Ultrasonics xxx (2014) xxx–xxx
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Ultrasonic monitoring of malolactic fermentation in red wines D. Novoa-Díaz a, J.M. Rodríguez-Nogales b, E. Fernández-Fernández b, J. Vila-Crespo c, J. García-Álvarez a, M.A. Amer d, J.A. Chávez a, A. Turó a, M.J. García-Hernández a, J. Salazar a,⇑ a
Sensor Systems Group, Department of Electronic Engineering, Universitat Politècnica de Catalunya, Jordi Girona 1-3, 08034 Barcelona, Spain Area of Food Technology, University of Valladolid, Agricultural Engineering College, Av. Madrid 44, 34071 Palencia, Spain c Area of Microbiology, University of Valladolid, Agricultural Engineering College, Av. Madrid 44, 34071 Palencia, Spain d Escola Universitària Salesiana de Sarrià, Passeig Sant Joan Bosco 74, 08017 Barcelona, Spain b
a r t i c l e
i n f o
Article history: Received 4 September 2013 Received in revised form 13 January 2014 Accepted 1 April 2014 Available online xxxx Keywords: Malolactic fermentation Ultrasound Ultrasonic velocity On-line monitoring
a b s t r a c t The progress of malolactic fermentation in red wines has been monitored by using ultrasonic techniques. The evolution of ultrasonic velocity of a tone burst 1 MHz longitudinal wave was measured, analyzed and compared to those parameters of oenological interest obtained simultaneously by analytical methods. Semi-industrial tanks were used during measurements pretending to be in real industrial conditions. Results showed that the ultrasonic velocity mainly changes as a result of the conversion by lactic acid bacteria of malic acid into lactic acid and CO2. Overall, the present study has demonstrated the potential of the ultrasonic technique in monitoring the malolactic fermentation process. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction The metabolism of malic acid, caused by the spontaneous or controlled action of lactic acid bacteria (LAB) and culminating principally in the formation of lactic acid and carbon dioxide (CO2), is known as malolactic fermentation (MLF). After alcoholic fermentation (AF), it is considered the most important biological phenomenon to occur during the production of the majority of red wine and some types of white wine [12]. Although MLF is not technically a mandatory element of winemaking, and was historically described as a capricious and harmful phenomenon that was difficult to understand, its contributions are vital to the development of the sensory characteristics of wine: it reduces acidity while increasing pH levels, adds microbiological stability and improves the organoleptic profile by producing a wide range of colors, flavors and aromas [4]. Thus, little by little this process has become practically indispensable to the winemaking industry. Until recently, winemakers tended to let the LAB act spontaneously, leaving them progress naturally throughout the entire production process. This generally resulted in a lack of control over the malolactic stage and interference in or alteration of the other stages, with uncertain end results that were not always as desired. Thus, there is a clear need to organize and control the action of the ⇑ Corresponding author. Tel.: +34 93 401 56 74. E-mail address: [email protected] (J. Salazar).
LAB in its entirety; a task which remains necessary today and, among other considerations, requires the use and setting up of instruments and tools that facilitate the monitoring of the changes experienced by the wine throughout the entire malolactic process. The task of monitoring the progress of MLF is mostly carried out by measuring the concentration of malic and lactic acid in samples taken from the wine, using methods and techniques that, among other drawbacks, can take a great deal of time to generate results and are not designed for working in real time. Otherwise, if either an improved precision, performance or speed is required then it would result in a dramatic increase in cost. Additionally, the bacterial growth is also monitored, along with other parameters of oenological interest such as pH level, total acidity, turbidity, alcohol content and volatile acidity. In this context, analytical techniques based on ultrasonic waves may be a suitable option to fill the gap in the current monitoring of MLF. Ultrasonic signals are information-rich and appropriate to use in determining the characteristics of liquids, semi-liquids, multiphase systems, optically opaque substances and dense suspensions, as they are able to penetrate containers and chamber walls without suffering significant degradation or being affected by a wide range of conditions specific to each case. They can form part of measurement systems that are non-invasive, hygienic, precise, rapid, low cost and suitable for automated processes [7]. Moreover, given that the propagation properties of ultrasonic waves are sensitive to physical and chemical changes in the propagation medium, it also follows that the alterations in density,
http://dx.doi.org/10.1016/j.ultras.2014.04.004 0041-624X/Ó 2014 Elsevier B.V. All rights reserved.
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compressibility, turbidity and viscosity produced in wine by the actions and development of LAB during MLF could be recorded and defined using techniques involving ultrasonic waves. Consequently, the main objective of this study is to assess the feasibility of the propagation speed of ultrasonic waves to monitor the different physical and chemical changes that occur in red wine during MLF process. By measuring variations in time of flight (TOF) in the wine, the ultrasonic-wave velocity has been calculated throughout the entirety of the malolactic stage for wine of the Tempranillo variety, to be subsequently compared and correlated with the various conventional parameters commonly used for monitoring the process.
2. Material and methods 2.1. Winemaking process The wines of this study were manufactured from Tempranillo grapes from the 2011 harvest in the experimental winery of the Agricultural Engineering College (University of Valladolid, Palencia, Spain) using stainless steel tanks of 100 l. Destemmed-crushed grapes were inoculated with 25 g/Hl of Saccharomyces cerevisiae (Viniferm RVA, Agrovin). The alcoholic fermentation-maceration process was carried out at 18–22 °C and went on for nine days.
Table 1 Analytical characteristics of the red wines before and after the malolactic fermentation (MLF). Before MLF
pH Volatile acidity (g/l acetic acid) Total acidity (g/l tartaric acid) Alcohol degree (%) Turbidity (NTU) Malic acid (g/l) Lactic acid (g/l)
3.58 0.24 4.74 14.2 59 1.53 0.34
After MLF Tank 1
Tank 2
3.82 0.49 3.91 14.2 23 0.03 1.37
3.80 0.41 3.93 14.2 23 0.01 1.33
After this time, the wines were racked off and inoculated with a commercial preparation of Oenococcus oeni (Lalvin VP41Ò, Lallemand, 1 g/Hl), the most desirable LAB for winemaking [13,5], in order to induce the MLF using two tanks. In Table 1 are the chemical characteristics of the wines before and after the MLF. 2.2. Analytical methods Parameters of oenological interest (pH, total acidity, turbidity, alcohol degree, and volatile acidity) were evaluated following official analysis methods [9]. Enzymatic test kits (Novakit, Barcelon, Spain) were employed for the determination of malic and lactic acids from samples. MRS agar was employed for enumeration of O. oeni during the MLF. Serial dilutions were performed in saline solutions, plating duplicates in statistically significant dilutions on MRS [9]. Petri plates were incubated at 28 °C for 5 days. All analysis were carried out in triplicate. 2.3. Technique used to measure the ultrasonic wave velocity An experimental set-up was prepared for the transmission, propagation, reception (in the form of echoes) and processing of ultrasonic signals through red wine (Fig. 1). The main component was a sensor based on the pulse-echo ultrasonic technique (Fig. 2 (top)). An emitter–receiver ultrasound transducer (1 MHz, Panametrics Accuscan A102S-RM) was attached (using Panametrics-NDT gel-type ultrasonic couplant) to one end of a high-density polyethylene (HDPE) buffer rod, the opposite end of which was in direct contact with the wine and separated by the fixed distance d from a wave reflector disc made from the same material as the tank (stainless steel). The working principle is as follows (Fig. 2): the ultrasonic transducer is excited by a sine-wave burst of 7 cycles and 10Vp-p of amplitude (using an Agilent 33250A signal generator), which generates an ultrasonic wave that propagates through the buffer rod. At the buffer rod-wine interface, part of the incident wave is echoed back onto the ultrasound transducer (ECHO11) and the other part is transmitted through the wine until it reaches the reflector
Fig. 1. Experimental set-up.
