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In-line characterization of a whey protein aggregation process: Aggregates size and rheological measurements
Journal of Food Engineering 115 (2013) 73–82
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Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
In-line characterization of a whey protein aggregation process: Aggregates size and rheological measurements Fatou Toutie Ndoye a,⇑, Nicolas Erabit a,b,c, Denis Flick b,c, Graciela Alvarez a a
Irstea, Refrigeration Processes Engineering Research Unit, Antony, France AgroParisTech, UMR1145 Ingénierie Procédés Aliments, Massy, France c INRA, UMR1145 Ingénierie Procédés Aliments, F-91300 Massy, France b
a r t i c l e
i n f o
Article history: Received 20 June 2012 Received in revised form 26 September 2012 Accepted 28 September 2012 Available online 6 October 2012 Keywords: In-line measurements FBRM Particle size analysis Tubular viscometer Whey protein Aggregation
a b s t r a c t Heat induced whey protein (WP) aggregates functionalities have been found to strongly depend on aggregates size distribution. The latter is affected by the time/temperature/shear rate history, which affects rheological properties. This work evaluates the potential of the focused beam reflectance measurement (FBRM), an in situ granulometric method, and of an in-house developed tube viscometer, to perform in situ characterization of a WP aggregation process. Potential of turbidity techniques to characterize the process was also explored by off-line measurements. Experiments were investigated with WP solutions of 6% b-lactoglobulin added with different concentrations of CaCl2, and heated at three different holding temperatures. Aggregates mean size, viscosity and turbidity increase with holding temperatures and CaCl2 concentration as expected. A good correlation was found between viscosity data and aggregates mean sizes. The latter were also well correlated with turbidity at 900 nm. FBRM and tube viscometer are shown to be promising and valuable tools to perform in-line characterization of heat induced WP aggregation process. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Whey proteins are widely used as ingredients in elaborated food products, not only for their nutritional properties but also for their functional properties. Among the functional properties of whey protein (WP), aggregation which are related to protein– protein interactions, are of great importance. When heated, WP molecule, particularly the b-lactoglobuline (b-lg), unfolds (denaturation) and aggregation then occurs due to sulfhydryl (–SH) binding, hydrophobic interactions and intermolecular proteinion-protein cross-linking (Morr and Josephson, 1968; Cayot and Lorient, 1998). Heat induced WP aggregates can be used for structure or sensory properties improvement of food, but these aggregates functionalities strongly depend on the aggregates size. Several studies have demonstrated that aggregates size is affected by physicochemical (pH, ionic strength, protein concentration) and processing (heating and cooling kinetics, flow, shear) conditions (Xiong, 1992; Hoffmann et al., 1996; Verheul et al., 1999; Foegeding et al., 2002; Simmons et al., 2007; De Wit, 2009). In particular, it has been recognised that the presence of divalent calcium ions enhances WP heat induced aggregation and strongly influenced WP aggregates size. The time/temperature/shear rate ⇑ Corresponding author. E-mail address: [email protected] (F.T. Ndoye). 0260-8774/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jfoodeng.2012.09.021
history also affects rheological properties (Ndoye et al., 2012), and aggregates size distribution (ASD). Therefore, ASD and viscosity monitoring and control are of major interest. Existing microparticulation processes consist on a heat treatment followed by a mechanical step of shearing (high pressure homogeneisation) to break aggregates for the desired size. Most of these processes have been developed through trials and errors, but an optimization of the current process is still possible, especially by in situ characterization of heat induced WP aggregation. Conventional granulometric and rheological methods need sampling which could damage the aggregates and introduce systematic errors. In situ measurements provide a key insight in the dominate process mechanisms and a quick assessment of the impact of process variables on ASD and viscosity. Furthermore, real time analytics make possible the following of the overall aggregation process with time and offer the possibility to rapidly act on the physicochemical and operating conditions. The objective of the work was to implement in situ sensors in a laboratory scale heat treatment device to monitor the aggregation of WP. In-line particles characterisation techniques and rheological characterisation methods have been selected. A feasibility study was performed to assess the potential of innovative in situ sensors to properly follow the WP aggregates properties (ASD, aggregates mean size, viscosity). Integration of the selected sensors in the heat treatment pilot allowed inline measurements of the WP aggregates
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Nomenclature A Lt dP Q R
absorbance (–) tube length (m) pressure drop (Pa) flow rate (m3 s1) tube radius (m)
l r
dynamic viscosity (Pa s) shear stress (Pa)
Indices w
tube wall
Greek letters c_ shear rate (s1)
size and of the viscosity of the suspension depending on various calcium concentrations and heating conditions. 2. Selection of in situ sensors for WP aggregation process characterization Techniques of characterization of WP aggregates are often based on various physical properties of aggregates. But the choice of the most suitable measurement technique must be made with consideration for the type of results desired and the practicalities of the situation. Multiple analytical techniques ensure that the true nature of the aggregates is determined and that the biases inherent in each measuring technique do not give a misleading representation. In this work, it was chosen to combine several techniques for the characterisation of heat induced b-lg aggregate: the Focused Beam Reflectance Measurement (FBRM) method and the tubular viscometry respectively for aggregates size and for rheological characterizations; a turbidity technique, by use of a spectrophotometer, to have global information about protein particles size and concentration of the suspension. 2.1. In situ aggregates size measurements Counting the number of particles and sizing them is the earliest method to follow aggregation and particles processes. An innovative in situ method of counting and sizing particles and aggregates in food technology is FBRM. Haddad Amamou et al. (2010) had given a detailed description of the FBRM measurement principle. The technology returns a chord length distribution (CLD) which depends on both the particle size distribution (PSD) and the shape of the particle. However, the relationship between chord length and particle size is not straightforward. This problem has been examined by many authors who proposed theoretical methods for the translation of a measured CLD into its PSD (Heath et al., 2002; Wynn, 2003; Li and Wilkinson, 2005; Kail et al., 2009). Several studies have been devoted to the comparison of FBRM response to conventional particle sizing techniques. Heath et al. (2002) used laser diffraction (Malvern Mastersizer) and electrical sensing zone technique (Coulter Multisizer) as alternative sizing techniques. They analysed the effects on the measured FBRM CLD of several factors such as the mode of weighting the distribution, the position of the focal point, the agitation speed, the particle properties and the suspension volume fraction. They concluded that the mean of square-weighted chord lengths compare well with the mean diameter obtained with the used conventional sizing techniques over the range from 50 to 400 lm. They also found that the square-weighted FBRM results were not affected by changes of the focal position, the suspension fluid flow velocity, or the solid fraction. Similar study achieved by Li et al. (2005); compared FBRM technique with laser diffraction (LD), ultrasonic attenuation spectroscopy (UAS) and image analysis (IA). They found a good agreement between PSDs obtained by LD, UAS and IA, but that particle shape strongly affects the result obtained by different techniques.
