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Use of shear wave elastography for monitoring enzymatic milk coagulation
Journal of Food Engineering 136 (2014) 73–79
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Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Use of shear wave elastography for monitoring enzymatic milk coagulation E. Budelli a,c, M. Bernal b, P. Lema a, M. Fink b, C. Negreira c, M. Tanter b,1, J.L. Gennisson b,⇑,1 a
Instituto de Ingeniería Química, Facultad de Ingeniería, Universidad de la República, Julio Herrera y Reissig 565, CP 11300 Montevideo, Uruguay Institut Langevin – Ondes et Images, ESPCI ParisTech, PSL Research University, CNRS UMR 7587, INSERM U979, 1 rue Jussieu, 75005 Paris, France c Laboratorio Acústica Ultrasonora, Instituto de Física, Facultad de Ingeniería, Universidad de la República, Iguá 4225, CP 11400 Montevideo, Uruguay b
a r t i c l e
i n f o
Article history: Received 4 September 2013 Received in revised form 6 March 2014 Accepted 19 March 2014 Available online 30 March 2014 Keywords: Shear wave elastography Milk coagulation Rheological properties Ultrafast ultrasound imaging
a b s t r a c t In the manufacturing of cheese, the cutting of the curd is an essential step which depends on the firmness of the curds and significantly affects the yield of the cheese and its quality. In this work, we present a technique to measure elastic properties of the curd during coagulation that could be used to quantitatively determine the cutting time. The technique uses ultrasound to generate and measure shear waves. These waves do not propagate in liquids and their velocity of propagation depends on the viscoelastic characteristics of the medium. Hence, they can be used to identify the beginning of coagulation and subsequently to monitor the evolution of the coagulum firmness. Our results showed this technique is sensitive to changes of the medium structure during coagulation. It also proved reproducible and sensitive to different coagulation conditions. Therefore this technique can be used to develop a system suitable for the dairy industry. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Milk coagulation is the process in which liquid milk is transformed into a viscoelastic semi-solid coagulum by specific enzymes called chymosins. These enzymes act upon milk’s j-caseins, partially fragmenting these molecules. This phenomenon causes a change in the micelle surface charge, increases their hydrophobicity and promotes their aggregation (Koc and Ozer, 2008). Micelle aggregation is responsible for an increase in milk’s viscosity. These characteristics are important in cheese making because they determine the coagulum’s cutting point (Fox et al., 2000). The analysis of the different stages in the formation of macromolecular networks is of major importance, since understanding the structure and properties of gels requires the understanding of their organization process (Nassar et al., 2001). Milk gelation is a major step in the cheese making process; determining the end of the coagulation stage (cutting time) affects the yield of the cheese production process significantly. If the curd is cut when the coagulum is too soft, the loss of fat and curd fines decreases the cheese yield sharply. If the curd is too firm, syneresis is retarded, resulting in a high moisture cheese, which can require a longer ripening stage (Benedito et al., 2002). The cutting time is generally predetermined ⇑ Corresponding author. Tel.: +33 1 80 96 30 79; fax: +33 1 80 96 33 55. 1
E-mail address: [email protected] (J.L. Gennisson). These authors are co-last authors.
http://dx.doi.org/10.1016/j.jfoodeng.2014.03.026 0260-8774/Ó 2014 Elsevier Ltd. All rights reserved.
based on previous batches information, which makes it difficult to maximize the yield for every particular batch. Therefore, a methodology that allows the assessment of the cutting time in real time is needed in order to standardise cheese yield and its physical and sensory characteristics (Koc and Ozer, 2008). In addition, the change in coagulation time is useful in comparing strengths of different enzyme solutions (Gan et al., 2006). However, the current on-line systems used in the prediction of the cutting time, are generally more sensitive to changes occurring during the gelation process rather than around the cutting point. This introduces a source of error in those factors which influence the rate of coagulum firming but may not influence the rate of gel formation in a similar fashion (O’Callaghan et al., 2000). Over the years, ultrasonic sensing devices have been used for coagulation monitoring because they provide non-destructive, quick and low cost measurements (Klandar et al., 2007). The most frequently used parameters in ultrasonic measurements are the ultrasonic velocity and attenuation of compressional waves (Wang et al., 2007). Several authors have successfully used low intensity ultrasonic measurements for studying the structural changes involved in milk coagulation and monitoring. Experiments have been carried out in casein solutions (Wang et al., 2007), reconstituted milk (Ay and Gunasekaran, 1994; Benguigui et al., 1994; Bakkali et al., 2001; Nassar et al., 2001, 2004) and whole milk (Koc and Ozer, 2008). These authors define different parameters to monitor the coagulation process. On the one hand, Ay and
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Gunasekaran (1994) define the coagulation time as the point in the ultrasound attenuation curve where there is a change in the slope; and the cutting time as a function of the coagulation time. On the other hand, Nassar et al. (2001, 2004) define the clotting time as the time when the ultrasound velocity starts to increase. A more recent approach to study gelation processes uses shear waves to characterize viscoelastic properties in coagulating blood (Bernal et al., 2012, 2013; Gennisson and Cloutier, 2006). The technique used, so called Supersonic Shear Imaging, uses ultrasound ultrafast imaging (until 20,000 frames/s) to catch propagation of shear wave generated by ultrasonic radiation force in real time (Bercoff et al., 2004). Since shear wave speed (VS) is directly related to the shear modulus (l) by: l = qV2S with q the density, the viscoelastic properties of the investigated medium can be retrieved. This approach could be useful for monitoring the milk coagulation process. As shear waves do not propagate in liquids, they can be used to identify the beginning of coagulation (time at which the shear waves begin to propagate in the milk sample) and subsequently the technique could be used to monitor the evolution of the coagulum firmness. In this work we implemented the Supersonic Shearwave Imaging (SSI) technique, within the milk sample. We further assessed the suitability, sensitivity and reproducibility of this technique for monitoring enzymatic milk coagulation.
