fermentation kinetics of commercial yeast strains rehydrated with different activator supplements Fig. 2. Residual sugars (g/L) vs vitality as detection time (DT, h1) at 8th (a) and 15th (b) days of the fermentation process for L1 (A), L2 (-), L3 (:), L4 (C),control for each strain () and the nine activators tested. (aC) L4 þ oleic acid, (b:) L3 þ nicotinic acid, (c:) L3 þ ergosterol, (d:) L3 þ ascorbic acid. Standard deviation was less than 10% in all cases.
An inverse correlation was observed between sugar concentrations and strain vitality. The strains displaying the greatest vitality after 15 days of fermentation (L1 and L2) failed to exhaust the sugar in the medium, whereas the least vital strains (L3 and L4) consumed almost all of the sugar. The inherent ability of a yeast strain to metabolize sugar is a fundamental consideration in winemaking; selecting the most suitable strain in this respect is
Fig. 3. Rf, relative metabolic activity assessed by fluorescence at the 15th day of the fermentation process for L1eL4 strains with the nine activators tested (a e pyruvate; b e FH4; c e manganese; d e oleic acid; e e nicotinic acid; f e glutathione; g e ascorbic acid; h e ergosterol; i e glycerol) L1 L2 L3 and L4 -. Positive values of Rf indicate low % of fluorescence activity compared to controls, whilst negative ones indicated greater activity than controls.
The most effective way of analysing fermentation behaviour is by measuring kinetic parameters such as CO2 production, the area under the curve (AUC) at a pre-established time, specific growth rate, lag phase and asymptotic value during the stationary phase, and by measuring levels of major fermentation compounds such as ethanol, glycerol and residual sugars. Experimental data displayed a good fit to Zwietering’s modification of the Gompertz model, based on the equation outlined in the materials and methods. The fitting of CO2 production curves adapted to this model is shown in Fig. 4, where the profile of each strain rehydrated with water alone is shown, together with the activators that yielded the best and worst results with that strain. The fermentation kinetics of strain L1 (Fig. 4A) were improved by the addition of nicotinic acid, whereas for strains L2 (Fig. 4B) and L3 (Fig. 4C) the best activators were glycerol and ergosterol, respectively. The fermentation kinetics for strains L1, L2 and L3 were all worsened by the addition of oleic acid. The addition of glycerol in the rehydration of strain L4 (Fig. 4D) had no effect on fermentation kinetics, whereas manganese increased the latent period and significantly reduced the area under the curve. The four yeast strains displayed considerable differences in fermentation behaviour. After 15 days of fermentation, residual sugar levels in the growth medium of strains L1 and L2 were unacceptably high. The addition of ergosterol and FH4 resulted in a marked increase in sugar consumption and conversion to glycerol, and a consequent decrease in alcohol production. However, none of the activators tested succeeded in reducing residual sugar content to acceptable levels (<2 g/L) (data not shown). Table 1 shows kinetic parameters such as maximum growth rate (mmax), asymptotic value (A) and area under the curve (AUC) together with data on chemical composition (residual sugars,
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Fig. 4. Fitting of CO2 production curves to the Gompertz model for strains L1 (a), L2 (b), L3 (c) and L4 (d). The figure shows the profile of each strain rehydrated with water alone (-), together with the activators that yielded the best (C) and worst ( ) results with that strain. L1: control (-) nicotinic acid (C) oleic acid ( ); L2: control (-) ergosterol (C) oleic acid ( ); L3: control (-) glycerol (C) oleic acid ( ); L4: control (-) glycerol (C) manganese ( ). Standard deviation was less than 10% in all cases.