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Red wine
HDPE buffer rod
AE2 Ai
A0
At
AE1
Ultrasonic transducer emitter-receiver
d
Stainless steel wave reflector
LBR Stainless steel tank
Amplitude A0 ECHO2
AE2 ECHO11
AE11
ECHO12
AE12
t
TOFBR
TOFRed-wine
Fig. 2. Basic configuration of the pulse-echo measurement set-up in a fermentation tank (top) and representation of the signals on the ultrasonic transducer (bottom).
disc; the disc ‘‘echoes’’ the signal back towards the buffer rod-wine interface, where once again part of the signal is reflected towards the wine and the other part (ECHO2) is transmitted via the buffer rod and detected by the ultrasonic transducer. The echoes (Fig. 2 (bottom)) is then acquired by a Tektronix DP02024 oscilloscope, which is configured to an average of 128 with a sampling frequency of 500 MHz, and stored on a computer via a universal serial bus (USB) connection. The system takes two consecutive samples (to be averaged out later) every twelve hours. The resultant signal, which has an SNR of approximately 34 dB, is then stored on a computer for processing. Thus, the propagation velocity for the ultrasonic signals through the red wine cred-wine can be obtained via the following equation:
cred-wine ¼
2d TOF red-wine
ð1Þ
where TOFred–wine corresponds to the TOF of the ultrasonic signal through the wine, that is, the time interval between ECHO11 and ECHO2 in Fig. 2 (bottom). Additionally, Fig. 2 (bottom) shows the presence of another signal (ECHO12). This signal corresponds to the subsequent echo of ECHO11 and was not used to obtain information: however, its presence prior to the reception of ECHO2 is useful in terms of resizing
the buffer rod, in accordance with the findings presented in an earlier paper [2]. This makes it possible to reduce the length of the buffer rod and minimize certain factors (such as weight) while increasing the signal-to-noise ratio (SNR), thereby improving the functionality of the component. The entire set-up is controlled by a Visual Basic application that enables complete manipulation and programming of the instruments’ parameters via computer. Finally, MATLAB is used to automatically process all the stored samples and provide the results of the TOF and ultrasonic velocity. The signals are analyzed using the Phase-Shift method (based on a fast Fourier transform algorithm) that was described in a previous paper [8]. Using this method, we calculated the time variations between consecutive signals that were subsequently used to obtain velocity variations, which in turn allowed us to calculate absolute velocities, on the basis on an initial reference value. Finally, given that, at the moment of introducing the LAB and instigating MLF, red wine can be categorized as a liquid mixture composed of a number of different elements. It is reasonable to assume that the propagation parameters for ultrasonic waves in the medium are a composite of the separate contributions made by each individual element [10]. Therefore, the analysis of the results for ultrasonic velocity presented in this paper is corroborated by previous measurements
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made within the same working group. Specifically, experimental tests were conducted on binary mixtures of base wine with malic acid, base wine with lactic acid, and base wine of varying levels of turbidity [3].
pH
Total acidity 4.8
3.90
3.85
3. Results and discussion
4.6
4.4
pH
malic acid
1.6
4.2 3.70 4.0
3.65
3.8
3.60 0
2
4
6
8
10
12
14
Fermentation time (days) Fig. 4. Evolution of the pH and total acidity during the malolactic fermentation.
Concentration of bacteria
Turbidity
7.0
60
6.6 50
Log UFC/ml
The evolution of the concentration of malic and lactic acids, pH, total acidity, turbidity and bacterial growth during the development of the MLF were studied. In Figs. 3–6, results from one of the fermentation tanks are shown as example only. Similar behavior was found for both fermentation tanks. Fig. 3 shows the monitoring of the concentration of malic and lactic acids during the MLF. During the first two days the changes in the concentration of malic acid were very slight. Maximum conversion of malic acid was observed between the second and the eighth days with a velocity of malic acid degradation of 0.26 g/l/ day. Then, the end of the MLF was achieved, reaching values of malic acid of 0.08–0.03 g/l. Parallel evolution was observed for the lactic acid production, observing slight changes of its concentration during the first days. For this parameter, the maximum velocity of lactic acid production was of 0.25 g/l/day, value very similar to that achieved for the malic acid degradation. Finally, the stationary phase was observed at the eighth day, reaching values of lactic acid of about 1.31 g/l. The theoretical balance of the MLF supposes that 1 g of malic acid is transformed into 0.67 g of lactic acid and 0.33 g of CO2 [14]. Taking this into account, the theoretical degradation of 1.50 g/l of malic acid will be transformed into 1.02 g/l of lactic acid, similar value to that achieved in this trial (1.37–0.34 g/l) (Table 1). In Fig. 4 is shown the evolution of the pH and total acidity during the MLF. Regarding the pH, no changes were observed during the first days. For the fifth day, an increase of the pH was recorded as a result of the decarboxylation of malic acid into lactic acid. Parallel to the increase of the pH, a drastic reduction of the total acidity was observed. The reason of this decline could be explained by three mechanisms (Hidalgo, 2011): (i) the transformation of a stronger acid (malic acid with two acid groups) into a weak acid (lactic acid with an acid group); (ii) the tartaric acid precipitation as potassium bitartrate or calcium tartrate, (iii) the decrease of the solubility of potassium bitartrate. Theoretically, the degradation of 1 g of malic acid provokes a decline of 0.6 g/l of total acidity
3.75
6.2 40
NTU
3.1. Chemical and microbial monitoring of MLF
g/l tartaric acid
3.80
5.8
30 5.4
5.0
20
0
2
4
6
8
10
12
14
Fermentation time (days) Fig. 5. Evolution of the turbidity and bacterial growth during the malolactic fermentation.
lactic acid
1586
Theoretical 2 1585
Ultrasonic velocity (m/s)
g/l
1.2
0.8
Experimental 1584
1583
Theoretical 1
1582
0.4 1581
1580
0
0.0
0
2
4
6
8
10
12
14
2
4
6
8
10
12
14
Fermentation time (days)
Fermentation time (days) Fig. 3. Evolution of the malic and lactic acids during the malolactic fermentation.