2. Methods 2.1. Milk samples Pasteurized whole milk was purchased from local market. In order to increase the amount of scatters, cellulose (Cellulose powder, Type 20, Sigma Chemical, St. Louis, MO, USA) was added in a concentration of 10 g/l to the milk samples before the experiment. Coagulation was initiated in the milk samples by adding calcium chloride (CaCl2, number C 5080, Sigma Chemical, St. Louis, MO, USA) and rennet (active chymosin P520 mg/L, COOPER, Melun, France), which was purchased from a local pharmacy. In order to test the sensibility of the technique to different coagulation conditions, the concentrations of CaCl2 and rennet added to the milk samples, were varied according to Table 1. During experiments, samples were held in 150 ml beakers and placed in a water bath at 35 °C (Fig. 1). The experimental procedure was as follows. 150 ml of milk with cellulose and CaCl2 were placed in the plastic beaker. The solution was preheated up to 30–32 °C and put inside the water bath at 35 °C in order to guarantee a stable temperature. While the milk reached 35 °C, the transducer was placed and aligned as shown in Fig. 1a. Once the sample achieved the desired temperature, the generation and acquisition of the shear waves was started. After 4 min of acquisition, coagulation was initiated by adding the ren-
Table 1 Rennet and CaCl2 concentrations used in the experiments. The signs adjacent to the values, symbolize the concentration levels; ‘‘+’’ and ‘‘‘‘ being high and low concentrations respectively, while ‘‘+ +’’ and ‘‘ ‘‘ correspond to doubling or cutting in half the concentrations. This notation is used in following graphs to simplify the notation. Concentrations
CaCl2 (g/l)
Rennet (ml/l)
1 2 3 4 5 6
0.100 0.050 0.100 0.050 0.200 0.025
3.00 3.00 1.50 1.50 6.00 0.75
(+) () (+) () (+ +) ( )
(+) (+) () () (+ +) ( )
net. Measurements for ultrasound images and shear wave propagation were recorded every minute for 120 min. 2.2. Rheological measurements Rheological measurements were made using a rheometer Anton Paar Physica 301 equipped with a peltier temperature control system. Oscillatory tests were made in bob cylinder geometry at 35 °C. Strain of 2% and 1 Hz was applied every 5 min. Data acquisition was started immediately after rennet addition. Storage and loss modulus (G0 , G00 ) were recorded and compared with results obtained by Supersonic Shear Imaging. As a preliminary study, the effect of cellulose addition in the coagulation process was evaluated. Evolution of G0 and G00 during coagulation of samples containing cellulose showed no significant differences with the results obtained for samples without cellulose addition. 2.3. Supersonic Shear Imaging and elasticity maps Supersonic Shear Wave Imaging (SSI) was performed by using an ultrafast ultrasound device (V12, Supersonic Imagine, Aix en Provence, France) which was the research prototype of a clinical commercial ultrasound device (Aixplorer, Supersonic Imagine, Aix en Provence, France). This prototype is a fully programmable system (256 channels, 60 MHz sampling rate) driving 8 MHz central frequency ultrasonic probe (256 piezoelectric elements, 0.2 mm pitch). SSI is an ultrasound technique that allows to recover elastic maps of biological tissues by measuring the shear wave speed which is directly related to stiffness by the relationship, E = 3qV2S (where E is the Young’s modulus, q the density of the medium and VS the shear wave speed, Tanter et al., 2008). It is based on two concepts: the generation of shear waves within the media, the monitoring of the propagation of such shear waves (Fink and Tanter, 2010). Both concepts are achieved by using an ultrafast ultrasound scanner classically used in medical imaging for breast cancer diagnosis (Berg et al., 2012; Tanter et al., 2008), liver fibrosis staging (Bavu et al., 2011) or muscle stiffness assessment (Gennisson et al., 2010). First the shear waves are generated by focusing ultrasound within the milk (Fig. 1b) along one line on the z axis during 100 ls. Then by using an ultrafast imaging mode, a movie of the propagating shear wave is acquired in a very short time (some tenth of ms) (Fig. 1c). Therefore from the shear wave movie the speed of shear waves is calculated giving access to the stiffness of the medium. In the present paper everything is presented in terms of shear wave speed. Finally this acquisition is done. At last this acquisition mode is repeated every minute or more depending the experiments needs. Dimensions of the field measured at each acquisition were 30.0 mm depth by 51.2 mm wide and the spatial resolution of the SSI technique to differentiate the elasticity properties was in the order of 1 mm (Figs. 2 and 3). At last, in order to show direct comparison between SSI and rheological measurements, parallel monitoring of coagulation using both techniques was made. 3. Results Changes in the propagation of the shear wave during coagulation are shown in Fig. 2. As the milk starts to coagulate (between minute 5 and 15) shear wave begin to propagate in the medium, and as the milk becomes firmer, the speed of the waves increases. This is directly visible on the images since the shear wave front (represented by the blue line along the z axis Fig. 2) is at a bigger distance from the source (represented by the red line along the z axis) as a function of the coagulation time for each acquisition
E. Budelli et al. / Journal of Food Engineering 136 (2014) 73–79
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Fig. 1. (a) Picture of the ultrasound setup. (b) and (c) Schematic of the shear wave experiment. The shear wave is first generated by ultrasound radiation force and then imaged by using an ultrafast ultrasound imaging mode.
Fig. 2. Shear wave propagation at different coagulation time points and at different times during the acquisition. The first row shows the wave propagation at minute 0, where Panels B, C and D represent different acquisition times (3, 15 and 30 ms). Panel A shows the corresponding B mode image at this coagulation time point. Similarly, the second row shows the wave propagation series after 6 min of coagulation. In the same fashion, the third and fourth row show the propagation at 30 and 120 min of coagulation. The dotted white lines represent the bottom of the plastic container in which the milk samples were hold. On the third colon the red line represents the shear wave source and the blue line the shear wave front. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. Two dimension shear velocity maps. The first row shows the elasticity maps at different time points during milk coagulation (0, 6, 30 and 120 min). The white rectangle shown in Panels B, C and D, represents the area analyzed for Fig. 4. The bottom row shows the corresponding ultrasound images at the same time points. The dotted white line represents the bottom of the plastic container.
