F Centeno

M Lourenço

M De Fátima Teixeira

P Strehaiano

M Alliet

Fermentation of sparkling wines with immobilized S. cerevisiae cells: comparative study with free cells and influence of temperature fermentation

Keywords: Immobilized yeast, sparkling wine, second fermentation, calcium alginate beads

Introduction

For over 30 years the use of immobilised yeast for sparkling winemaking has been widely tested and discussed amongst the scientific community [START_REF] Yokotsuka | Production of bottle fermented sparkling wine using yeast immobilized in double layer gel beads or strands[END_REF][START_REF] Gòdia | Application of immobilized yeast cells to sparkling wine fermentation[END_REF][START_REF] Fumi | Immobilization of Sacharomyces cerevisiae in calcium alginate gel and its application to bottle-fermented sparkling wine production[END_REF]. The main advantage of immobilized yeast for sparkling winemaking is the suppression of the riddling step which requires costly equipment that takes up a lot of space in the wineries. In spite of all the academic work and potential industrial interest very few processes have successfully passed from the laboratory into an industrial scale. Proenol together with the Laboratoire de Génie Chimique de Toulouse developed an industrial immobilization process using the vibrating nozzle technology. In this process the yeasts are immobilized in double layer beads made of calcium alginate and afterwards are dried in order to increase the shelf-life. The objectives of this work were to evaluate the fermentative performance of the active dried encapsulated yeast in different base wines and to make a comparative study with the fermentation with the free cells.

Material and Methods

Yeast and wine

The strain of S. cerevisiae used for the alcoholic fermentation of the sparkling wines was the QA23. The propagation and immobilization of the yeast was carried out following the industrial protocol developed by Proenol. The immobilization process makes use of the vibrating nozzle technology, which allows for the production of double layered calcium alginate beads. After immobilization the beads were dried in order to guarantee a minimum of 6 months shelf-life. Three different base wines (MF, TN and MM) were supplied by Murganheira. The ethanol content and pH of each base wine was: MF -11.5%, 3.05; TN -11.2%, 2.94; MM -11.7, 2.93. Sugar was added to the wines in order to have a final concentration of 24g/L. Prior to bottling the wines were filtered through 0,45 m cartridges. The bottles were then inoculated with the encapsulated yeast (1,2g/bottle which corresponds to 4 -5.10 6 cel/mL) or with the free yeast (2-4. 10 6 cel/mL).

Fermentation and tasting

The fermentations with the immobilized yeast were carried out at 14ºC and 10ºC in controlled temperature chambers. With the free yeast only fermentations at 14ºC were done. The fermentation was assessed by periodic measurements of pressure and sugars in the bottles. 3 months after the tirage several physical-chemical parameters were evaluated: ethanol, sugar, glycerol, volatile acidity, total polysaccharides. Four months after the end of the second fermentation the wines were tasted by a trained panel of 13 professional tasters from the Comissão de Viticultura da Região dos Vinhos Verdes (Portugal).

Analyses

The pressure generated by the alcoholic fermentation inside the bottles was measured with pressure gauges. The sugars (glucose and fructose), L-malic acid and glycerol were determined with an automatic enzymatic analyzer from Horiba (ABX Pentra 400). The enzymatic kits were provided by Megazyme, reference K-FRUGL for sugars, K-LMALR for the L-malic acid and K-GCROL for the glycerol. Alcohol concentration was evaluated by ebulliometry. Volatile acidity and pH were determined using recommended methods (International Organization of Vine and Wine, OIV-2009). The determination of total polysacharydes was done by the Instituto de Fermentaciones Industriales -CSIC (Madrid, Spain) according to their internal procedures. The viability of the yeast cells inside the beads was assessed by dissolving the calcium alginate in a citrate solution, and then by staining the cells with methylene blue and counting it in an optical microscope.