They also showed that the CLD measured by FBRM is complex, depending not only on the PSD, but also on particle optical properties and shape, especially for particles diameters <20 lm. 2.2. In situ rheological characterization Rheological data can be used as a quantitative indicator of product microstructure with associated links to quality attributes. Measurements of viscosity data from a single point, as it is often done, give no information about the flow behaviour. The viscosity of a fluid during processing can increase in linear or a non linear way with a transition from Newtonian to non-Newtonian behaviour as the total solids concentration is increased (Walstra and Jenness, 1984). WP aggregation phenomena induce a modification of the protein volume fraction and, consequently of the apparent viscosity of the fluid. Aggregate size seems to influence the apparent viscosity. Walkenström et al. (1999) pointed out the dependence of the WP aggregates suspension viscosity on the microstructure (various sizes and compactness) which is determined by process parameters. A decreased viscosity related to a decreased aggregate size is observed with an increasing shear rate. The challenge is to perform online rheological measurements and not only a viscosity measurement after aggregation treatment, in order to characterize the fluid flow behaviour and monitor changes occurring according to process conditions. Tube viscometers have been used by several authors to measure the flow behaviour of gelatinized starch dispersions at high temperature (Lagarrigue and Alvarez, 2001). Online rheometry system composed of a set of pipes of several diameters have been successfully used to follow a crystallization process (Cerecero-Enriquez, 2003). This type of viscometer is based on Rabinowitsch–Mooney equation (Eq. (1)) relating wall shear stress rw (Eq. (2)) to wall shear rate c_ w for laminar and steady flow (Steffe, 1996):
c_ w ¼ f ðrw Þ ¼
rw ¼
3Q
pR3
" # dðQ =ðpR3 ÞÞ þ rw drw
ðdPÞR 2Lt
ð1Þ
ð2Þ
The pressure drop dP is measured over a fixed length Lt of a horizontal tube of inside radius R for a given volumetric flow rate Q. Plotting the apparent wall shear rate versus the wall shear stress for different operating conditions and each diameter of tube allow to determine the rheological behaviour of the fluid. 2.3. Turbidity characterization The properties of suspended particles or aggregates can also be derived by measurement of turbidity. A light beam illuminating a suspension of particles is partially scattered. The intensity of light scattering depends on particles size, shape concentration and refractive index of the surrounding liquid, as well as on the light wavelength and the angle of detection. An increase in particle size
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makes a very large increase in scattered light intensity and an increase in the proportion of light scattered at low angles (Gregory, 2009). Scattering by particles causes a reduction of transmitted light intensity through the suspension. The change in turbidity might be a useful measure of the degree of WP aggregation. Turbidity is often measured as absorbance with a spectrophotometer at wavelengths where the attenuation of the light beam is essentially due to scattering and absorption is negligible (Ju and Kilara, 1998; Simons et al., 2002; Giroux et al., 2010). 3. Materials and methods 3.1. Experimental procedure The experimental procedure adopted in this work is as following: The first step of the work is a feasibility study devoted to the evaluation of FBRM as an innovative sensor technology to detect and measure WP aggregation, and of the ability of spectrophotometer to characterize WP aggregation. The second step of the study is the integration of in situ sensors (FBRM, and tube viscometer) in a heat treatment pilot for inline characterization of the b-lg heat induced aggregation process. Off-line measurements were performed with the spectrophotometer for correlation with FBRM and viscosity measurements, and with a rheometer to compare with in-line viscosity data. 3.2. Whey protein A commercial b-lg powder containing 88.9% of b-lg was used for experiments. b-lg is the major WP in milk and its aggregation kinetic is known to govern the one of the whole WPs. The protein was rehydrated at 6% w/w in distilled water at 40 °C. Calcium was added to the protein solution during the preparation using a 1 M CaCl2 solution. Concentrations used in this study were varied between 5.0 and 7.0 mM of CaCl2. The solution was kept at 40 °C at least 2 h to achieve a complete rehydration. Measurement of the pH solution gives a value of 6.8. 3.3. Heat treatments Two heat treatment methods have been used in this work: a static system for the feasibility study and a dynamic device consisting on a heat treatment laboratory scale plant for in situ measurements. For static heating, 6% b-lg solutions with 6.6 mM CaCl2 were placed in sealed glass tubes (1 cm internal diameter) fitted with thermocouple to allow the acquisition of temperature during heating. Samples were first heated in boiling water for 1 min before being held in a thermostatically controlled bath (Fluke 7340 – USA). Samples removed from the bath, were immediately placed in water at room temperature (20–25 °C) to stop aggregation process. Investigated conditions are temperatures of 75 °C, 80 °C, 85 °C and 90 °C for 1, 3 and 5 min of holding time. This system has been used to assess the applicability of FBRM and spectrophotometer to WP aggregation. For in situ characterisation of aggregates, experiments were performed using an ultra high temperature (UHT) laboratory scale plant (Armfield FT174X, Ringwood, UK) schematized on Fig. 1. The heat treatment system has a nominal capacity of 20–50 L/h where the product is fed by a volumetric pump. In situ sensors were implemented at the outlet of the cooler to characterize the process in real time. Experiments in the UHT device have been performed with 6% b-lg solutions with 5 different concentrations of CaCl2 (5.0, 6.0, 6.4, 6.6 and 7.0 mM) and at 3 different temperatures (75 °C,