time. At time zero, it is possible to see that there is no propagation of the shear waves due to the liquid state of the medium (shear waves do not propagate in liquids). In the second row (minute 6 of coagulation) it can be observed that the wave only propagates a few mm, even 30 ms after the push. The 3rd and 4th rows show more advanced coagulation times (30 and 120 min, respectively), and it is evident in that the wave propagates longer distances at these stages (Panels L and P). Panels A, E, I and M show the Bmode images at 0, 6, 30 and 120 min, respectively. The dotted lines in all the figures represent the bottom of the plastic beaker. The 2D shear wave velocity maps are shown in Fig. 3. As mentioned in the methods section, the shear wave speed was calculated using the shear waves generated by the ultrasound radiation force at elements 64, 128 and 192. The white arrows in Fig. 3B show the location of the pushes. Three excitations were required to characterize the whole 2D field. Panel A shows no data since the shear waves cannot propagate in liquids. The beginning of the coagulation process can be observed in Panel B, where only part of the 2D field has information due to the limited propagation of the shear waves. The rest of the field was discarded because the correlation algorithms did not provide reliable velocity values. Panels C and D show a more complete 2D field as coagulation develops and an increase shear velocity values. Panels E, F, G and H show the ultrasound images, also called Bmode images, of the corresponding 2D fields at times (0, 6, 30 and 120 min of coagulation). These results show that the stiffness is linked to shear waves and not to ultrasound. The use of ultrasound only cannot help to characterize the medium stiffness as we see no differences between each images on panels E, F G and H. Fig. 4 shows the average value of shear wave speed within the white square shown in Fig. 3(panels B, C and D). In this case the mean value was done excluding all those pixels in which the correlation algorithms failed to provide a velocity value. In this sample, the coagulation time was around 10 min, and the shear wave speed shows a well behaved increase that seems to plateau close to 120 min. To evaluate the reproducibility of our technique, we tested three milk samples under the same coagulation conditions (temperature, CaCl2 and rennet concentrations). The results of the mean shear wave speeds are shown in Fig. 5. It is evident that our technique has a good reproducibility, capturing the same coagulation time for all 3 samples as well as the behavior in time, with a coefficient of variation between the 3 curves of 0.16.
Fig. 4. Evolution of the shear wave velocity during coagulation. Arrow represents the coagulation time.
Fig. 5. Three experiments using the same CaCl2 and rennet concentrations (0.1 g/l and 3.0 ml/l, respectively) to evaluate the reproducibility of our technique. The coefficient of variation between the curves was 0.16. Arrow represents the coagulation time.
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Fig. 7. (a) Mean values with standard deviation of shear wave speed as a function of time over three milk samples acquired with SSI technique. (b) Mean values with standard deviation of conservation modulus as a function of time over three milk samples acquired with classical rheology.
A direct comparison between classical rheometry and SSI measurements was performed on three different samples. In Fig. 7 are presented mean values with standard deviations as a function of time over three samples. The coagulation process was initiated with 0.1 g/l of CaCl2 and 1.5 ml/l of rennet in a commercial pasteurized whole milk. The change of slope can be used to determine the coagulation time which is quite the same in both experiments. Finally the echogenicity of the raw milk was tested on a small sample and presented in Fig. 8.
Fig. 6. Evolution of shear wave speed for the different concentrations for CaCl2 and rennet presented in Table 1. Arrows represent the coagulation time.
4. Discussion
Fig. 6 shows the evolution of the shear wave speed for the different concentrations of CaCl2 and rennet presented in Table 1. In Panel A are the curves for 0.1 g/l of CaCl2 (+) and 3.0 ml/l of rennet (+), versus 0.05 of CaCl2 () and 1.5 ml/l of rennet (). In Panel B are the curves for 0.1 g/l of CaCl2 (+) and 1.5 ml/l of rennet (), versus 0.05 of CaCl2 () and 3.0 ml/l of rennet (+). Panel C shows the curves for 0.2 g/l of CaCl2 (++) and 6.0 ml/l of rennet (++) against the 0.025 of CaCl2 ( ) and 0.75 ml/l of rennet ( ). To evaluate the influence of the CaCl2 and rennet concentrations on the coagulation dynamics, we compared the shear wave speed at selected times (20, 40 and 100 min) for the different concentrations. Table 2 shows the velocities calculated for different times after adding the rennet. It also shows the coagulation times, (t0 time when shear waves begin to propagate).
As it was mentioned before, the speed of the shear waves increases as the gelation of the milk takes place. This was easily identifiable in Fig. 2, where the shear waves clearly propagate further after 30 min and even more after 120 of coagulation. It is interesting to note that in the first few minutes of coagulation, even though the medium is starting to change, it behaves as a liquid. At these times it is possible to identify the push from the radiation force, but it is clear that there is no propagation. This figure also allows us to see the quasi plane wave generated by the multiple pushes at different depths in a rapid succession. These pushes sum up to generate one long push and therefore a plane front. It is also worth mentioning that while the changes in the mechanical properties are significant, there seems to be no significant changes in the Bmode images.
Table 2 Coagulation time (t0), (V0) shear wave speed at (t0), velocities of propagation for minutes 20, 40, 60, 100 and velocity after reaching the plateau for the different coagulation conditions. Concentrations (rennet, CaCl2)
t0 (min)
V0 (m/s)
V20 (m/s)
V40 (m/s)
V60 (m/s)
V100 (m/s)
Vplateau (m/s)
1 2 3 4 5 6
10 ± 1 15 ± 1 11 ± 1 11 ± 1 7±1 15 ± 1
0.35 0.35 0.34 0.35 0.35 0.35
0.54 0.42 0.48 0.52 0.54 0.41
0.65 0.57 0.63 0.63 0.63 0.60
0.69 0.63 0.68 0.68 0.67 0.67
0.74 0.71 0.72 0.71 0.73 0.74
0.77 0.74 0.74 0.73 0.73 0.75
(+, +) (, ) (+, ) (, +) (++, ++) (, )
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Fig. 8. Ultrasound image of raw milk showing scatterrers within the medium.