Fig. 6. Evolution of the experimental and theoretical ultrasonic velocities during the malolactic fermentation.
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(expressed as tartaric acid) (Hidalgo, 2011). In our case, a decrease of total acidity of 0.83 g/l (4.74–3.91 for tank 1) was observed (Table 1), value very close to the theoretical value (0.90 g/l). Finally, a monitoring of the turbidity and bacteria account was carried out during the MLF (Fig. 5). After the inoculation of the bacteria into the wine, a rapid multiplication of the bacteria was observed with a specific growth rate of 0.174 (expressed as log ufc/ml/day). At the eighth day of fermentation, the stationary phase was achieved. In this phase, no microbial degradation of malic acid was detected. On the other hand, the increase of the bacteria account was not accompanied by a rise of the wine turbidity. On the contrary, it was observed a drop of the wine turbidity from 59 to 23 NTU (Table 1), probably due to a wine clarification process. Pearson’s correlation coefficient and the p-value were analyzed to explore the relationship among the chemical and microbial parameters. Only the statistical significant correlations (p < 0.005) for tank 1 and 2 are summarized in Table 2. Similar results were found for both tanks. It should be noted the high correlation between malic acid and lactic acid (0.994 and 0.986, respectively for tank 1 and tank 2). On the other hand, the pH and total acidity are also highly correlated with the concentrations of malic acid (0.903, 0.995, respectively for tank 1) and lactic acid (0.899, 0.996, respectively for tank 1). No statistical significant correlations were found among turbidity and the other chemical parameters, except for turbidity and bacteria account (0.737 for tank 1 and 0.714 for tank 2). Finally, the bacteria account shows high correlations with malic and lactic acids, pH, and total acidity (0.909, 0.914, 0.796 and 0.898 for tank 1, respectively). As it has been mentioned above, the microbial degradation of malic acid involves the production of lactic acid, with a parallel increase in the wine pH and a decrease in the total acidity. 3.2. Ultrasonic monitoring of malolactic fermentation The evolution of the ultrasonic velocity on the tank 1 during the MLF was measured on-line and represented in Fig. 6
5
(Experimental). Similar behavior was found for the second tank. An increase of the ultrasonic velocity was monitored during the development of the MLF, reaching a stationary phase at approximately the sixth day and following a similar trend to that of some of the conventional parameters. Moreover, as can be seen in Table 2, the ultrasonic velocity was highly correlated with malic acid (0.888 and 0.907 for tank 1 and 2, respectively), and with lactic acid (0.909 and 0.885 for tank 1 and 2, respectively). Significant correlations were also found between the ultrasonic velocity and those variables related with the wine acidity, i.e., pH and total acidity (Table 2). The link between the acidic variables and the ultrasonic velocity can be found mainly in the decarboxylation of the malic acid during the MLF. Finally, a high significant positive correlation was found between ultrasonic velocity and bacteria account (Table 2). This result agrees with previous measurements of the ultrasonic velocity during monitoring of lactic acid fermentation using bacteria [10]. These data showed a linear relationship between bacterial growth and ultrasonic velocity with positive slope. These authors observed that an increase of 6 109 ufc/ml corresponds to a low increase in ultrasonic velocity of 0.1 m/s. During our MLF, the biomass increased from about 105–107 ufc/ml, whereby the incidence of bacterial growth could be negligible. Thus, the observed correlations would mainly indicate the capability of the ultrasonic velocity to detect changes during the MLF. In order to have a better understanding of what happens with ultrasonic velocity during the MLF, the sensitivity of ultrasonic velocity to malic and lactic acids concentrations, hydroalcoholic solutions (12% ethanol) were prepared at different concentrations of malic acid and lactic acid (Fig. 7). These solutions were thermostatated at 22 °C. Ultrasonic velocity decreases with the increase of malic acid concentration at a rate of 0.2 m/s per g/l (for the interval of 0–8 g/l malic acid). Moreover, ultrasonic velocity increases with the lactic acid concentration, providing a rate of 0.3 m/s per g/l (for the interval of 0–8 g/l lactic acid). Therefore, the increase on the ultrasonic velocity monitored during the MLF could be explained, at least in part, by the modification on the concentrations of malic and lactic acids.
Table 2 Pearson’s correlation coefficients and the p-values among ultrasonic velocity (Vel), malic and lactic acid concentrations, pH, total acidity (At) concentration of bacteria (Bact) and turbidity (Tur).a Tanks #1 Vel
Malic
Lactic
pH
At
Tur
Bact
0.888 (0.000)
0.909 (0.000) 0.994 (0.000)
0.754 (0.021) 0.903 (0.000) 0.899 (0.000)
0.872 (0.002) 0.995 (0.000) 0.996 (0.000) 0.909 (0.000)
0.843 (0.000)
0.917 (0.000) 0.909 (0.000) 0.914 (0.000) 0.796 (0.001) 0.898 (0.000) 0.737 (0.004)
0.907 (0.000)
0.885 (0.000) 0.986 (0.000)
0.719 (0.028) 0.863 (0.000) 0.879 (0.000)
0.870 (0.001) 0.987 (0.000) 0.986 (0.000) 0.898 (0.000)
0.844 (0.000)
0.934 (0.000) 0.930 (0.000) 0.888 (0.000) 0.780 (0.003) 0.888 (0.000) 0.714 (0.009)
Malic Lactic pH At Tur #2 Vel Malic Lactic pH At Tur a
In parenthesis are the p-values.
Fig. 7. Ultrasonic velocity variation in hydroalcoholic solutions (12% ethanol) at different malic acid (a) and lactic acid (b) concentrations.