75
87 °C and 95 °C) with a constant holding time of 60 s and at a constant flow rate of 20 L/h. 3.4. Instrumentation 3.4.1. FBRM instrument The LasentecÒ FBRM S400-8 (Mettler Toledo, USA) was used to measure CLDs in experiments. FBRM instrument is composed of a measurement probe, an electronic measurement unit and a computer for data acquisition and analysis. The probe, schematized on Fig. 2 is in stainless steel and has an external diameter of 9.5 mm and a length of 120 mm. The laser beam is projected through the sapphire window at a wavelength of 780 nm. For batch analysis, the probe is inserted into the agitated suspension with an inclination of 45° using a dedicated stand (LasentecÒ). The data analysis software gives the counts number per second for each selected size class of chord length. CLDs are given in different formats such as non weighted, square weighted and cubic weighted distribution in 100 predefined size classes. Thousands of chords can be measured per second, giving a CLD from 1 lm to more than 1 mm. Square-weighted mean chord length can be compared to the Sauter mean diameter defined as the diameter of monodispersed spheres that would have the same volume and the same surface area as the measured polydispersed particles. A dedicated device was designed in order to integrate FBRM probe on the UHT pilot for in-line measurements (Fig. 1). It comprises a derivation for the dilution of the aggregates suspensions outgoing the cooling section of the pilot with non-heated b-lg solution. A flowmeter is used in order to set the derivated flux. The probe is inserted in counter-current into the tube where circulates the diluted suspension, with an inclination of 45 °C. This inclination allows the renewal of the flux near the probe window. Arellano et al. (2012) has compared the FBRM probe used in this work with an IA method using Polyamid Seeding Particles (PSP) of known mean diameter. Very good agreement was found between the Sauter mean chord lengths obtained by FBRM measurements and those given by IA. This result validated the FBRM technique as a method of particle size measurement. 3.4.2. Tubular viscometer A tubular viscometer has been designed and manufactured in order to follow indirectly the protein aggregation process (Fig. 1). The system is formed of 4 PVC ducts of a length of 100 cm and of several diameters placed in series. In order to maintain a laminar flow regime inside the ducts, diameters of 5.8, 10, 13 and 16.7 mm have been retained. Each duct is equipped with two piezometric rings separated by 50 cm and at 25 cm of the extremity of the tube. These piezometric rings allow a homogeneous pressure and constitute the measurement point of pressure drop. Preliminary study with water has been performed in order to validate the system. A difference <3% was found between the measured water viscosity and the given values in the literature for temperature ranging from 4 °C to 25 °C. For online measurements with the UHT Armfield pilot, the tubular viscometer is placed at the outlet of the cooling exchanger. Aggregates suspension flowed inside the tubes at room temperature. Variation on temperature was continuously recorded using temperature sensors (Pt100) placed at the inlet and at the outlet of the set of pipes. Raw data of this online rheological measurement unit are pressure drop and flow rate continuously recorded respectively with a differential pressure drop transmitter and a flowmeter transmitter. These data allow calculating the wall shear stress and the apparent wall shear rate in order to represent the fluid behaviour using the Rabinowitsch–Mooney equation (Eq. (1). For Newtonian behaviour, the viscosity of the aggregates suspension can be obtained using the Poiseuille–Hagen law (Eq. (3)):
l¼
pdPR4 8Lt Q
ð3Þ
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Fig. 1. UHT pilot plant and in-line measurements instrumentation.
Off-line viscosity measurements were achieved systematically after each experiment in the pilot with a rheometer (ReoRox G2, Medirox AB, Sweden) based on the patented FOR (Free Oscillation Rheometry) technology. This device, calibrated for low viscosity (0.5–4 mPa s) measurements, uses a torsion wire system to set a sample into oscillation. The viscosity of the sample is calculated by analysing the frequency change and the amplitude damping from free oscillation. The technology is especially suitable for low viscous material. The FOR principle is widely used to study the kinetic of milk coagulation (Hennessy, 2011). Assays were done at 20 °C and in triplicate. The goal was to compare these measurements with the in situ ones and to validate the tubular viscometer. 3.4.3. Turbidimetric technique A spectrophotometer (Beckman Coulter DU 730, Fullerton, USA) was used for turbidity measurements. Spectrophotometer is employed to measure the amount of light that a sample absorbs or
scatters at a given wavelength, this amount of light being linked with the suspended particles sizes and concentration. The instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching the photometer. Measurements were carried out by wavelength scan with 1 nmwavelength intervals in the range of 190 and 1100 nm, giving absorbance spectra. 4. Results and discussion 4.1. Offline whey protein aggregation characterisation 4.1.1. Offline FBRM analyzes The ability of FBRM technique to characterize WP aggregation process has been assessed by investigating the effect of heating temperatures and times on b-lg solutions. Fig. 3-a represents the aggregates square weighted cumulative CLD obtained at heating
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77
Square wieghted cumulative frequency (%)
Fig. 2. Photography and cutaway of the FBRM probe tip.