Regarding ultrasound images Fig. 8 shows that it is not necessary to add cellulose when working with raw milk. In the present paper we chose to use homogenized milk and cellulose because the combination would allow us to study the sensitivity of our methodology to changes in rennet and calcium and it was easier to provide. This was a first work to study the potential of the SSI technique. Nevertheless this is a limitation of the technique, since without any speckle, i.e. an ultrasound amplitude strong enough, it is not possible to recover shear wave displacements and then to quantify shear wave speed. Further investigations over different type of milk should be done to define if the SSI technique is suitable to follow milk coagulation. Dynamics of milk coagulation seem to occur very fast in the initial stages with a fast increase of the shear wave speed, and a slower increase after 40 min (Fig. 4). A plateau seems to occur beyond 2 h of coagulation. A similar coagulation process was described by our group during blood coagulation (Bernal et al., 2012, 2013). Curves of shear wave velocity showed similar behaviors to those obtained by classical rheological measurements (Fig. 7). The change in slope of both parameters occurred at the same time (t = 15 min). Thus as well as classical rheology, the SSI technique could be used to determine on line the cutting time. The beginning of the gelation process in the milk can be correlated with the beginning of the propagation of the shear wave (Gennisson and Cloutier, 2006). As the medium starts to jellify, shear waves are able to propagate (shear waves do not propagate in liquids); this stage in the coagulation process can be identified as the beginning of gelation. Future studies should focus on the relating the evolution of the shear modulus with the cheese consistency and maturity. Even though several groups have reported coagulation times, it is important to notice that its definition is not always the same as discussed in the introduction. Some authors propose the detection of the cutting time as function of the coagulation time while other assign an arbitrary value. Although these determinations can be useful, it would be desirable to determine the cutting time independently of the coagulation time as differences in this value may not correspond to differences in the optimum cutting time. Precise definition of the cutting time is of great importance for the industry as it affects cheese quality and yield. Gunasekaran and Ay (1995) define the cutting time empirically as 20 min after the coagulation time, while Koc and Ozer (2008) define the standard coagulum cut time as 4 times the observed coagulation start time. In both cases the cutting times determined were around 40 min which in our experiments correspond to the change in the slope of the shear wave velocity curves (Figs. 4–6).
The results of parallel monitoring using SSI and rheology (Fig. 7) showed a good correspondence between both techniques. As classical rheology is used to determine coagulation and cutting points in parallel (Fox et al., 2000), a correlation between both techniques would allow the use of SSI for on-line cutting point detection. Nevertheless, the SSI technique cannot determine shear wave speed at very early stage of coagulation, since propagation of shear waves is not possible. The velocity of propagation of the shear waves is directly proportional to the elasticity of the medium; therefore it can be related with the firmness of the coagulum, providing a useful tool to determine the cutting time. Analysis of the shear wave velocity curve can potentially provide the optimum cutting time, allowing this time to be defined for each industrial process individually depending on the desired characteristics. In order to do this, the technique should be calibrated against the expertise of the cheese maker. The SSI measurements should be compared with the detection of the cutting point by the cheese maker. After calibration SSI could be used to determine when the coagulum is ready to cut. The methodology proved to be repetitive as can be seen in Fig. 5 where three repetitions for the same coagulation conditions are presented. Shear wave elastography was sensitive to changes in the coagulation conditions as it is shown in Figs. 6 and 7. Nevertheless, the different concentrations of CaCl2 and rennet used in our experiments did not seem to have a great impact on the dynamics of coagulation. The differences between adding 0.025 g/l of CaCl2 and 0.75 ml/l of rennet ( , ) and 0.2 g/l of CaCl2 and 6.00 ml/l of rennet (+ +, ++) showed the biggest differences with a coagulation time difference of 10 min. A similar result was found when comparing additions of 0.1 g/l of CaCl2 and 3.00 ml/l of rennet (+, +) and 0.05 g/l of CaCl2 and 1.50 ml/l of rennet (, ), with a coagulation time difference of 7 min. The CaCl2 and rennet combinations of 0.1 g/l and 1.5 ml/l (+, ) and 0.05 g/l and 3.00 ml/l (, +) seemed compensate each other, because there was no difference between two conditions; with an identical coagulation time of 11 min and very similar coagulation dynamics. It is important to note that the gap in curve for CaCl2 ( ) and rennet ( ) in Fig. 6C, was due to a computer malfunction, which resulted in 10 min of lost data. In Fig. 6 it can be observed that changes in the coagulation conditions (CaCl2 and rennet concentrations) affected mainly the beginning of the coagulation, changing the slope and time of coagulation within the first 40 min. After which, all the samples reached similar velocities toward the end of the experiment. This indicates that despite differences in the beginning of the coagulation process and the initial conditions tested, the final firmness of the coagulum was not affected. Table 2 showed the importance of the initial conditions, showing marked differences in the shear wave speed for the 20 and 40 min time points, while these differences become less significant after 100 min. This results suggest that tracking the shear wave velocity (shear elasticity) of the milk curd, would be beneficial to better determine the appropriate cutting time regardless of the coagulation dynamics due to variations in the initial conditions. Even though the cutting time was not actually measured, the changing stiffness of the gel can be related to the industrial determination of the cutting time. With this purpose it would be necessary to correlate the results of this technique with the desired characteristics of the coagulum when it is ready to cut. These characteristics depend on the process considered and are determined by the cheesemaker.
5. Conclusions Shear wave elastography was sensitive to the changes in structure occurring during milk coagulation. The method was repetitive
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and showed significant differences when coagulation conditions were changed. The curves showed similar behaviors to those reported in literature for coagulation monitoring using rheological methods (Fox et al., 2000). The addition of cellulose makes this technique unsuitable for measurements inside the coagulation vat. However, it could be used for monitoring the coagulation process in industrial setting by taking a sample from the coagulation vat at the beginning of the process. The evolution of shear velocity in this sample would provide information about the structure of the coagulum inside the vat. Preliminary tests showed that when working with raw milk (unhomogenized) the addition of cellulose could be avoided. This would allow the use of the technique on line. Comparison with rheological measurements showed good correlation of both techniques. This correlation could be used to calibrate the SSI technique. However, further research is needed in order to correlate the shear wave elastography with determinations of cutting time. Acknowledgements This work is supported by LABEX WIFI (Laboratory of Excellence ANR-10-LABX-24 within the French Program ‘‘Investments for the Future’’) under reference ANR-10-IDEX-0001-02 PSL*. References Ay, C., Gunasekaran, S., 1994. Ultrasonic attenuation measurements for estimating milk coagulation time. Am. Soc. Agric. Eng. 37 (3), 857–862. Bakkali, F., Moudden, A., Faiz, B., Amghar, A., Maze, G., Montero de espinosa, F., Akhnak, M., 2001. Ultrasonic measurement of milk coagulation time. Meas. Sci. Technol. 12, 2154–2159. Bavu, E., Gennisson, J.L., Couade, M., Bercoff, J., Mallet, V., Badel, A., Fink, M., Nalpas, B., Tanter, M., Pol, S., 2011. Noninvasive in vivo liver fibrosis evaluation using supersonic shear imaging : a clinical study on 113 hepatitis C virus patients. Ultrasound Med. Biol. 37 (9), 1365–1373. Benedito, J., Carcel, J.A., Gonzalez, R., Mulet, A., 2002. Application of low intensity ultrasonics to cheese manufacturing processes. Ultrasonics 40, 19–23. Benguigui, L., Emery, J., Durand, D., Bunsel, J.P., 1994. Ultrasonic study of milk clotting. Lait 74, 197–206.