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On the other hand, wine turbidity could also affect the measurement of the ultrasonic velocity since the ultrasonic velocity is significantly correlated with it, 0.843 and 0.844 for tank 1 and 2, respectively. In this sense, García-Álvarez et al. [3] demonstrated a linear reduction in the ultrasonic velocity when there are major variations in the wine turbidity (0.008 m/s per NTU for the interval of 0–500 NTU). As turbidity varied very little during the MLF (36 NTU for both tanks), the influence of this parameter on the ultrasonic velocity was small, of around +0.29 m/s. A theoretical ultrasonic velocity was plotted only taking into account the relative increase rate of 0.2 m/s per g/l of malic acid converted into lactic acid, 0.3 m/s per g/l of lactic acid produced and 0.008 m/s per NTU (see Fig. 6, Theoretical 1). The evolution of experimental and theoretical signals was similar; however, an appreciable difference in the magnitude of ultrasonic velocity (about 2.5 m/s) and a delay of approximately 1.5 days to achieve the stationary phase were observed between both plots. These discrepancies could be attributed to the high complexity of the MLF process compared to the empirical ultrasonic approach, which does consider neither carbon dioxide production nor minor products of MLF (such as acetic acid, diacetyl and acetoin) and other physicochemical transformations (such as salt and polyphenolic precipitations, and release of bacteria cell components). In addition, for the theoretical ultrasonic velocity only have been taken into account the malic and lactic acid concentrations while other factors or parameters were maintained constant, such as temperature and alcoholic degree. During the MLF a production of CO2 from the decarboxylation of malic acid takes place. The gas is dissolved into the wine until the CO2 saturation limit is reached, then CO2 bubbles appear. According to [6], at 20 °C saturation is reached at 0.73 l of CO2 per l of wine. After saturation, the excessive CO2 eludes as bubbles. The sensitivity of ultrasonic velocity to dissolved CO2 was studied in distilled water showing that the velocity increases when increasing the concentration of dissolved CO2 [1] unlike what happens with liquids containing gas bubbles, which possess higher attenuation properties and lower velocity than pure liquids [11]. In particular, coming back to our experiment, for each mole of malic acid, one mole of CO2 is produced. According to the ideal gas law, the fermentation of 1.53 g of malic acid generates 0.25 l of CO2 at the standard reference conditions of temperature and pressure. Therefore, the CO2 produced during the MLF (0.25 l of CO2 per l of wine) is dissolved into the wine, causing the increase of the ultrasonic velocity. However, this increase would be even less than that shown by the experimental ultrasonic velocity plot and therefore cannot be excluded that other factors also influence the ultrasonic velocity increase. One of the main factors to take always into account when working with ultrasound is temperature. In order to justify the difference in values between both plots, a third plot has been represented (see Fig. 6, Theoretical 2), derived from the theoretical data to which has been added the ultrasonic velocity variation due to the effect of temperature variations. The temperature inside the tank 1 was also monitored throughout the MLF process and the ultrasonic velocity variation with temperature (0.865 m/s per °C) in an hydroalcoholic solution containing 14.2% ethanol was determined empirically. As a result, Fig. 6, now the theoretical ultrasonic velocity with temperature compensation shows good agreement with the experimental ultrasonic velocity, thus validating the results. If the plot of the experimental ultrasonic velocity is used to predict the end of the MLF, it is observed that its maximum does not match the end of the reaction but is in advance between 1.5 and 2 days. Probably, this is because the maximum ultrasonic velocity corresponds to the maximum temperature achieved along the reaction but that does not match the end of the reaction. The fact
is that, the maximum temperature corresponds to a high population of bacteria together with the highest level of their metabolic activity but, after the sixth day there is still a residual metabolic activity due to the existing population which continues the development of the MLF to its end. 4. Conclusions Based on the achieved results, we can affirm that ultrasonic velocity in the wine is an adequate parameter for the on-line monitoring of the MLF. Significant correlations were observed between ultrasonic velocity and malic acid, lactic acid and UFC parameters. It was also observed that when the FML comes to an end the ultrasonic velocity stops increasing. In this sense, this technique would be of great help for enologists, improving the management and planning of the staff and also enological practices which take place immediately after the end of MLF, such as the wine racking and sulphiting. Despite the good results obtained in experiments using a single variety of wine, it should be noted that the MLF is a very complex process in which many factors can take part. So, further research with different varieties of wines and fermentation conditions must be done in order to develop an ultrasonic sensor for on-line supervision of the FML process. Acknowledgements This work is financially supported by the Spanish Ministerio de Economía y Competitividad Project with reference DPI200914468-C02-01. Authors would like to thank Beatriz Sanchez Montolío for the analytical work. References [1] T. Becker, M. Mitzscherling, A. Delgado, Ultrasonic velocity – a noninvasive method for the determination of density during beer fermentation, Eng. Life Sci. 1 (2001) 61–67. [2] J. García-Álvarez, M.J. García-Hernández, D.F. Novoa-Díaz, A. Turó Peroy, J.A. Chávez Domínguez, J. Salazar Soler, Resizing buffer rods for ultrasound testing of food products with acoustic noise considerations, Ultrasonics 53 (2013) 294–301. [3] J. García-Álvarez, D.F. Novoa-Díaz, E. Bertran, J.A. Chávez, A. Puig-Pujol, A. Turó, S. Mínguez, M.J. García-Hernández, J. Salazar, Ultrasonic study of red wine properties: preliminary measurements, in: Proceedings of the 34th World Congress of Vine and Wine: The Wine Construction, 20–27th June, Porto, Portugal (2011) pp. 1–7. [4] E. Lerm, L. Engelbrecht, M. du Toit, Malolactic fermentation: the ABC’s of MLF, S. Afr. J. Enol. Viticulture 31 (2010) 186–212. [5] S.-Q. Liu, A review malolactic fermentation in wine – beyond deacidification, J. Appl. Microbiol. 92 (2002) 589–601. [6] Lonvaud-Funel, N. Matsumoto, Le coefficient de solubilité du gaz carbonique dans les vins, Vitis 18 (1979) 137–147. [7] D.J. McClements, Ultrasonic characterization of foods and drinks: Principles, methods and applications, Crit. Rev. Food Sci. Nutr. 37 (1) (1997) 1–46. [8] D. Novoa-Díaz, J. García-Álvarez, J.A. Chávez, A. Turó, M. García-Hernández, J. Salazar, Comparison of methods for measuring ultrasonic velocity variations during ageing or fermentation of food materials, IET Sci. Meas. Technol. 6 (4) (2012) 205–212. [9] OIV, Recueil des Méthodes Internationales d’Analyse des Vins et des mouts, Office International de la Vigne et du vin, Paris, France, 2012. [10] P. Resa, T. Bolumar, L. Elvira, G. Pérez, F. Montero de Espinosa, Monitoring of lactic acid fermentation in culture broth using ultrasonic velocity, J. Food Eng. 78 (2007) 1083–1091. [11] P. Resa, L. Elvira, F. Montero de Espinosa, J. Barcenilla, On-line ultrasonic velocity monitoring of alcoholic fermentation kinetics, Bioprocess Biosyst. Eng. 32 (2009) 321–331. [12] J. Vila-Crespo, J.M. Rodriguez-Nogales, E. Fernández-Fernández, M.C. HernanzMoral, Strategies for the enhancement of malolactic fermentation in the new climate conditions, in: A. Méndez-Vilas (Ed.), Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology, Microbiology Series No. 2. Formatex. Badajoz. (2010). [13] D. Wibowo, R. Eschenbruch, C.R. Davis, G.H. Fleet, T.H. Lee, Ocurrence and Growth of lactic acid bacteria in wine: a review, Am. J. Enol. Viticulture 36 (1985) 302–313. [14] J. Hidalgo. Tratado de Enología, Ed. Mundi-Prensa, Madrid, España, (2011).