100 94%
(a)
80
90°C 80°C 77°C
60
85°C
40 26%
20
0 1
10
100
Chord length (µm)
Sauter mean chord length (µm)
20 18
(b)
16 14 12 10 8 6 4 2 0 75
80
85
90
95
90
95
Temperature (°C) 60000
(c)
-1
% counts number (s )
50000
40000
30000
20000
10000
0 75
80
85
Temperature (°C)
Fig. 3. Effect of heating temperature on a 6% b-lg solution aggregation (6.6 mM CaCl2 – 5 min of holding time); (a) square weighted cumulative CLD; (b) sauter mean chord length and (c) total counts number.
temperatures of 77 °C, 80 °C, 85 °C and 90 °C during 5 min of holding. Investigations at 75 °C are not reported because the FBRM measurements did not give significant values (counts number <300 s1). It is shown a remarkable difference between the four distributions. Large aggregates are obtained with increased heating temperature. Measurements have underlined that 94% of aggregates formed at 77 °C have a chord length between 1 and 10 lm while this rate decreases at 24% at 90 °C in favour of larger aggregates. This tendency is confirmed by the calculation of aggregates Sauter mean chord length as shown in Fig. 3-b, on which it can be seen an increasing in mean aggregates size with temperature. Total counts number of aggregates also increased with increasing temperature as can be seen in Fig. 3-c. These results are in agreement with the previous findings that heat induced WP aggregates size increase with heating temperature and time (Simmons et al., 2007). Results are also inline with earlier studies which reported that irreversible WP aggregation only occurs at temperatures above 75 °C (De Wit and Klarenbeek, 1984; Hoffmann and van Mil, 1999; Tolkach and Kulozik, 2007). FBRM measurements also showed the effect of heating time. A slight increase of the aggregate mean chord length was found as the holding time is extended. These preliminary measurements showed that FBRM is a valuable tool for characterizing aggregation of WPs in solution. The effect of aggregates fraction on FBRM measurements has been investigated. Aggregates concentration has been varied by diluting pure aggregated suspensions with the raw solution (non aggregated solution of 6% b-lg – 6.6 mM CaCl2) at protein mass fraction ranging from 0.15% w/w to 6% w/w. Experiments were performed with two aggregated samples produced at the UHT pilot at different conditions (87 °C and 18 s of holding time and 87 °C and 60 s of holding time). Fig. 4-a and b represent respectively the unweighted and the square weighted cumulative CLDs obtained with a b-lg aggregates suspension produced on the UHT pilot at a heating temperature of 87 °C for 18 s of holding time. Plots in Fig. 4-a reveal important differences between the pure suspension (6% w/w) CLD and the diluted suspensions CLDs. It seems that the sensitivity of FBRM to fine particles is increased in concentrated solutions. Results show that FBRM oversizes small particles (<10 lm) and undersizes larger ones (>10 lm) in the pure sample of b-lg aggregates. This is probably due to the overlapping of aggregates at high concentration or to fines overcrowding in the measurement zone near the window. Ruf et al. (2000) reported a masking effect of fines particles yielding to their overestimation by FBRM. However, by applying a normalized chord square weighting to the distributions, it can be seen in Fig. 4-b a progressive shift of the CLDs to the large sizes zone, with the concentration
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5 6% w/w 2% w/w 1.2% w/w 0.6% w/w 0.3% w/w 0.15% w/w
(a) 80
60
40
(a) Absorbance (AU)
Number cumulative frequency (%)
78
90°C
4
85°C 80°C
3
75°C
2
1
20
0 0
0 1
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600 800 Wavelength (nm)
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5
100 6% w/w 2% w/w 1.2% w/w 0.6% w/w 0.3% w/w 0.15% w/w
(b) 80
60
(b) Absorbance (AU)
Square weigthed cumulative frequency (%)
Chord length (µm)
4
6% w/w 2% w/w
3 1.2% w/w
2
0.6% w/w
1
0.3% w/w
40 0.15% w/w
20 0 0 0
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400
600
800
1000
1200
Wavelength (nm) 1
10
100
1000
Chord length (µm) Fig. 4. Effect of the aggregates mass fraction on the (a) unweighted and the (b) square weighted cumulative CLD (6% b-lg; 6.6 mM CaCl2; 87 °C/18 s; 20 L/h).
decreasing. For the most diluted solutions (0.15% w/w, 0.3% w/w and 0.6% w/w, respectively a dilution rate of 0.025, 0.05 and 0.1), the three distributions are tightening with a constant Sauter mean chord lengths of about 57 lm, while the 6% w/w solution had a Sauter mean chord lengths of 21 lm. Comparative results were found with aggregated suspension produced at 87 °C and 60 s of holding time. Heath et al. (2002) have also reported similar results for aluminum particles showing that the solid volume fraction largely affects the CLD. On the basis of these results, it seems that a dilution rate of 0.1 is a threshold value to overcome the effect of particles volume fraction. This result suggests the use of a derivative system in the UHT pilot for the installation of FBRM sensor for in situ measurements, in order to make dilution, as shown on Fig. 1. 4.1.2. Offline turbidimetric measurements To assess spectrophotometry as a technique of characterization of WP aggregates, turbidity measurements were carried out with WP aggregated samples produced by static heat treatment at four different temperatures (75 °C, 80 °C, 85 °C and 90 °C) during 5 min. Fig. 5-a shows the influence of heating temperature on absorbance spectra of b-lg aggregates suspensions. Between 190 and 500 nm, there is overlap between spectra obtained at 85 °C and 90 °C, while spectra obtained at 75 °C and 80 °C showed noticeable difference. Above the overlapping region, absorbance increases with heating temperature. The difference is the highest at about 900 nm. It is known that the transmitted light is reduced when the particle size increases, then increasing the turbidity of the analyzed suspension (Gregory, 2009). So variations obtained on turbidity data may be explained by the aggregates size increasing with heating temperature as seen above with FBRM measurements. These results have proven the ability of the turbidity technique to characterize WP aggregation process. The dependence of turbidity on aggregates fraction was studied. Fig. 5-b shows an effect of the protein mass fraction on the
Fig. 5. Absorbance spectra of aggregates suspension; (a) effect of the heating temperature (6% b-lg; 6.6 mM CaCl2), (b) effect of the aggregate mass fraction (87 °C/18 s; 20 L/h).