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Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Use of shear wave elastography for monitoring enzymatic milk coagulation E. Budelli a,c, M. Bernal b, P. Lema a, M. Fink b, C. Negreira c, M. Tanter b,1, J.L. Gennisson b,⇑,1 a
Instituto de Ingeniería Química, Facultad de Ingeniería, Universidad de la República, Julio Herrera y Reissig 565, CP 11300 Montevideo, Uruguay Institut Langevin – Ondes et Images, ESPCI ParisTech, PSL Research University, CNRS UMR 7587, INSERM U979, 1 rue Jussieu, 75005 Paris, France c Laboratorio Acústica Ultrasonora, Instituto de Física, Facultad de Ingeniería, Universidad de la República, Iguá 4225, CP 11400 Montevideo, Uruguay b
a r t i c l e
i n f o
Article history: Received 4 September 2013 Received in revised form 6 March 2014 Accepted 19 March 2014 Available online 30 March 2014 Keywords: Shear wave elastography Milk coagulation Rheological properties Ultrafast ultrasound imaging
a b s t r a c t In the manufacturing of cheese, the cutting of the curd is an essential step which depends on the firmness of the curds and significantly affects the yield of the cheese and its quality. In this work, we present a technique to measure elastic properties of the curd during coagulation that could be used to quantitatively determine the cutting time. The technique uses ultrasound to generate and measure shear waves. These waves do not propagate in liquids and their velocity of propagation depends on the viscoelastic characteristics of the medium. Hence, they can be used to identify the beginning of coagulation and subsequently to monitor the evolution of the coagulum firmness. Our results showed this technique is sensitive to changes of the medium structure during coagulation. It also proved reproducible and sensitive to different coagulation conditions. Therefore this technique can be used to develop a system suitable for the dairy industry. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Milk coagulation is the process in which liquid milk is transformed into a viscoelastic semi-solid coagulum by specific enzymes called chymosins. These enzymes act upon milk’s j-caseins, partially fragmenting these molecules. This phenomenon causes a change in the micelle surface charge, increases their hydrophobicity and promotes their aggregation (Koc and Ozer, 2008). Micelle aggregation is responsible for an increase in milk’s viscosity. These characteristics are important in cheese making because they determine the coagulum’s cutting point (Fox et al., 2000). The analysis of the different stages in the formation of macromolecular networks is of major importance, since understanding the structure and properties of gels requires the understanding of their organization process (Nassar et al., 2001). Milk gelation is a major step in the cheese making process; determining the end of the coagulation stage (cutting time) affects the yield of the cheese production process significantly. If the curd is cut when the coagulum is too soft, the loss of fat and curd fines decreases the cheese yield sharply. If the curd is too firm, syneresis is retarded, resulting in a high moisture cheese, which can require a longer ripening stage (Benedito et al., 2002). The cutting time is generally predetermined ⇑ Corresponding author. Tel.: +33 1 80 96 30 79; fax: +33 1 80 96 33 55. 1
E-mail address: [email protected] (J.L. Gennisson). These authors are co-last authors.
http://dx.doi.org/10.1016/j.jfoodeng.2014.03.026 0260-8774/Ó 2014 Elsevier Ltd. All rights reserved.
based on previous batches information, which makes it difficult to maximize the yield for every particular batch. Therefore, a methodology that allows the assessment of the cutting time in real time is needed in order to standardise cheese yield and its physical and sensory characteristics (Koc and Ozer, 2008). In addition, the change in coagulation time is useful in comparing strengths of different enzyme solutions (Gan et al., 2006). However, the current on-line systems used in the prediction of the cutting time, are generally more sensitive to changes occurring during the gelation process rather than around the cutting point. This introduces a source of error in those factors which influence the rate of coagulum firming but may not influence the rate of gel formation in a similar fashion (O’Callaghan et al., 2000). Over the years, ultrasonic sensing devices have been used for coagulation monitoring because they provide non-destructive, quick and low cost measurements (Klandar et al., 2007). The most frequently used parameters in ultrasonic measurements are the ultrasonic velocity and attenuation of compressional waves (Wang et al., 2007). Several authors have successfully used low intensity ultrasonic measurements for studying the structural changes involved in milk coagulation and monitoring. Experiments have been carried out in casein solutions (Wang et al., 2007), reconstituted milk (Ay and Gunasekaran, 1994; Benguigui et al., 1994; Bakkali et al., 2001; Nassar et al., 2001, 2004) and whole milk (Koc and Ozer, 2008). These authors define different parameters to monitor the coagulation process. On the one hand, Ay and
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Gunasekaran (1994) define the coagulation time as the point in the ultrasound attenuation curve where there is a change in the slope; and the cutting time as a function of the coagulation time. On the other hand, Nassar et al. (2001, 2004) define the clotting time as the time when the ultrasound velocity starts to increase. A more recent approach to study gelation processes uses shear waves to characterize viscoelastic properties in coagulating blood (Bernal et al., 2012, 2013; Gennisson and Cloutier, 2006). The technique used, so called Supersonic Shear Imaging, uses ultrasound ultrafast imaging (until 20,000 frames/s) to catch propagation of shear wave generated by ultrasonic radiation force in real time (Bercoff et al., 2004). Since shear wave speed (VS) is directly related to the shear modulus (l) by: l = qV2S with q the density, the viscoelastic properties of the investigated medium can be retrieved. This approach could be useful for monitoring the milk coagulation process. As shear waves do not propagate in liquids, they can be used to identify the beginning of coagulation (time at which the shear waves begin to propagate in the milk sample) and subsequently the technique could be used to monitor the evolution of the coagulum firmness. In this work we implemented the Supersonic Shearwave Imaging (SSI) technique, within the milk sample. We further assessed the suitability, sensitivity and reproducibility of this technique for monitoring enzymatic milk coagulation.