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Ultrasonic monitoring of malolactic fermentation in red wines D. Novoa-Díaz a, J.M. Rodríguez-Nogales b, E. Fernández-Fernández b, J. Vila-Crespo c, J. García-Álvarez a, M.A. Amer d, J.A. Chávez a, A. Turó a, M.J. García-Hernández a, J. Salazar a,⇑ a
Sensor Systems Group, Department of Electronic Engineering, Universitat Politècnica de Catalunya, Jordi Girona 1-3, 08034 Barcelona, Spain Area of Food Technology, University of Valladolid, Agricultural Engineering College, Av. Madrid 44, 34071 Palencia, Spain c Area of Microbiology, University of Valladolid, Agricultural Engineering College, Av. Madrid 44, 34071 Palencia, Spain d Escola Universitària Salesiana de Sarrià, Passeig Sant Joan Bosco 74, 08017 Barcelona, Spain b
a r t i c l e
i n f o
Article history: Received 4 September 2013 Received in revised form 13 January 2014 Accepted 1 April 2014 Available online xxxx Keywords: Malolactic fermentation Ultrasound Ultrasonic velocity On-line monitoring
a b s t r a c t The progress of malolactic fermentation in red wines has been monitored by using ultrasonic techniques. The evolution of ultrasonic velocity of a tone burst 1 MHz longitudinal wave was measured, analyzed and compared to those parameters of oenological interest obtained simultaneously by analytical methods. Semi-industrial tanks were used during measurements pretending to be in real industrial conditions. Results showed that the ultrasonic velocity mainly changes as a result of the conversion by lactic acid bacteria of malic acid into lactic acid and CO2. Overall, the present study has demonstrated the potential of the ultrasonic technique in monitoring the malolactic fermentation process. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction The metabolism of malic acid, caused by the spontaneous or controlled action of lactic acid bacteria (LAB) and culminating principally in the formation of lactic acid and carbon dioxide (CO2), is known as malolactic fermentation (MLF). After alcoholic fermentation (AF), it is considered the most important biological phenomenon to occur during the production of the majority of red wine and some types of white wine [12]. Although MLF is not technically a mandatory element of winemaking, and was historically described as a capricious and harmful phenomenon that was difficult to understand, its contributions are vital to the development of the sensory characteristics of wine: it reduces acidity while increasing pH levels, adds microbiological stability and improves the organoleptic profile by producing a wide range of colors, flavors and aromas [4]. Thus, little by little this process has become practically indispensable to the winemaking industry. Until recently, winemakers tended to let the LAB act spontaneously, leaving them progress naturally throughout the entire production process. This generally resulted in a lack of control over the malolactic stage and interference in or alteration of the other stages, with uncertain end results that were not always as desired. Thus, there is a clear need to organize and control the action of the ⇑ Corresponding author. Tel.: +34 93 401 56 74. E-mail address: [email protected] (J. Salazar).
LAB in its entirety; a task which remains necessary today and, among other considerations, requires the use and setting up of instruments and tools that facilitate the monitoring of the changes experienced by the wine throughout the entire malolactic process. The task of monitoring the progress of MLF is mostly carried out by measuring the concentration of malic and lactic acid in samples taken from the wine, using methods and techniques that, among other drawbacks, can take a great deal of time to generate results and are not designed for working in real time. Otherwise, if either an improved precision, performance or speed is required then it would result in a dramatic increase in cost. Additionally, the bacterial growth is also monitored, along with other parameters of oenological interest such as pH level, total acidity, turbidity, alcohol content and volatile acidity. In this context, analytical techniques based on ultrasonic waves may be a suitable option to fill the gap in the current monitoring of MLF. Ultrasonic signals are information-rich and appropriate to use in determining the characteristics of liquids, semi-liquids, multiphase systems, optically opaque substances and dense suspensions, as they are able to penetrate containers and chamber walls without suffering significant degradation or being affected by a wide range of conditions specific to each case. They can form part of measurement systems that are non-invasive, hygienic, precise, rapid, low cost and suitable for automated processes [7]. Moreover, given that the propagation properties of ultrasonic waves are sensitive to physical and chemical changes in the propagation medium, it also follows that the alterations in density,
http://dx.doi.org/10.1016/j.ultras.2014.04.004 0041-624X/Ó 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: D. Novoa-Díaz et al., Ultrasonic monitoring of malolactic fermentation in red wines, Ultrasonics (2014), http://dx.doi.org/ 10.1016/j.ultras.2014.04.004
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compressibility, turbidity and viscosity produced in wine by the actions and development of LAB during MLF could be recorded and defined using techniques involving ultrasonic waves. Consequently, the main objective of this study is to assess the feasibility of the propagation speed of ultrasonic waves to monitor the different physical and chemical changes that occur in red wine during MLF process. By measuring variations in time of flight (TOF) in the wine, the ultrasonic-wave velocity has been calculated throughout the entirety of the malolactic stage for wine of the Tempranillo variety, to be subsequently compared and correlated with the various conventional parameters commonly used for monitoring the process.
2. Material and methods 2.1. Winemaking process The wines of this study were manufactured from Tempranillo grapes from the 2011 harvest in the experimental winery of the Agricultural Engineering College (University of Valladolid, Palencia, Spain) using stainless steel tanks of 100 l. Destemmed-crushed grapes were inoculated with 25 g/Hl of Saccharomyces cerevisiae (Viniferm RVA, Agrovin). The alcoholic fermentation-maceration process was carried out at 18–22 °C and went on for nine days.
Table 1 Analytical characteristics of the red wines before and after the malolactic fermentation (MLF). Before MLF
pH Volatile acidity (g/l acetic acid) Total acidity (g/l tartaric acid) Alcohol degree (%) Turbidity (NTU) Malic acid (g/l) Lactic acid (g/l)
3.58 0.24 4.74 14.2 59 1.53 0.34
After MLF Tank 1
Tank 2
3.82 0.49 3.91 14.2 23 0.03 1.37
3.80 0.41 3.93 14.2 23 0.01 1.33
After this time, the wines were racked off and inoculated with a commercial preparation of Oenococcus oeni (Lalvin VP41Ò, Lallemand, 1 g/Hl), the most desirable LAB for winemaking [13,5], in order to induce the MLF using two tanks. In Table 1 are the chemical characteristics of the wines before and after the MLF. 2.2. Analytical methods Parameters of oenological interest (pH, total acidity, turbidity, alcohol degree, and volatile acidity) were evaluated following official analysis methods [9]. Enzymatic test kits (Novakit, Barcelon, Spain) were employed for the determination of malic and lactic acids from samples. MRS agar was employed for enumeration of O. oeni during the MLF. Serial dilutions were performed in saline solutions, plating duplicates in statistically significant dilutions on MRS [9]. Petri plates were incubated at 28 °C for 5 days. All analysis were carried out in triplicate. 2.3. Technique used to measure the ultrasonic wave velocity An experimental set-up was prepared for the transmission, propagation, reception (in the form of echoes) and processing of ultrasonic signals through red wine (Fig. 1). The main component was a sensor based on the pulse-echo ultrasonic technique (Fig. 2 (top)). An emitter–receiver ultrasound transducer (1 MHz, Panametrics Accuscan A102S-RM) was attached (using Panametrics-NDT gel-type ultrasonic couplant) to one end of a high-density polyethylene (HDPE) buffer rod, the opposite end of which was in direct contact with the wine and separated by the fixed distance d from a wave reflector disc made from the same material as the tank (stainless steel). The working principle is as follows (Fig. 2): the ultrasonic transducer is excited by a sine-wave burst of 7 cycles and 10Vp-p of amplitude (using an Agilent 33250A signal generator), which generates an ultrasonic wave that propagates through the buffer rod. At the buffer rod-wine interface, part of the incident wave is echoed back onto the ultrasound transducer (ECHO11) and the other part is transmitted through the wine until it reaches the reflector
Fig. 1. Experimental set-up.