absorbance of the WP suspension obtained with a b-lg aggregates suspension produced on the UHT pilot at a heating temperature of 87 °C for 18 s of holding time. Aggregates solutions at 6% w/w, 2% w/w and 1.2% w/w showed spectrum with absorbencies essentially constant with the wavelength. Important absorbance variations with wavelength were found for the most diluted solution (0.6% w/w, 0.3% w/w and 0.15% w/w). Turbidity decreased with the particles mass fraction decreasing. It may be attributed to a difference of sensitivity according to the wavelength (Ryan et al., 2012). It was chosen to use absorbance at 900 nm (A900) in order to give exhaustive comparisons in further sections and to be sure that measured absorbance data are only due to light scattering, in other words, due to turbidity (negligible protein light absorption at 900 nm). It was also chosen to work at a dilution rate of 0.1 for turbidity off-line measurements, in coherence with FBRM measurements. 4.2. In-line whey protein aggregation characterization Aggregates size and viscosity measurements were performed in-line on the UHT pilot plant. Off-line turbidity and viscosity measurements were systematically carried out for each experiment, respectively with the spectrophotometer and the rheometer in order to compare with in situ measurements. Two variables: the heating temperature and the CaCl2 concentration were investigated to measure the in-line sensors sensibility on WP aggregation process. 4.2.1. Aggregates size FBRM measurements were continuously performed during the aggregation process and allow the observation of real-time changes in aggregates counts and size. Fig. 6-a showed the evolution of Sauter aggregates mean chord size with temperature and calcium concentration. It can be seen an increasing of aggregates mean size with both heating temperature and concentration in
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CaCl2. These results suggest that aggregation is promoted by temperature on the one hand and by calcium ions on the other hand, or by their combination, as it was widely recognized in the literature (Morr and Josephson, 1968; Donovan and Mulvihill, 1987; Mulvihill and Donovan, 1987; Galani and Owusu Apenten, 1999; Simons et al., 2002; Mounsey and O’Kennedy, 2007; Simmons et al., 2007; Tolkach and Kulozik, 2007; Mercadé-Prieto et al., 2009). Upon heating above 70 °C near neutral pH, b-lg native molecules undergo conformational changes exposing buried thiol groups and hydrophobic residues (Verheul et al., 1998; Schokker et al., 1999; Sava et al., 2005). The so denaturated molecules aggregate according to a two-consecutive-steps mechanism: (1) formation of b-lg oligomers through the establishment of intermolecular thiol-disulphide interactions and thiol–thiol oxidation, (2) formation of b-lg polymers via non-covalent interactions between hydrophobic residues (Sawyer, 1968; Roefs and Kruif, 1994; Schokker et al., 1999). Increasing temperature increases the accessibility of the thiol groups and then the rate of aggregation via thiol-disulphide exchange reaction resulting in larger aggregates (De Wit, 2009). Results of this work are inline with findings of Simmons et al. (2007) who reported that small and weakly bonded aggregates of WP are formed at temperature below 75 °C while large and strong aggregates are obtained at higher temperatures. Furthermore, at elevated temperature, the presence of salts like CaCl2 result in more stable hydrophobic interactions inducing aggregates size increasing (Xiong et al., 1993; Sherwin and Foegeding, 1997; Simons et al., 2002; Mounsey and O’Kennedy, 2007; O’Kennedy and Mounsey, 2009). However, investigating the role of calcium in b-lg aggregation, more recent studies of Simons et al. (2002) and Mounsey and O’Kennedy (2007) have questioned the order of the two-steps aggregation mechanism
FBRM Sauter chord length (µm)
35
(a)
30 25 20 15 10
75°C 87°C 95°C
5 0 4
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8
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Aggrgates counts number /sec
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79
described above. They rather suggested that non-covalent physical interactions were mainly responsible for the heat-induced primary aggregation in presence of calcium at neutral pH. Secondary formation of covalent bonds through thiol-disulphide interchange reactions would then proceed leading to irreversible aggregate formation. Simons et al. (2002) reported that calcium acts in the screening of surface charges and promotes aggregation by binding to carboxylates with a threshold affinity. This result have been confirmed by Mounsey and O’Kennedy (2007) who also found that a stoichiometric ratio of calcium to protein was required to initiate aggregate formation. This result is consistent with that showed on Fig. 6-b representing the evolution of the aggregates counts number/s with heating temperature and CaCl2 concentration. It can be seen on Fig. 6-b that the number of particles increases with temperature but decreases slightly with calcium concentration. The decrease in aggregates concentration when the calcium concentration increases may be explain by the promoting effect of calcium in the aggregation of the denaturated molecules, resulting in larger aggregates and reducing the total number of aggregates in suspension. Giroux et al. (2010) have calculated WP aggregates voluminosity and reported that the protein concentration in aggregates (which is the reciprocal of voluminosity) increased with calcium chloride concentration, giving more compact and less porous aggregates. An increase of protein concentration in aggregates results on a decrease in aggregates concentration in the suspension. Reduction in particles number is more frank above 6 mM of CaCl2, corresponding to an excess of calcium. The increase in aggregates concentration with temperature can be linked to the denaturation level which increase fastly with heating temperature as mentioned in previous works (Petit et al., 2011). 4.2.2. Viscosity In situ viscosity characterization has been carried out by direct measurement of differential pressure drop with an in-house developed tubular viscometer. Results showed a Newtonian behaviour in the investigated conditions, and the viscosity of the aggregates suspension was derived using the Poiseuille–Hagen law (Eq. (3)). Evolution of the viscosity of the aggregates suspension as a function of heating temperature and CaCl2 concentration is shown in Fig. 7-a. The doted line represents the viscosity of the native b-lg solution (no thermal treatment). As expected, increased heating temperature resulted in increased viscosity. At a given heating temperature, the b-lg aggregates solution became more viscous as the CaCl2 concentration increased. Maximum viscosity was reached at 95 °C and 6.6 mM of CaCl2, increasing the viscosity almost by a factor 2. The increase in aggregates size induces an increase particles volume fraction resulting in a more viscous solution (Vardhanabhuti and Foegeding, 1999). In-line viscosity measurements were compared to the off-line ones in Fig. 7-b. The parity plot showed that all data points are distributed below and above the parity line without significant deviation, indicating a good agreement between off-line and in-line viscosity measurements. These results showed that the in situ viscosity measurements gave reliable data of the small variations with the operating conditions. In Fig. 8, viscosity data measured at the three heating temperatures and the five CaCl2 concentrations are plotted against the corresponding Sauter mean chord length measured by FBRM. The graph shows a good correlation (R2 = 0.9593) between in situ size and viscosity measurements, with a non-linear evolution of the aggregates mean size in function of the viscosity of the aggregates solution. This correlation confirms the close relation between the WP aggregates size and the rheology of the WP aggregates solution.