Results and Discussion

The fermentation profile of the base wines inoculated with the immobilized yeast (figure 1) shows that different wines can have different profiles even with the same yeast batch. The wines TN and MF had a very similar progression while the wine MM had a slower pressure evolution and accordingly, sugar consumption. The fermentation was considered finished when the pressure was stable and the sugar concentration was below 2 g/L. For the TN and MF wines it took approximately 80 days to reach the end of the fermentation, for the MM the time needed to achieve the end was 110days. 2. From the beginning of the fermentation there's in all the wines a small linear decrease of the cells viability. When the pressure reaches 5 to 6 bar the cell viability which was is until that moment superior to 80%, suffers a sharp decrease. This lost of viability is coincident with the exhaustion of the sugars. This results shows that during the fermentation the immobilized cells can have high viability (>80%) but once the fermentation reaches an end there's a quick loss of viability.

Figure 2 -Evolution of immobilized yeast viability as a function of the pressure inside the bottle

The pressure profiles of the free and immobilized cells are presented in figure 3. They are very similar in the wines MF and MM. This result was not expected since immobilized cells tend to ferment slower due to mass transfer limitations, especially in static conditions as it is the case of sparkling wine fermentation. On the other hand the wine MF shows very different fermentation profiles between the free and the immobilized cells. The pressure at the end of the fermentation is superior for the free yeast and the residual sugars were less then 0.5 g/L in both cases, this suggests there was an over dosage of sugar in the wine inoculated with the free yeast. Overall these results show that the fermentation with the immobilized yeast can be, in certain cases, as fast as with the free yeast. base wines (MF, TN and MM) Analyses of alcohol and volatile acidity made 3 months after the tirage do not show any significant difference between the wine fermented with the immobilized and with the free yeast. These results are presented in figure 4. The same was observed for the L-malic acid and glycerol concentration (data not shown). Total polysaccharide analysis (figure 5) shows that the fermented wines tend to have the same or higher concentrations than the base wine. It is also possible to see that the wines fermented with the immobilized yeasts have the same or higher concentration of polysaccharides than the wines fermented with the free yeast. with immobilized and free yeast Finally a comparison of the fermentation profile at 14ºC and at 10ºC (figure 7) shows that a decrease in the temperature has a strong influence on the kinetics. The fermentation at 14ºC had 0.4 g/L of sugar at day 95, while the bottles fermenting at 10ºC still had 6,5g/L of sugar after 180 days. Raising the dose of beads per bottle from 1.2g to 1.6g and 1.8g allows, for the same conditions, a reduction of the fermentation time to 140 days. The residual sugars at that time were 1.8g per bottle, which indicates that there's no advantage of using doses superior to 1.6g.

Conclusion

The results showed that the base wine exerts the same effect in the kinetics of the free and of the immobilized yeast. The cells viability decreased slowly during the fermentation to values around 80% but once the pressure is around 5 to 6 bar there's a sharp loss of viability. The physicalchemical parameters analysed at the end of the fermentation were very similar for free and immobilized yeast. The taste also didn't show significant differences between the wines fermented with free or immobilized cells. The trials also showed that fermentations at 10ºC are significantly slower than the ones at 14ºC. When using immobilized yeast this difference can be easily attenuated if the dose of beads per bottle is raised. Nonetheless we observed that above a certain dose there's no improvement in the kinetic. These results show that the use of the immobilized yeasts in double layer calcium alginate is a valide alternative to the traditional second bottle fermentation with the free cells for the industrial production of sparkling wines.

Figure 1 -

 1 Figure 1 -Follow up of the sparkling wine fermentation with immobilized cells for the 3 base wines a) Pressure evolution and b) sugar consumption as a function of fermentation time The evolution of the cells viability as a function of the pressure generated inside the bottle is presented in figure2. From the beginning of the fermentation there's in all the wines a small linear decrease of the cells viability. When the pressure reaches 5 to 6 bar the cell viability which was is until that moment superior to 80%, suffers a sharp decrease. This lost of viability is coincident with the exhaustion of the sugars. This results shows that during the fermentation the immobilized cells can have high viability (>80%) but once the fermentation reaches an end there's a quick loss of viability.