glycerol and ethanol) for two of the strains rehydrated in the presence of all the activators tested (L3 and L4). In strain L3, mmax ranged from 0.066 to 0.105 h1. Cells rehydrated in the presence of ergosterol, glycerol and the antioxidants ascorbic acid and glutathione displayed the greatest maximum growth rates. In particular, ergosterol contributed to a larger AUC and a higher asymptotic value compared to the other additives. In contrast, the addition of oleic acid resulted in the lowest maximum growth rates, AUC, and asymptotic values. A similar trend towards higher mmax (ranging from 0.090 to 0.175 h1) in the presence of ergosterol, glycerol and glutathione was observed in L4. After 15 days of fermentation, strain L4 rehydrated in water alone had a sugar content of 1.1 g/L. This was not improved by activator supplementation, although a decline in alcohol content was observed. The sugar content following supplementation with glycerol, nicotinic acid and oleic acid was similar to the control, but manganese content increased, and ascorbic acid, pyruvic acid, glutathione and alcohol content decreased. The use of additives may help improve sugar consumption when rehydrating strains associated with high levels of sugar consumption (Table 1). Ascorbic acid and ergosterol improved sugar metabolism, yielding values of 2.2 and 1.7 g/L, respectively, but also modified glycerol and ethanol values with respect to the control. In summary, these data show that kinetic and major chemical parameters are useful for evaluating activator supplements. If the
Table 1 Kinetics parameters and residual sugar concentration, glycerol and ethanol of L3 (a) and L4 (b) strains at the 15th day of fermentation. Maximum specific rate (mmax); asymptote value (A); under curve value (AUC); glucose þ fructose (Residual Sugars, R.S.); glycerol (Glyc.) and alcoholic grade ( Alcoholic). n ¼ 2 replicates. SD in all values was 15%.
a Control Glycerol Ergosterol Ascorbic Ac. Glutathione Nicotinic Ac. Oleic Acid Manganese FH4 Pyruvate b Control Glycerol Ergosterol Ascorbic Ac. Glutathione Nicotinic Ac. Oleic Acid Manganese FH4 Pyruvate
mmax (h1) A (g) AUC (g*h) R.S. (g/L) Glyc. (g/L)
0.083 0.103 0.105 0.092 0.098 0.097 0.066 0.082 0.080 0.079
17.4 17.5 19.4 17.6 17.2 17.2 16.4 15.6 17.4 17.9
2714.6 3856.8 4326.0 3599.2 2901.4 3512.4 2471.0 2658.4 3280.8 3280.8
3.0 4.3 1.7 2.2 3.4 2.7 2.3 4.8 5.7 8.9
5.5 4.7 4.1 6.6 5.8 5.9 6.4 6.3 5.4 5.3
13.4 15.4 11.4 13.4 12.6 14.9 13.3 14.2 12.6 13.6
0.149 0.175 0.170 0.126 0.162 0.124 0.090 0.099 0.137 0.137
18.7 18.0 18.8 17.4 18.8 17.2 18.5 12.5 17.0 18.3
4690.3 4832.6 4955.0 4149.0 4100.7 4018.5 3373.2 2545.6 4268.7 4723.5
1.1 1.4 1.6 1.4 1.4 1.7 2.0 1.2 1.4 1.7
7.6 6.0 6.6 6.7 7.2 5.8 9.5 6.9 7.1 6.0
14.6 14.4 12.8 12.6 12.5 14.5 14.5 14.8 12.8 12.3
Alcoholic
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aim is to achieve less aggressive kinetics e as measured by the area under the curve e together with acceptable vitality (Fig. 1B) and appropriate residual sugar, ethanol and glycerol levels, ascorbic acid may be the activator of choice in combination with yeast strain L4. In contrast, nicotinic acid had an adverse effect on the vitality of L3 and L4 after 15 days, although the kinetic and chemical data were acceptable. 4. Conclusions The inherent ability of a yeast strain to metabolize sugar is a fundamental consideration in winemaking. Given that fermentation problems tend to arise in the latter third of the process, it is important that the yeast cells completely exhaust the sugar and retain as much vitality as possible in order to avoid additional complications. Choosing the most suitable strain is therefore of immense importance. The use of certain adjuvants in difficult winemaking conditions has the potential to greatly increase residual vitality, as demonstrated in this study by the 40% increase in L4 vitality following the addition of oleic acid. However, the results obtained for vitality, kinetics, major compounds, viability and sugar metabolism using the various fermentation activators tested varied considerably, indicating a high degree of strain-specificity. These results suggest that there is no “universal” activator apart from ascorbic acid and that formulation of a “tailor-made” activator is likely to be required for each commercial yeast strain. Acknowledgements This research was supported by the Regional Project Programme (PII-2109-0178-4509) from the government of Castilla La Mancha.
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