[CaCl2] (mM)
Fig. 6. Dependence of the aggregates size on the heating temperature and the CaCl2 concentration; (a) evolution of aggregates Sauter mean chord length; (b) evolution of FBRM aggregates counts number.
4.2.3. Correlations with turbidity measurements In situ turbidity measurements were considered at the beginning of the work, but due to technical difficulties related to its
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3 75°C 87°C 95°C Native solution
3
75°C 87°C 95°C
2.5 2
A900
Viscosity (mPa s)
(a)
2
1.5 1
1 0.5 0
0 4
5
6
7
4
8
5
6
[CaCl2] (mM) 3
8
Fig. 9. Evolution of aggregates solutions absorbance at 900 nm depending on the heating temperature and the CaCl2 concentration.
2.5
2
75°C 87°C 95°C
1.5 1.5
2
2.5
3
In-line viscosity (mPa s)
Fig. 7. Dependence of the aggregates solutions viscosity on the heating temperature and the CaCl2 concentration; (a) evolution of viscosity data; (b) parity plot of in-line and off-line viscosity data.
implementation, it was decided to still carry out offline measurements. WP aggregated samples were collected at the outlet of the cooling exchanger. Before performing turbidity measurements, collected sample was diluted with the 6% b-lg native solution added with the corresponding calcium concentration, at a dilution rate of 0.1. Evolution of A900 with heating temperature and calcium concentration is plotted in Fig. 9. A clear increase in the turbidity is found with heating temperature and calcium concentration. At 95 °C, turbidity quickly increased from 0.8 to 2.13 between 5 and 6 mM of CaCl2 and seems to reach a saturation level for the highest calcium concentrations. The turbidity increasing may be related to the larger aggregates produced with increasing heating temperatures and calcium concentration. Simons et al. (2002) reported a turbidity increase of a 20 mg/mL b-lg solution heated at 70 °C for 2 h when calcium concentration was increased from 0 to 15 mM,
while the turbidity decreased at higher calcium concentrations (50 and 100 mM). This decrease may be explained by the precipitation of very large aggregates as observed by other authors (Ju and Kilara, 1998). Using a microheater able to mimick industrial time temperature profiles as in the UHT pilot used in this work, Purwanti et al., (2009) studied the effect of heating temperature (50–95 °C) and NaCl concentration (0, 20 and 50 mM) on the turbidity of a WP solution containing 3.2 g/L of b-lg. The authors reported an increase of the turbidity with the temperature of treatment and mentioned that this increase was more pronounced at temperatures above 75 °C. The turbidity also increased quickly with the NaCl concentration, inline with the findings of this work with the CaCl2 concentration. Like calcium ions, sodium ions have been found to promote heat induced aggregation of WP (Xiong, 1992; Majhi et al., 2006; Abhyankar et al., 2010), albeit to a lesser extent. 35
FBRM Sauter chord length(µm)
(b) Off-lineviscosity (mPas)
7
[CaCl2] (mM)
(a)
30 25 20 15 10
75°C 87°C 95°C Regression
5 0 0
0,5
1
1,5
2
2,5
3
A900
3.0
(b)
75°C 87°C 95°C Regression
30
Viscosity (mPa s)
FBRM Sauter chord length (µm)
35
25 20 15
2.5
2.0
1.5
10
75°C 87°C 95°C Regression
5 1.0 0
0.0 1.0
1.5
2.0
2.5
3.0
0.5
1.0
1.5 A 900
2.0
2.5
3.0
Viscosity (mPa s) Fig. 8. Correlation of aggregates Sauter mean chord length with the viscosity.
Fig. 10. Correlation of aggregates (a) Sauter mean chord length and (b) viscosity with the absorbance at 900 nm.