Figure 3 -

 3 Figure 3 -Pressure evolution of the fermentation with immobilized and free cells for the 3base wines (MF, TN and MM) Analyses of alcohol and volatile acidity made 3 months after the tirage do not show any significant difference between the wine fermented with the immobilized and with the free yeast. These results are presented in figure 4. The same was observed for the L-malic acid and glycerol concentration (data not shown). Total polysaccharide analysis (figure5) shows that the fermented wines tend to have the same or higher concentrations than the base wine. It is also possible to see that the wines fermented with the immobilized yeasts have the same or higher concentration of polysaccharides than the wines fermented with the free yeast.

Figure 4 -

 4 Figure 4 -Concentration of a) alcohol b) volatile acidity measured 3 months after the tirage in the three base wines (MF, TN and MM) fermented with immobilized and free cells

Figure 6 -

 6 Figure 6 -Results of sensorial analysis for the sparkling wines (MF, TN and MM) producedwith immobilized and free yeast Finally a comparison of the fermentation profile at 14ºC and at 10ºC (figure7) shows that a decrease in the temperature has a strong influence on the kinetics. The fermentation at 14ºC had

Figure 7 -

 7 Figure 7 -Pressure evolution for fermentations at 14ºC and 10ºC with immobilized yeast


Maciej Jaroszewski
email: maciej.jaroszewski@pwr.edu.pl

Abderrahmane Beroual
email: abderrahmane.beroual@ec-lyon.fr

Paweł Rózga
email: pawel.rozga@p.lodz.pl

Electrostatic charging tendency of natural transformer ester -impact of the surface state of solid on electrification currents

Keywords: flow electrification, natural ester, transformer

This paper deals with the static electrification phenomena of natural ester for the use in power transformers. The spinning disk method was used in the study. The rate of oil flow on the surface of solid material is simulated by the rotational speed of the disk. The dependency of the electrification current of the natural ester versus the flow velocity of the oil and the type of roughness of the surface of the solid material has been investigated; the different roughnesses are obtained by making different scratches' patterns on the rotating disc. As with mineral oils, the static electrification current of natural ester oil increases with the flow rate and/or the surface roughness.

I. INTRODUCTION

When a liquid comes into contact with a solid dielectric, as a result of physicochemical reactions at the interphase, the originally electrically neutral system becomes polarized. A socalled double charge layer appears. The phenomenon of space charge formation on the surface of a solid body and in the subsurface layer of a liquid was initially described by Helmholtz. This model was then further developed by Gouy and Chapman, who took into account the fact that the movement of ions in a liquid creates a diffusion layer of finite thickness. Stern's model, which takes into account the existence of both Helmholtz and Gouy-Chapman layers, is based on the two models mentioned above. A schematic representation of the double layer structure according to the models mentioned above can be found in [START_REF] Jaroszewski | Electrification currents measurements of natural ester obtained by rotating disc method[END_REF].

When there is movement of the oil in relation to the insulation system and structural elements, caused by the transformer cooling system, the ions of the diffusion layer move with the oil flow, generating thence the electrification phenomenon. The development and intensity of this phenomenon are influenced by many factors, such as the type of oil and its flow rate, the type of solid material with which the oil comes into contact and its surface condition. To this must be added other factors which change during the operation of the transformer, in particular the aging of oil and the fluctuations of the water content.

The history of static electrification resulting from the forced flow of oil in power transformers began in the 1970s, when transformer manufacturers and their users began to suspect the phenomenon of static electrification as being at the origin of unexplained failures of power transformers.

The static electrification processes caused by the flow of oil inside power transformers may lead to the initiation of partial discharges and breakdown of the transformer insulation. Failures of power transformers filled with mineral oil caused by this phenomenon have been known and studied since the mid-twentieth century. Currently, due to the increased requirements for environmental protection, units filled with transformer organic esters occupy a growing share in the production of transformers. Physical phenomena at the solid-liquid interface are obviously dependent on the physicochemical properties of the liquid. Thus, the level of static electrification depends on the kind of insulating oils. Therefore, the use of a new type of insulating oil requires among others the knowledge of its properties with respect to static electrification.