2. Methods 2.1. Milk samples Pasteurized whole milk was purchased from local market. In order to increase the amount of scatters, cellulose (Cellulose powder, Type 20, Sigma Chemical, St. Louis, MO, USA) was added in a concentration of 10 g/l to the milk samples before the experiment. Coagulation was initiated in the milk samples by adding calcium chloride (CaCl2, number C 5080, Sigma Chemical, St. Louis, MO, USA) and rennet (active chymosin P520 mg/L, COOPER, Melun, France), which was purchased from a local pharmacy. In order to test the sensibility of the technique to different coagulation conditions, the concentrations of CaCl2 and rennet added to the milk samples, were varied according to Table 1. During experiments, samples were held in 150 ml beakers and placed in a water bath at 35 °C (Fig. 1). The experimental procedure was as follows. 150 ml of milk with cellulose and CaCl2 were placed in the plastic beaker. The solution was preheated up to 30–32 °C and put inside the water bath at 35 °C in order to guarantee a stable temperature. While the milk reached 35 °C, the transducer was placed and aligned as shown in Fig. 1a. Once the sample achieved the desired temperature, the generation and acquisition of the shear waves was started. After 4 min of acquisition, coagulation was initiated by adding the ren-
Table 1 Rennet and CaCl2 concentrations used in the experiments. The signs adjacent to the values, symbolize the concentration levels; ‘‘+’’ and ‘‘‘‘ being high and low concentrations respectively, while ‘‘+ +’’ and ‘‘ ‘‘ correspond to doubling or cutting in half the concentrations. This notation is used in following graphs to simplify the notation. Concentrations
CaCl2 (g/l)
Rennet (ml/l)
1 2 3 4 5 6
0.100 0.050 0.100 0.050 0.200 0.025
3.00 3.00 1.50 1.50 6.00 0.75
(+) () (+) () (+ +) ( )
(+) (+) () () (+ +) ( )
net. Measurements for ultrasound images and shear wave propagation were recorded every minute for 120 min. 2.2. Rheological measurements Rheological measurements were made using a rheometer Anton Paar Physica 301 equipped with a peltier temperature control system. Oscillatory tests were made in bob cylinder geometry at 35 °C. Strain of 2% and 1 Hz was applied every 5 min. Data acquisition was started immediately after rennet addition. Storage and loss modulus (G0 , G00 ) were recorded and compared with results obtained by Supersonic Shear Imaging. As a preliminary study, the effect of cellulose addition in the coagulation process was evaluated. Evolution of G0 and G00 during coagulation of samples containing cellulose showed no significant differences with the results obtained for samples without cellulose addition. 2.3. Supersonic Shear Imaging and elasticity maps Supersonic Shear Wave Imaging (SSI) was performed by using an ultrafast ultrasound device (V12, Supersonic Imagine, Aix en Provence, France) which was the research prototype of a clinical commercial ultrasound device (Aixplorer, Supersonic Imagine, Aix en Provence, France). This prototype is a fully programmable system (256 channels, 60 MHz sampling rate) driving 8 MHz central frequency ultrasonic probe (256 piezoelectric elements, 0.2 mm pitch). SSI is an ultrasound technique that allows to recover elastic maps of biological tissues by measuring the shear wave speed which is directly related to stiffness by the relationship, E = 3qV2S (where E is the Young’s modulus, q the density of the medium and VS the shear wave speed, Tanter et al., 2008). It is based on two concepts: the generation of shear waves within the media, the monitoring of the propagation of such shear waves (Fink and Tanter, 2010). Both concepts are achieved by using an ultrafast ultrasound scanner classically used in medical imaging for breast cancer diagnosis (Berg et al., 2012; Tanter et al., 2008), liver fibrosis staging (Bavu et al., 2011) or muscle stiffness assessment (Gennisson et al., 2010). First the shear waves are generated by focusing ultrasound within the milk (Fig. 1b) along one line on the z axis during 100 ls. Then by using an ultrafast imaging mode, a movie of the propagating shear wave is acquired in a very short time (some tenth of ms) (Fig. 1c). Therefore from the shear wave movie the speed of shear waves is calculated giving access to the stiffness of the medium. In the present paper everything is presented in terms of shear wave speed. Finally this acquisition is done. At last this acquisition mode is repeated every minute or more depending the experiments needs. Dimensions of the field measured at each acquisition were 30.0 mm depth by 51.2 mm wide and the spatial resolution of the SSI technique to differentiate the elasticity properties was in the order of 1 mm (Figs. 2 and 3). At last, in order to show direct comparison between SSI and rheological measurements, parallel monitoring of coagulation using both techniques was made. 3. Results Changes in the propagation of the shear wave during coagulation are shown in Fig. 2. As the milk starts to coagulate (between minute 5 and 15) shear wave begin to propagate in the medium, and as the milk becomes firmer, the speed of the waves increases. This is directly visible on the images since the shear wave front (represented by the blue line along the z axis Fig. 2) is at a bigger distance from the source (represented by the red line along the z axis) as a function of the coagulation time for each acquisition
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Fig. 1. (a) Picture of the ultrasound setup. (b) and (c) Schematic of the shear wave experiment. The shear wave is first generated by ultrasound radiation force and then imaged by using an ultrafast ultrasound imaging mode.
Fig. 2. Shear wave propagation at different coagulation time points and at different times during the acquisition. The first row shows the wave propagation at minute 0, where Panels B, C and D represent different acquisition times (3, 15 and 30 ms). Panel A shows the corresponding B mode image at this coagulation time point. Similarly, the second row shows the wave propagation series after 6 min of coagulation. In the same fashion, the third and fourth row show the propagation at 30 and 120 min of coagulation. The dotted white lines represent the bottom of the plastic container in which the milk samples were hold. On the third colon the red line represents the shear wave source and the blue line the shear wave front. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. Two dimension shear velocity maps. The first row shows the elasticity maps at different time points during milk coagulation (0, 6, 30 and 120 min). The white rectangle shown in Panels B, C and D, represents the area analyzed for Fig. 4. The bottom row shows the corresponding ultrasound images at the same time points. The dotted white line represents the bottom of the plastic container.