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Red wine
HDPE buffer rod
AE2 Ai
A0
At
AE1
Ultrasonic transducer emitter-receiver
d
Stainless steel wave reflector
LBR Stainless steel tank
Amplitude A0 ECHO2
AE2 ECHO11
AE11
ECHO12
AE12
t
TOFBR
TOFRed-wine
Fig. 2. Basic configuration of the pulse-echo measurement set-up in a fermentation tank (top) and representation of the signals on the ultrasonic transducer (bottom).
disc; the disc ‘‘echoes’’ the signal back towards the buffer rod-wine interface, where once again part of the signal is reflected towards the wine and the other part (ECHO2) is transmitted via the buffer rod and detected by the ultrasonic transducer. The echoes (Fig. 2 (bottom)) is then acquired by a Tektronix DP02024 oscilloscope, which is configured to an average of 128 with a sampling frequency of 500 MHz, and stored on a computer via a universal serial bus (USB) connection. The system takes two consecutive samples (to be averaged out later) every twelve hours. The resultant signal, which has an SNR of approximately 34 dB, is then stored on a computer for processing. Thus, the propagation velocity for the ultrasonic signals through the red wine cred-wine can be obtained via the following equation:
cred-wine ¼
2d TOF red-wine
ð1Þ
where TOFred–wine corresponds to the TOF of the ultrasonic signal through the wine, that is, the time interval between ECHO11 and ECHO2 in Fig. 2 (bottom). Additionally, Fig. 2 (bottom) shows the presence of another signal (ECHO12). This signal corresponds to the subsequent echo of ECHO11 and was not used to obtain information: however, its presence prior to the reception of ECHO2 is useful in terms of resizing
the buffer rod, in accordance with the findings presented in an earlier paper [2]. This makes it possible to reduce the length of the buffer rod and minimize certain factors (such as weight) while increasing the signal-to-noise ratio (SNR), thereby improving the functionality of the component. The entire set-up is controlled by a Visual Basic application that enables complete manipulation and programming of the instruments’ parameters via computer. Finally, MATLAB is used to automatically process all the stored samples and provide the results of the TOF and ultrasonic velocity. The signals are analyzed using the Phase-Shift method (based on a fast Fourier transform algorithm) that was described in a previous paper [8]. Using this method, we calculated the time variations between consecutive signals that were subsequently used to obtain velocity variations, which in turn allowed us to calculate absolute velocities, on the basis on an initial reference value. Finally, given that, at the moment of introducing the LAB and instigating MLF, red wine can be categorized as a liquid mixture composed of a number of different elements. It is reasonable to assume that the propagation parameters for ultrasonic waves in the medium are a composite of the separate contributions made by each individual element [10]. Therefore, the analysis of the results for ultrasonic velocity presented in this paper is corroborated by previous measurements
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made within the same working group. Specifically, experimental tests were conducted on binary mixtures of base wine with malic acid, base wine with lactic acid, and base wine of varying levels of turbidity [3].
pH
Total acidity 4.8
3.90
3.85
3. Results and discussion
4.6
4.4
pH
malic acid
1.6
4.2 3.70 4.0
3.65
3.8
3.60 0
2
4
6
8
10
12
14
Fermentation time (days) Fig. 4. Evolution of the pH and total acidity during the malolactic fermentation.
Concentration of bacteria
Turbidity
7.0
60
6.6 50
Log UFC/ml
The evolution of the concentration of malic and lactic acids, pH, total acidity, turbidity and bacterial growth during the development of the MLF were studied. In Figs. 3–6, results from one of the fermentation tanks are shown as example only. Similar behavior was found for both fermentation tanks. Fig. 3 shows the monitoring of the concentration of malic and lactic acids during the MLF. During the first two days the changes in the concentration of malic acid were very slight. Maximum conversion of malic acid was observed between the second and the eighth days with a velocity of malic acid degradation of 0.26 g/l/ day. Then, the end of the MLF was achieved, reaching values of malic acid of 0.08–0.03 g/l. Parallel evolution was observed for the lactic acid production, observing slight changes of its concentration during the first days. For this parameter, the maximum velocity of lactic acid production was of 0.25 g/l/day, value very similar to that achieved for the malic acid degradation. Finally, the stationary phase was observed at the eighth day, reaching values of lactic acid of about 1.31 g/l. The theoretical balance of the MLF supposes that 1 g of malic acid is transformed into 0.67 g of lactic acid and 0.33 g of CO2 [14]. Taking this into account, the theoretical degradation of 1.50 g/l of malic acid will be transformed into 1.02 g/l of lactic acid, similar value to that achieved in this trial (1.37–0.34 g/l) (Table 1). In Fig. 4 is shown the evolution of the pH and total acidity during the MLF. Regarding the pH, no changes were observed during the first days. For the fifth day, an increase of the pH was recorded as a result of the decarboxylation of malic acid into lactic acid. Parallel to the increase of the pH, a drastic reduction of the total acidity was observed. The reason of this decline could be explained by three mechanisms (Hidalgo, 2011): (i) the transformation of a stronger acid (malic acid with two acid groups) into a weak acid (lactic acid with an acid group); (ii) the tartaric acid precipitation as potassium bitartrate or calcium tartrate, (iii) the decrease of the solubility of potassium bitartrate. Theoretically, the degradation of 1 g of malic acid provokes a decline of 0.6 g/l of total acidity
3.75
6.2 40
NTU
3.1. Chemical and microbial monitoring of MLF
g/l tartaric acid
3.80
5.8
30 5.4
5.0
20
0
2
4
6
8
10
12
14
Fermentation time (days) Fig. 5. Evolution of the turbidity and bacterial growth during the malolactic fermentation.
lactic acid
1586
Theoretical 2 1585
Ultrasonic velocity (m/s)
g/l
1.2
0.8
Experimental 1584
1583
Theoretical 1
1582
0.4 1581
1580
0
0.0
0
2
4
6
8
10
12
14
2
4
6
8
10
12
14
Fermentation time (days)
Fermentation time (days) Fig. 3. Evolution of the malic and lactic acids during the malolactic fermentation.