F.T. Ndoye et al. / Journal of Food Engineering 115 (2013) 73–82
Turbidity data can be correlated to aggregates mean size and viscosity. Fig. 10-a and b represented, respectively Sauter mean aggregates size and viscosity data against A900. Both aggregates mean size and viscosity increased non-linearly with turbidity. In the turbidity range between 0 and about 1.5, FBRM Sauter mean chord length and viscosity varied very few. Above a turbidity value of 1.5 (data corresponding to high heating temperatures and high calcium concentrations), aggregates Sauter mean chord length and viscosity increased significantly with turbidity. This result may be linked to the presence of a majority of small particles (<1–2 lm) generated by the protein denaturation and few aggregation at low heating temperatures and low calcium concentrations. These fine aggregates can modify the turbidity (interaction with light at 900 nm) without significantly change the viscosity at the same time. Moreover, particles of this size cannot be detected by the FBRM, so the aggregates Sauter mean size did not change with turbidity. 5. Conclusion The study showed that FBRM and tubular viscometer are promising sensors for in situ characterization of WP aggregation processes. Pronounced sensitivity to heating temperature and calcium concentration has been found for both sensors. Furthermore, aggregates size data obtained with FBRM correlated well with viscosity measurements. Turbidity measurements at 900 nm were also correlated to FBRM and viscosity data and could be use as an indicator of protein aggregation. Results obtained in this work show that in-line characterization of WP aggregation processes changes with short response time is possible. Real time command-control based on such measurements can then permit the development of optimized and more efficient WP aggregation processes. Acknowledgement The authors would like to acknowledge financial support from ANR (Agence Nationale de la Recherche) within the framework of the GLOBULE Project (ANR–08–ALIA–08). References Abhyankar, A.R., Mulvihill, D.M., Fenelon, M.A., Auty, M.A.E., 2010. Microstructural characterization of b-lactoglobulin–konjac glucomannan systems: effect of NaCl concentration and heating conditions. Food Hydrocolloids 24 (1), 18–26. Arellano, M., Benkhelifa, H., Flick, D., Alvarez, G., 2012. Online ice crystal size measurements during sorbet freezing by means of the focused beam reflectance measurement (FBRM) technology. Influence of operating conditions. Journal of Food Engineering 113 (2), 351–359. Cayot, P., Lorient, D., 1998. Effets des traitements thermiques sur la structure des proteines. Structures et Technofonctions des Proteines du Lait. T. Doc. Paris, Lavoisier, pp. 107–129. Cerecero-Enriquez, R., 2003. Etude des Transferts Thermiques et du Comportement à l’écoulement lors de la production d’un sorbet au sein d’un échangeur à surface raclée, Ph.D. INAPG – CEMAGREF, Paris. De Wit, J.N., 2009. Thermal behaviour of bovine beta-lactoglobulin at temperatures up to 150 °C. A review. Trends in Food Science & Technology 20 (1), 27–34. De Wit, J.N., Klarenbeek, G., 1984. Effects of various heat treatments on structure and solubility of whey proteins. Journal of Dairy Science 67 (11), 2701–2710. Donovan, M., Mulvihill, D.M., 1987. Thermal denaturation and aggregation of whey proteins. Irish Journal of Food Science and Technology 11, 87–100. Foegeding, E.A., Davis, J.P., Doucet, D., McGuffey, M.K., 2002. Advances in modifying and understanding whey protein functionality. Trends in Food Science & Technology 13 (5), 151–159. Galani, D., Owusu Apenten, R.K., 1999. Heat-induced denaturation and aggregation of beta-lactoglobulin: kinetics of formation of hydrophobic and disulphidelinked aggregates. International Journal of Food Science & Technology 34 (5–6), 467–476. Giroux, H.J., Houde, J., Britten, M., 2010. Preparation of nanoparticles from denatured whey protein by pH-cycling treatment. Food Hydrocolloids 24 (4), 341–346. Gregory, J., 2009. Monitoring particle aggregation processes. Advances in Colloid and Interface Science 147–148, 109–123.
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Haddad Amamou, A., Benkhelifa, H., Alvarez, G., Flick, D., 2010. Study of crystal size evolution by focused-beam reflectance measurement during the freezing of sucrose/water solutions in a scraped-surface heat exchanger. Process Biochemistry 45 (11), 1821–1825. Heath, A.R., Fawell, P.D., Bahri, P.A., Swift, J.D., 2002. Estimating average particle size by Focused Beam Reflectance Measurement (FBRM). Particle & Particle Systems Characterization 19 (2), 84–95. Hennessy, R.J., 2011. Studying milk coagulation kinetics with laser scanning confocal microscopy Image Processing and Computational modeling. Biomedical Engineering, Master of Science, California Polytechnic State University, San Luis Obispo, p. 89. Hoffmann, M.A.M., Roefs, S.P.F.M., Verheul, M., van Mil, P.J.J.M., De Kruif, K.G., 1996. Aggregation of beta-lactoglobulin studied by in situ light scattering. Journal of Dairy Research 63 (03), 423–440. Hoffmann, M.A.M., van Mil, P.J.J.M., 1999. Heat-induced aggregation of betalactoglobulin as a function of pH. Journal of Agricultural and Food Chemistry 47 (5), 1898–1905. Ju, Z.Y., Kilara, A., 1998. Aggregation induced by calcium chloride and subsequent thermal gelation of whey protein isolate. Journal of Dairy Science 81 (4), 925– 931. Kail, N., Marquardt, W., Briesen, H., 2009. Estimation of particle size distributions from focused beam reflectance measurements based on an optical model. Chemical Engineering Science 64 (5), 984–1000. Lagarrigue, S., Alvarez, G., 2001. The rheology of starch dispersions at high temperatures and high shear rates: a review. Journal of Food Engineering 50 (4), 189–202. Li, M., Wilkinson, D., 2005. Determination of non-spherical particle size distribution from chord length measurements. Part 1. Theoretical analysis. Chemical Engineering Science 60 (12), 3251–3265. Li, M., Wilkinson, D., Patchigolla, K., 2005. Determination of non-spherical particle size distribution from chord length measurements. Part 2. Experimental validation. Chemical Engineering Science 60 (18), 4992–5003. Majhi, P.R., Ganta, R.R., Vanam, R.P., Seyrek, E., Giger, K., Dubin, P.L., 2006. Electrostatically driven protein aggregation: b-lactoglobulin at low ionic strength. Langmuir 22 (22), 9150–9159. Mercadé-Prieto, R., Paterson, W.R., Wilson, D.I., 2009. Effect of salts on the alkaline degradation of b-lactoglobulin gels and aggregates: existence of a dissolution threshold. Food Hydrocolloids 23 (6), 1587–1595. Morr, C.V., Josephson, R.V., 1968. Effect of