time. At time zero, it is possible to see that there is no propagation of the shear waves due to the liquid state of the medium (shear waves do not propagate in liquids). In the second row (minute 6 of coagulation) it can be observed that the wave only propagates a few mm, even 30 ms after the push. The 3rd and 4th rows show more advanced coagulation times (30 and 120 min, respectively), and it is evident in that the wave propagates longer distances at these stages (Panels L and P). Panels A, E, I and M show the Bmode images at 0, 6, 30 and 120 min, respectively. The dotted lines in all the figures represent the bottom of the plastic beaker. The 2D shear wave velocity maps are shown in Fig. 3. As mentioned in the methods section, the shear wave speed was calculated using the shear waves generated by the ultrasound radiation force at elements 64, 128 and 192. The white arrows in Fig. 3B show the location of the pushes. Three excitations were required to characterize the whole 2D field. Panel A shows no data since the shear waves cannot propagate in liquids. The beginning of the coagulation process can be observed in Panel B, where only part of the 2D field has information due to the limited propagation of the shear waves. The rest of the field was discarded because the correlation algorithms did not provide reliable velocity values. Panels C and D show a more complete 2D field as coagulation develops and an increase shear velocity values. Panels E, F, G and H show the ultrasound images, also called Bmode images, of the corresponding 2D fields at times (0, 6, 30 and 120 min of coagulation). These results show that the stiffness is linked to shear waves and not to ultrasound. The use of ultrasound only cannot help to characterize the medium stiffness as we see no differences between each images on panels E, F G and H. Fig. 4 shows the average value of shear wave speed within the white square shown in Fig. 3(panels B, C and D). In this case the mean value was done excluding all those pixels in which the correlation algorithms failed to provide a velocity value. In this sample, the coagulation time was around 10 min, and the shear wave speed shows a well behaved increase that seems to plateau close to 120 min. To evaluate the reproducibility of our technique, we tested three milk samples under the same coagulation conditions (temperature, CaCl2 and rennet concentrations). The results of the mean shear wave speeds are shown in Fig. 5. It is evident that our technique has a good reproducibility, capturing the same coagulation time for all 3 samples as well as the behavior in time, with a coefficient of variation between the 3 curves of 0.16.
Fig. 4. Evolution of the shear wave velocity during coagulation. Arrow represents the coagulation time.
Fig. 5. Three experiments using the same CaCl2 and rennet concentrations (0.1 g/l and 3.0 ml/l, respectively) to evaluate the reproducibility of our technique. The coefficient of variation between the curves was 0.16. Arrow represents the coagulation time.
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Fig. 7. (a) Mean values with standard deviation of shear wave speed as a function of time over three milk samples acquired with SSI technique. (b) Mean values with standard deviation of conservation modulus as a function of time over three milk samples acquired with classical rheology.
A direct comparison between classical rheometry and SSI measurements was performed on three different samples. In Fig. 7 are presented mean values with standard deviations as a function of time over three samples. The coagulation process was initiated with 0.1 g/l of CaCl2 and 1.5 ml/l of rennet in a commercial pasteurized whole milk. The change of slope can be used to determine the coagulation time which is quite the same in both experiments. Finally the echogenicity of the raw milk was tested on a small sample and presented in Fig. 8.
Fig. 6. Evolution of shear wave speed for the different concentrations for CaCl2 and rennet presented in Table 1. Arrows represent the coagulation time.
4. Discussion
Fig. 6 shows the evolution of the shear wave speed for the different concentrations of CaCl2 and rennet presented in Table 1. In Panel A are the curves for 0.1 g/l of CaCl2 (+) and 3.0 ml/l of rennet (+), versus 0.05 of CaCl2 () and 1.5 ml/l of rennet (). In Panel B are the curves for 0.1 g/l of CaCl2 (+) and 1.5 ml/l of rennet (), versus 0.05 of CaCl2 () and 3.0 ml/l of rennet (+). Panel C shows the curves for 0.2 g/l of CaCl2 (++) and 6.0 ml/l of rennet (++) against the 0.025 of CaCl2 ( ) and 0.75 ml/l of rennet ( ). To evaluate the influence of the CaCl2 and rennet concentrations on the coagulation dynamics, we compared the shear wave speed at selected times (20, 40 and 100 min) for the different concentrations. Table 2 shows the velocities calculated for different times after adding the rennet. It also shows the coagulation times, (t0 time when shear waves begin to propagate).
As it was mentioned before, the speed of the shear waves increases as the gelation of the milk takes place. This was easily identifiable in Fig. 2, where the shear waves clearly propagate further after 30 min and even more after 120 of coagulation. It is interesting to note that in the first few minutes of coagulation, even though the medium is starting to change, it behaves as a liquid. At these times it is possible to identify the push from the radiation force, but it is clear that there is no propagation. This figure also allows us to see the quasi plane wave generated by the multiple pushes at different depths in a rapid succession. These pushes sum up to generate one long push and therefore a plane front. It is also worth mentioning that while the changes in the mechanical properties are significant, there seems to be no significant changes in the Bmode images.
Table 2 Coagulation time (t0), (V0) shear wave speed at (t0), velocities of propagation for minutes 20, 40, 60, 100 and velocity after reaching the plateau for the different coagulation conditions. Concentrations (rennet, CaCl2)
t0 (min)
V0 (m/s)
V20 (m/s)
V40 (m/s)
V60 (m/s)
V100 (m/s)
Vplateau (m/s)
1 2 3 4 5 6
10 ± 1 15 ± 1 11 ± 1 11 ± 1 7±1 15 ± 1
0.35 0.35 0.34 0.35 0.35 0.35
0.54 0.42 0.48 0.52 0.54 0.41
0.65 0.57 0.63 0.63 0.63 0.60
0.69 0.63 0.68 0.68 0.67 0.67
0.74 0.71 0.72 0.71 0.73 0.74
0.77 0.74 0.74 0.73 0.73 0.75
(+, +) (, ) (+, ) (, +) (++, ++) (, )
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Fig. 8. Ultrasound image of raw milk showing scatterrers within the medium.
Regarding ultrasound images Fig. 8 shows that it is not necessary to add cellulose when working with raw milk. In the present paper we chose to use homogenized milk and cellulose because the combination would allow us to study the sensitivity of our methodology to changes in rennet and calcium and it was easier to provide. This was a first work to study the potential of the SSI technique. Nevertheless this is a limitation of the technique, since without any speckle, i.e. an ultrasound amplitude strong enough, it is not possible to recover shear wave displacements and then to quantify shear wave speed. Further investigations over different type of milk should be done to define if the SSI technique is suitable to follow milk coagulation. Dynamics of milk coagulation seem to occur very fast in the initial stages with a fast increase of the shear wave speed, and a slower increase after 40 min (Fig. 4). A plateau seems to occur beyond 2 h of coagulation. A similar coagulation process was described by our group during blood coagulation (Bernal et al., 2012, 2013). Curves of shear wave velocity showed similar behaviors to those obtained by classical rheological measurements (Fig. 7). The change in slope of both parameters occurred at the same time (t = 15 min). Thus as well as classical rheology, the SSI technique could be used to determine on line the cutting time. The beginning of the gelation process in the milk can be correlated with the beginning of the propagation of the shear wave (Gennisson and Cloutier, 2006). As the medium starts to jellify, shear waves are able to propagate (shear waves do not propagate in liquids); this stage in the coagulation process can be identified as the beginning of gelation. Future studies should focus on the relating the evolution of the shear modulus with the cheese consistency and maturity. Even though several groups have reported coagulation times, it is important to notice that its definition is not always the same as discussed in the introduction. Some authors propose the detection of the cutting time as function of the coagulation time while other assign an arbitrary value. Although these determinations can be useful, it would be desirable to determine the cutting time independently of the coagulation time as differences in this value may not correspond to differences in the optimum cutting time. Precise definition of the cutting time is of great importance for the industry as it affects cheese quality and yield. Gunasekaran and Ay (1995) define the cutting time empirically as 20 min after the coagulation time, while Koc and Ozer (2008) define the standard coagulum cut time as 4 times the observed coagulation start time. In both cases the cutting times determined were around 40 min which in our experiments correspond to the change in the slope of the shear wave velocity curves (Figs. 4–6).