During the expertise of damaged transformers, the presence of traces caused by electrical discharges was revealed. These were traces of electric trees, burned holes or the presence of carbon on the internal elements of solid insulation. Thus, one could assume that the flow electrification process occurring at the interface between oil and solid insulator seems to be responsible for these events [START_REF] Shimizu | Electrostatics in power transformers[END_REF][START_REF] Crofts | The static electrification phenomena in power transformers[END_REF][START_REF] Peyraque | Static electrification induced by oil flow in power transformers[END_REF]. These experiments made it possible to design/imagine a hypothetical transformer failure scenario [START_REF] Kędzia | Threat to the insulation of power transformers caused by static electricity[END_REF]. It includes the electrification processes caused by the flow of oil resulting consequently in:

• generation of space charge

• transferring the space charge in the direction of the forced oil flow

• accumulation of space charge in privileged places of the transformer structure

• deformation of the electric field distribution due to the accumulated space charge

• the possibility of the appearance of a constant component of the electric field

• initiation and development of various forms of partial discharges from places of accumulation of spatial charge

• catastrophic electric breakdown under the action of AC operating voltage.

The problem of static electrification of mineral transformer oils is quite well recognized and documented in literature. Despite this, current articles on this phenomenon can still be found in recognized international journals [START_REF] Talhi | Impact of Local Overheating and Electrical Discharge on the Streaming Electrification of Transformer Oil[END_REF][START_REF] Lin | Flow Electrification Characteristics of Typical Plane Oil-Pressboard Insulation Under AC/DC Voltage[END_REF][START_REF] Zdanowski | The analysis and selection of the spin-ning disk system parameters for the measurement of static electrification of insulation oils[END_REF]. This proves that not all problems related to the static electrification of mineral oils have been completely solved. In the case of a new class of transformer oils based on organic esters, with a chemical structure different from that of mineral oils, this issue is still open and requires intensive research [START_REF] Zdanowski | Streaming electrification of mineral insulating oil and synthetic ester MIDEL 7131[END_REF][START_REF] Beroual | Investigation on Static Electrification Phenomenon of Ester Liquids and Mineral oil[END_REF][START_REF] Zdanowski | Research on the electrostatic properties of liquid dielectric mixtures[END_REF].

The study of the effect of the surface roughness of a dielectric (pressboard, textolite) disk on the electrification of biodegradable transformer oil showed that increasing the surface roughness resulted in higher current values compared to disk with a smoother surface [START_REF] Jaroszewski | Electrification currents measurements of natural ester obtained by rotating disc method[END_REF]. However, it is problematic in this case to make the pressboard surface roughness with defined patterns. Earlier works on the phenomenon of flow electrification in a system with a rotating disk showed that the electrification currents measured in a system with a conductive disk exhibit very similar behaviour as in the case of disks made of dielectric materials [START_REF] Jaroszewski | Electrification currents measurements of natural ester obtained by rotating disc method[END_REF][START_REF] Zdanowski | The analysis and selection of the spin-ning disk system parameters for the measurement of static electrification of insulation oils[END_REF][START_REF] Zdanowski | Influence of Composition of Dielectric Liquid Mixtures on Electrostatic Charge Tendency[END_REF]. In particular, it should be noted that the use of a metal electrode in the spinning disk method allows to demonstrate the differences in the intensity of electrification for fresh and aged oils [START_REF] Zdanowski | Electrostatic Charging Tendency Analysis Concerning Retrofilling Power Transformers with Envirotemp FR3 Natural Ester[END_REF] or for mixtures of different oils [START_REF] Zdanowski | Streaming Electrification of Nycodiel 1255 Synthetic Ester and Trafo EN Mineral Oil Mixtures by Using Rotating Disc Method[END_REF]. The study of the flow electrification of dielectric liquids, in particular transformer oils, can therefore also be carried out using metal electrodes. It is not without significance here that roughening the electrode surface with a specific pattern is possible for metal electrodes.