Fig. 6. Evolution of the experimental and theoretical ultrasonic velocities during the malolactic fermentation.
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(expressed as tartaric acid) (Hidalgo, 2011). In our case, a decrease of total acidity of 0.83 g/l (4.74–3.91 for tank 1) was observed (Table 1), value very close to the theoretical value (0.90 g/l). Finally, a monitoring of the turbidity and bacteria account was carried out during the MLF (Fig. 5). After the inoculation of the bacteria into the wine, a rapid multiplication of the bacteria was observed with a specific growth rate of 0.174 (expressed as log ufc/ml/day). At the eighth day of fermentation, the stationary phase was achieved. In this phase, no microbial degradation of malic acid was detected. On the other hand, the increase of the bacteria account was not accompanied by a rise of the wine turbidity. On the contrary, it was observed a drop of the wine turbidity from 59 to 23 NTU (Table 1), probably due to a wine clarification process. Pearson’s correlation coefficient and the p-value were analyzed to explore the relationship among the chemical and microbial parameters. Only the statistical significant correlations (p < 0.005) for tank 1 and 2 are summarized in Table 2. Similar results were found for both tanks. It should be noted the high correlation between malic acid and lactic acid (0.994 and 0.986, respectively for tank 1 and tank 2). On the other hand, the pH and total acidity are also highly correlated with the concentrations of malic acid (0.903, 0.995, respectively for tank 1) and lactic acid (0.899, 0.996, respectively for tank 1). No statistical significant correlations were found among turbidity and the other chemical parameters, except for turbidity and bacteria account (0.737 for tank 1 and 0.714 for tank 2). Finally, the bacteria account shows high correlations with malic and lactic acids, pH, and total acidity (0.909, 0.914, 0.796 and 0.898 for tank 1, respectively). As it has been mentioned above, the microbial degradation of malic acid involves the production of lactic acid, with a parallel increase in the wine pH and a decrease in the total acidity. 3.2. Ultrasonic monitoring of malolactic fermentation The evolution of the ultrasonic velocity on the tank 1 during the MLF was measured on-line and represented in Fig. 6
5
(Experimental). Similar behavior was found for the second tank. An increase of the ultrasonic velocity was monitored during the development of the MLF, reaching a stationary phase at approximately the sixth day and following a similar trend to that of some of the conventional parameters. Moreover, as can be seen in Table 2, the ultrasonic velocity was highly correlated with malic acid (0.888 and 0.907 for tank 1 and 2, respectively), and with lactic acid (0.909 and 0.885 for tank 1 and 2, respectively). Significant correlations were also found between the ultrasonic velocity and those variables related with the wine acidity, i.e., pH and total acidity (Table 2). The link between the acidic variables and the ultrasonic velocity can be found mainly in the decarboxylation of the malic acid during the MLF. Finally, a high significant positive correlation was found between ultrasonic velocity and bacteria account (Table 2). This result agrees with previous measurements of the ultrasonic velocity during monitoring of lactic acid fermentation using bacteria [10]. These data showed a linear relationship between bacterial growth and ultrasonic velocity with positive slope. These authors observed that an increase of 6 109 ufc/ml corresponds to a low increase in ultrasonic velocity of 0.1 m/s. During our MLF, the biomass increased from about 105–107 ufc/ml, whereby the incidence of bacterial growth could be negligible. Thus, the observed correlations would mainly indicate the capability of the ultrasonic velocity to detect changes during the MLF. In order to have a better understanding of what happens with ultrasonic velocity during the MLF, the sensitivity of ultrasonic velocity to malic and lactic acids concentrations, hydroalcoholic solutions (12% ethanol) were prepared at different concentrations of malic acid and lactic acid (Fig. 7). These solutions were thermostatated at 22 °C. Ultrasonic velocity decreases with the increase of malic acid concentration at a rate of 0.2 m/s per g/l (for the interval of 0–8 g/l malic acid). Moreover, ultrasonic velocity increases with the lactic acid concentration, providing a rate of 0.3 m/s per g/l (for the interval of 0–8 g/l lactic acid). Therefore, the increase on the ultrasonic velocity monitored during the MLF could be explained, at least in part, by the modification on the concentrations of malic and lactic acids.
Table 2 Pearson’s correlation coefficients and the p-values among ultrasonic velocity (Vel), malic and lactic acid concentrations, pH, total acidity (At) concentration of bacteria (Bact) and turbidity (Tur).a Tanks #1 Vel
Malic
Lactic
pH
At
Tur
Bact
0.888 (0.000)
0.909 (0.000) 0.994 (0.000)
0.754 (0.021) 0.903 (0.000) 0.899 (0.000)
0.872 (0.002) 0.995 (0.000) 0.996 (0.000) 0.909 (0.000)
0.843 (0.000)
0.917 (0.000) 0.909 (0.000) 0.914 (0.000) 0.796 (0.001) 0.898 (0.000) 0.737 (0.004)
0.907 (0.000)
0.885 (0.000) 0.986 (0.000)
0.719 (0.028) 0.863 (0.000) 0.879 (0.000)
0.870 (0.001) 0.987 (0.000) 0.986 (0.000) 0.898 (0.000)
0.844 (0.000)
0.934 (0.000) 0.930 (0.000) 0.888 (0.000) 0.780 (0.003) 0.888 (0.000) 0.714 (0.009)
Malic Lactic pH At Tur #2 Vel Malic Lactic pH At Tur a
In parenthesis are the p-values.
Fig. 7. Ultrasonic velocity variation in hydroalcoholic solutions (12% ethanol) at different malic acid (a) and lactic acid (b) concentrations.