The results of parallel monitoring using SSI and rheology (Fig. 7) showed a good correspondence between both techniques. As classical rheology is used to determine coagulation and cutting points in parallel (Fox et al., 2000), a correlation between both techniques would allow the use of SSI for on-line cutting point detection. Nevertheless, the SSI technique cannot determine shear wave speed at very early stage of coagulation, since propagation of shear waves is not possible. The velocity of propagation of the shear waves is directly proportional to the elasticity of the medium; therefore it can be related with the firmness of the coagulum, providing a useful tool to determine the cutting time. Analysis of the shear wave velocity curve can potentially provide the optimum cutting time, allowing this time to be defined for each industrial process individually depending on the desired characteristics. In order to do this, the technique should be calibrated against the expertise of the cheese maker. The SSI measurements should be compared with the detection of the cutting point by the cheese maker. After calibration SSI could be used to determine when the coagulum is ready to cut. The methodology proved to be repetitive as can be seen in Fig. 5 where three repetitions for the same coagulation conditions are presented. Shear wave elastography was sensitive to changes in the coagulation conditions as it is shown in Figs. 6 and 7. Nevertheless, the different concentrations of CaCl2 and rennet used in our experiments did not seem to have a great impact on the dynamics of coagulation. The differences between adding 0.025 g/l of CaCl2 and 0.75 ml/l of rennet ( , ) and 0.2 g/l of CaCl2 and 6.00 ml/l of rennet (+ +, ++) showed the biggest differences with a coagulation time difference of 10 min. A similar result was found when comparing additions of 0.1 g/l of CaCl2 and 3.00 ml/l of rennet (+, +) and 0.05 g/l of CaCl2 and 1.50 ml/l of rennet (, ), with a coagulation time difference of 7 min. The CaCl2 and rennet combinations of 0.1 g/l and 1.5 ml/l (+, ) and 0.05 g/l and 3.00 ml/l (, +) seemed compensate each other, because there was no difference between two conditions; with an identical coagulation time of 11 min and very similar coagulation dynamics. It is important to note that the gap in curve for CaCl2 ( ) and rennet ( ) in Fig. 6C, was due to a computer malfunction, which resulted in 10 min of lost data. In Fig. 6 it can be observed that changes in the coagulation conditions (CaCl2 and rennet concentrations) affected mainly the beginning of the coagulation, changing the slope and time of coagulation within the first 40 min. After which, all the samples reached similar velocities toward the end of the experiment. This indicates that despite differences in the beginning of the coagulation process and the initial conditions tested, the final firmness of the coagulum was not affected. Table 2 showed the importance of the initial conditions, showing marked differences in the shear wave speed for the 20 and 40 min time points, while these differences become less significant after 100 min. This results suggest that tracking the shear wave velocity (shear elasticity) of the milk curd, would be beneficial to better determine the appropriate cutting time regardless of the coagulation dynamics due to variations in the initial conditions. Even though the cutting time was not actually measured, the changing stiffness of the gel can be related to the industrial determination of the cutting time. With this purpose it would be necessary to correlate the results of this technique with the desired characteristics of the coagulum when it is ready to cut. These characteristics depend on the process considered and are determined by the cheesemaker.
5. Conclusions Shear wave elastography was sensitive to the changes in structure occurring during milk coagulation. The method was repetitive
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and showed significant differences when coagulation conditions were changed. The curves showed similar behaviors to those reported in literature for coagulation monitoring using rheological methods (Fox et al., 2000). The addition of cellulose makes this technique unsuitable for measurements inside the coagulation vat. However, it could be used for monitoring the coagulation process in industrial setting by taking a sample from the coagulation vat at the beginning of the process. The evolution of shear velocity in this sample would provide information about the structure of the coagulum inside the vat. Preliminary tests showed that when working with raw milk (unhomogenized) the addition of cellulose could be avoided. This would allow the use of the technique on line. Comparison with rheological measurements showed good correlation of both techniques. This correlation could be used to calibrate the SSI technique. However, further research is needed in order to correlate the shear wave elastography with determinations of cutting time. Acknowledgements This work is supported by LABEX WIFI (Laboratory of Excellence ANR-10-LABX-24 within the French Program ‘‘Investments for the Future’’) under reference ANR-10-IDEX-0001-02 PSL*. References Ay, C., Gunasekaran, S., 1994. Ultrasonic attenuation measurements for estimating milk coagulation time. Am. Soc. Agric. Eng. 37 (3), 857–862. Bakkali, F., Moudden, A., Faiz, B., Amghar, A., Maze, G., Montero de espinosa, F., Akhnak, M., 2001. Ultrasonic measurement of milk coagulation time. Meas. Sci. Technol. 12, 2154–2159. Bavu, E., Gennisson, J.L., Couade, M., Bercoff, J., Mallet, V., Badel, A., Fink, M., Nalpas, B., Tanter, M., Pol, S., 2011. Noninvasive in vivo liver fibrosis evaluation using supersonic shear imaging : a clinical study on 113 hepatitis C virus patients. Ultrasound Med. Biol. 37 (9), 1365–1373. Benedito, J., Carcel, J.A., Gonzalez, R., Mulet, A., 2002. Application of low intensity ultrasonics to cheese manufacturing processes. Ultrasonics 40, 19–23. Benguigui, L., Emery, J., Durand, D., Bunsel, J.P., 1994. Ultrasonic study of milk clotting. Lait 74, 197–206.
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