This paper deals with the static electrification phenomena of natural ester through the electrification current using a rotating disk method. The electrification current is especially investigated versus the flow velocity of the oil and the type of roughness to the surface of the solid material; the different roughness are obtained by making different scratches' patterns on the rotating disc.

II. MEASURING SYSTEM

The experiments were performed using the rotating disk method. This method involves immersing a rotating electrode in a conductive vessel filled with transformer oil. The rotation of the electrode disk with respect to the oil causes the charge of the bilayer to break and flow to the walls of the vessel. The measuring vessel is grounded through a pico-ammeter and the current flow is recorded in the external circuit. The schematic diagram and photo of the circuit are given in Figures 1 and2, respectively. Fig. 1. Scheme of the system for measuring static electrification of oils with the spinning disk method: (1) measuring vessel, (2) oil, (3) target, (4) Teflon supports, (5) motor, (6) Teflon coupling / electrical separator, and (7) Faraday's cage.

Photo of test cell

The disc rotational speed was regulated by changing the voltage supplying the motor. The rotational speed was measured with a DMT 21 tachometer from POLMATIK with an accuracy of ± 0.2%, using an optical converter. The measurement of the current generated by the rotating disk was carried out using a KEITHLEY type 485 pico-ammeter with an accuracy of ± 0.2%.

Measurements were made for target electrodes made of copper. Shields with a smooth and rough surface were used. The smooth reference electrode (electrode A) was cut out of the rolled sheet, not subjected to additional treatment. Rough electrodes have undergone additional surface treatment by: Electrode B -applying scratches by rotary polishing with 600 grit sandpaper; electrode C -applying scratches by longitudinal polishing with 600 grit sandpaper; electrode D -applying scratches by radial polishing with 600 grit sandpaper. Fig. 6. Electrification current for a smooth electrode (A) and for electrode (B) with additional surface treatment by applying scratches by radial polishing. In the cases of rotary and longitunal polishing the increase The general trend observed from these figures is that increasing the disc roughness results in an increase in the electrification current. In the cases of rotary and longitudinal polishing, the increase is much less than in the case of polishing. This difference is all the more important as the rotational speed of the disc increases. These results confirm those reported elsewhere [START_REF] Jaroszewski | Electrification currents measurements of natural ester obtained by rotating disc method[END_REF][START_REF] Zdanowski | The analysis and selection of the spin-ning disk system parameters for the measurement of static electrification of insulation oils[END_REF].

As for mineral oils, the electrification current in transformer organic esters increases with the increase of the rotational speed of the disc. However, the obtained current values are higher than those in mineral oils [START_REF] Kędzia | Electrification Current in the Spinning Disk System[END_REF].

IV. CONCLUSION

The disk surface roughness has a similar effect on the static electrification of biodegradable transformer oil as it does on mineral oils. Increasing the surface roughness of the discs results in higher currents compared to disks with a smoother surface. The increase in electrification current in tests performed with electrodes with modified surface roughness may be related both to the increase in the specific surface of the electrode (disk) and the appearance of "thresholds" that may support the detachment of the diffusion layer charges from the surface of the solid material.

Fig. 3

 3 Fig. 3 shows the diagram of the surface treatment of electrodes.

Fig. 3 .

 3 Fig. 3. Diagram of the surface treatment of the electrodes Before the tests, each disc was cleaned in order to remove any contamination from the surface. Five electrification current characteristics were made as a function of rotational speed ω at intervals of at least 20 minutes. From the data obtained in this way, mean values were calculated and the characteristics presented in the figures were plotted.

Figures 4

 4 Figures 4 to 6 show the evolution of the electrification current for the three types of surface treatment.

Fig. 4 .Fig. 5 .

 45 Fig. 4. Electrification current for a smooth electrode (A) and for electrode (B) with additional surface treatment by applying scratches by rotary polishing.

ACKNOWLEDGMENT M.J. thanks Mr. Partyk Widoniak for his help in performing measurements.