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On the other hand, wine turbidity could also affect the measurement of the ultrasonic velocity since the ultrasonic velocity is significantly correlated with it, 0.843 and 0.844 for tank 1 and 2, respectively. In this sense, García-Álvarez et al. [3] demonstrated a linear reduction in the ultrasonic velocity when there are major variations in the wine turbidity (0.008 m/s per NTU for the interval of 0–500 NTU). As turbidity varied very little during the MLF (36 NTU for both tanks), the influence of this parameter on the ultrasonic velocity was small, of around +0.29 m/s. A theoretical ultrasonic velocity was plotted only taking into account the relative increase rate of 0.2 m/s per g/l of malic acid converted into lactic acid, 0.3 m/s per g/l of lactic acid produced and 0.008 m/s per NTU (see Fig. 6, Theoretical 1). The evolution of experimental and theoretical signals was similar; however, an appreciable difference in the magnitude of ultrasonic velocity (about 2.5 m/s) and a delay of approximately 1.5 days to achieve the stationary phase were observed between both plots. These discrepancies could be attributed to the high complexity of the MLF process compared to the empirical ultrasonic approach, which does consider neither carbon dioxide production nor minor products of MLF (such as acetic acid, diacetyl and acetoin) and other physicochemical transformations (such as salt and polyphenolic precipitations, and release of bacteria cell components). In addition, for the theoretical ultrasonic velocity only have been taken into account the malic and lactic acid concentrations while other factors or parameters were maintained constant, such as temperature and alcoholic degree. During the MLF a production of CO2 from the decarboxylation of malic acid takes place. The gas is dissolved into the wine until the CO2 saturation limit is reached, then CO2 bubbles appear. According to [6], at 20 °C saturation is reached at 0.73 l of CO2 per l of wine. After saturation, the excessive CO2 eludes as bubbles. The sensitivity of ultrasonic velocity to dissolved CO2 was studied in distilled water showing that the velocity increases when increasing the concentration of dissolved CO2 [1] unlike what happens with liquids containing gas bubbles, which possess higher attenuation properties and lower velocity than pure liquids [11]. In particular, coming back to our experiment, for each mole of malic acid, one mole of CO2 is produced. According to the ideal gas law, the fermentation of 1.53 g of malic acid generates 0.25 l of CO2 at the standard reference conditions of temperature and pressure. Therefore, the CO2 produced during the MLF (0.25 l of CO2 per l of wine) is dissolved into the wine, causing the increase of the ultrasonic velocity. However, this increase would be even less than that shown by the experimental ultrasonic velocity plot and therefore cannot be excluded that other factors also influence the ultrasonic velocity increase. One of the main factors to take always into account when working with ultrasound is temperature. In order to justify the difference in values between both plots, a third plot has been represented (see Fig. 6, Theoretical 2), derived from the theoretical data to which has been added the ultrasonic velocity variation due to the effect of temperature variations. The temperature inside the tank 1 was also monitored throughout the MLF process and the ultrasonic velocity variation with temperature (0.865 m/s per °C) in an hydroalcoholic solution containing 14.2% ethanol was determined empirically. As a result, Fig. 6, now the theoretical ultrasonic velocity with temperature compensation shows good agreement with the experimental ultrasonic velocity, thus validating the results. If the plot of the experimental ultrasonic velocity is used to predict the end of the MLF, it is observed that its maximum does not match the end of the reaction but is in advance between 1.5 and 2 days. Probably, this is because the maximum ultrasonic velocity corresponds to the maximum temperature achieved along the reaction but that does not match the end of the reaction. The fact
is that, the maximum temperature corresponds to a high population of bacteria together with the highest level of their metabolic activity but, after the sixth day there is still a residual metabolic activity due to the existing population which continues the development of the MLF to its end. 4. Conclusions Based on the achieved results, we can affirm that ultrasonic velocity in the wine is an adequate parameter for the on-line monitoring of the MLF. Significant correlations were observed between ultrasonic velocity and malic acid, lactic acid and UFC parameters. It was also observed that when the FML comes to an end the ultrasonic velocity stops increasing. In this sense, this technique would be of great help for enologists, improving the management and planning of the staff and also enological practices which take place immediately after the end of MLF, such as the wine racking and sulphiting. Despite the good results obtained in experiments using a single variety of wine, it should be noted that the MLF is a very complex process in which many factors can take part. So, further research with different varieties of wines and fermentation conditions must be done in order to develop an ultrasonic sensor for on-line supervision of the FML process. Acknowledgements This work is financially supported by the Spanish Ministerio de Economía y Competitividad Project with reference DPI200914468-C02-01. Authors would like to thank Beatriz Sanchez Montolío for the analytical work. References [1] T. Becker, M. Mitzscherling, A. Delgado, Ultrasonic velocity – a noninvasive method for the determination of density during beer fermentation, Eng. Life Sci. 1 (2001) 61–67. [2] J. García-Álvarez, M.J. García-Hernández, D.F. Novoa-Díaz, A. Turó Peroy, J.A. Chávez Domínguez, J. Salazar Soler, Resizing buffer rods for ultrasound testing of food products with acoustic noise considerations, Ultrasonics 53 (2013) 294–301. [3] J. García-Álvarez, D.F. Novoa-Díaz, E. Bertran, J.A. Chávez, A. Puig-Pujol, A. Turó, S. Mínguez, M.J. García-Hernández, J. Salazar, Ultrasonic study of red wine properties: preliminary measurements, in: Proceedings of the 34th World Congress of Vine and Wine: The Wine Construction, 20–27th June, Porto, Portugal (2011) pp. 1–7. [4] E. Lerm, L. Engelbrecht, M. du Toit, Malolactic fermentation: the ABC’s of MLF, S. Afr. J. Enol. Viticulture 31 (2010) 186–212. [5] S.-Q. Liu, A review malolactic fermentation in wine – beyond deacidification, J. Appl. Microbiol. 92 (2002) 589–601. [6] Lonvaud-Funel, N. Matsumoto, Le coefficient de solubilité du gaz carbonique dans les vins, Vitis 18 (1979) 137–147. [7] D.J. McClements, Ultrasonic characterization of foods and drinks: Principles, methods and applications, Crit. Rev. Food Sci. Nutr. 37 (1) (1997) 1–46. [8] D. Novoa-Díaz, J. García-Álvarez, J.A. Chávez, A. Turó, M. García-Hernández, J. Salazar, Comparison of methods for measuring ultrasonic velocity variations during ageing or fermentation of food materials, IET Sci. Meas. Technol. 6 (4) (2012) 205–212. [9] OIV, Recueil des Méthodes Internationales d’Analyse des Vins et des mouts, Office International de la Vigne et du vin, Paris, France, 2012. [10] P. Resa, T. Bolumar, L. Elvira, G. Pérez, F. Montero de Espinosa, Monitoring of lactic acid fermentation in culture broth using ultrasonic velocity, J. Food Eng. 78 (2007) 1083–1091. [11] P. Resa, L. Elvira, F. Montero de Espinosa, J. Barcenilla, On-line ultrasonic velocity monitoring of alcoholic fermentation kinetics, Bioprocess Biosyst. Eng. 32 (2009) 321–331. [12] J. Vila-Crespo, J.M. Rodriguez-Nogales, E. Fernández-Fernández, M.C. HernanzMoral, Strategies for the enhancement of malolactic fermentation in the new climate conditions, in: A. Méndez-Vilas (Ed.), Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology, Microbiology Series No. 2. Formatex. Badajoz. (2010). [13] D. Wibowo, R. Eschenbruch, C.R. Davis, G.H. Fleet, T.H. Lee, Ocurrence and Growth of lactic acid bacteria in wine: a review, Am. J. Enol. Viticulture 36 (1985) 302–313. [14] J. Hidalgo. Tratado de Enología, Ed. Mundi-Prensa, Madrid, España, (2011).
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