calcium, N-ethylmaleimide and casein upon heat-induced whey protein aggregation. Journal of Dairy Science 51 (9), 1349–1355. Mounsey, J.S., O’Kennedy, B.T., 2007. Conditions limiting the influence of thioldisulphide interchange reactions on the heat-induced aggregation kinetics of blactoglobulin. International Dairy Journal 17 (9), 1034–1042. Mulvihill, D.M., Donovan, M., 1987. Whey proteins and their thermal denaturation – a review. Irish Journal of Food Science and Technology 11, 43–75. Ndoye, F.T., Erabit, N., Alvarez, G., Flick, D., 2012. Influence of whey protein aggregation on the residence time distribution in a tubular heat exchanger and a helical holding tube during heat treatment process. Journal of Food Engineering 112 (3), 158–167. O’Kennedy, B.T., Mounsey, J.S., 2009. The dominating effect of ionic strength on the heat-induced denaturation and aggregation of [beta]-lactoglobulin in simulated milk ultrafiltrate. International Dairy Journal 19 (3), 123–128. Petit, J., Herbig, A.L., Moreau, A., Delaplace, G., 2011. Influence of calcium on blactoglobulin denaturation kinetics: implications in unfolding and aggregation mechanisms. Journal of Dairy Science 94 (12), 5794–5810. Purwanti, N., Fox, M., Schroën, K., De Jong, P., 2009. The microheater: a new tool for rapid determination of food kinetics. Journal of Food Engineering 91 (1), 78–84. Roefs, S.P.F.M., Kruif, K.G., 1994. A model for the denaturation and aggregation of beta-lactoglobulin. European Journal of Biochemistry 226 (3), 883–889. Ruf, A., Worlitschek, J., Mazzotti, M., 2000. Modeling and experimental analysis of PSD measurements through FBRM. Particle & Particle Systems Characterization 17 (4), 167–179. Ryan, K.N., Vardhanabhuti, B., Jaramillo, D.P., van Zanten, J.H., Coupland, J.N., Foegeding, E.A., 2012. Stability and mechanism of whey protein soluble aggregates thermally treated with salts. Food Hydrocolloids 27 (2), 411–420. Sava, N., Van der Plancken, I., Claeys, W., Hendrickx, M., 2005. The kinetics of heatinduced structural changes of b-lactoglobulin. Journal of Dairy Science 88 (5), 1646–1653. Sawyer, W.H., 1968. Heat denaturation of bovine beta-lactoglobulins and relevance of disulfide aggregation. Journal of Dairy Science 51 (3), 323–329. Schokker, E.P., Singh, H., Pinder, D.N., Norris, G.E., Creamer, L.K., 1999. Characterization of intermediates formed during heat-induced aggregation of beta-lactoglobulin AB at neutral pH. International Dairy Journal 9 (11), 791–800. Sherwin, C.P., Foegeding, E.A., 1997. The effects of CaCl2 on aggeregation of whey proteins. Milchwissenschaft 52, 93–96. Simmons, M.J.H., Jayaraman, P., Fryer, P.J., 2007. The effect of temperature and shear rate upon the aggregation of whey protein and its implications for milk fouling. Journal of Food Engineering 79 (2), 517–528. Simons, J.-W.F.A., Kosters, H.A., Visschers, R.W., de Jongh, H.H.J., 2002. Role of calcium as trigger in thermal b-lactoglobulin aggregation. Archives of Biochemistry and Biophysics 406 (2), 143–152. Steffe, J.F., 1996. Tube Viscometry. Rheological Methods in Food Process Engineering. F. Press., East lansing MI, USA, pp. 94–156. Tolkach, A., Kulozik, U., 2007. 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Walstra, P., Jenness, R., 1984. Dairy Chemistry and Physics. John Wiley and Sons, New York. Wynn, E.J.W., 2003. Relationship between particle-size and chord-length distributions in focused beam reflectance measurement: stability of direct inversion and weighting. Powder Technology 133 (1–3), 125–133. Xiong, Y.L., 1992. Influence of pH and ionic environment on thermal aggregation of whey proteins. Journal of Agricultural and Food Chemistry 40 (3), 380–384. Xiong, Y.L., Dawson, K.A., Wan, L., 1993. Thermal aggregation of b-lactoglobulin: effect of pH, ionic environment, and thiol reagent. Journal of Dairy Science 76 (1), 70–77.
Contents lists available at SciVerse ScienceDirect
Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
In-line characterization of a whey protein aggregation process: Aggregates size and rheological measurements Fatou Toutie Ndoye a,⇑, Nicolas Erabit a,b,c, Denis Flick b,c, Graciela Alvarez a a
Irstea, Refrigeration Processes Engineering Research Unit, Antony, France AgroParisTech, UMR1145 Ingénierie Procédés Aliments, Massy, France c INRA, UMR1145 Ingénierie Procédés Aliments, F-91300 Massy, France b
a r t i c l e
i n f o
Article history: Received 20 June 2012 Received in revised form 26 September 2012 Accepted 28 September 2012 Available online 6 October 2012 Keywords: In-line measurements FBRM Particle size analysis Tubular viscometer Whey protein Aggregation
a b s t r a c t Heat induced whey protein (WP) aggregates functionalities have been found to strongly depend on aggregates size distribution. The latter is affected by the time/temperature/shear rate history, which affects rheological properties. This work evaluates the potential of the focused beam reflectance measurement (FBRM), an in situ granulometric method, and of an in-house developed tube viscometer, to perform in situ characterization of a WP aggregation process. Potential of turbidity techniques to characterize the process was also explored by off-line measurements. Experiments were investigated with WP solutions of 6% b-lactoglobulin added with different concentrations of CaCl2, and heated at three different holding temperatures. Aggregates mean size, viscosity and turbidity increase with holding temperatures and CaCl2 concentration as expected. A good correlation was found between viscosity data and aggregates mean sizes. The latter were also well correlated with turbidity at 900 nm. FBRM and tube viscometer are shown to be promising and valuable tools to perform in-line characterization of heat induced WP aggregation process. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Whey proteins are widely used as ingredients in elaborated food products, not only for their nutritional properties but also for their functional properties. Among the functional properties of whey protein (WP), aggregation which are related to protein– protein interactions, are of great importance. When heated, WP molecule, particularly the b-lactoglobuline (b-lg), unfolds (denaturation) and aggregation then occurs due to sulfhydryl (–SH) binding, hydrophobic interactions and intermolecular proteinion-protein cross-linking (Morr and Josephson, 1968; Cayot and Lorient, 1998). Heat induced WP aggregates can be used for structure or sensory properties improvement of food, but these aggregates functionalities strongly depend on the aggregates size. Several studies have demonstrated that aggregates size is affected by physicochemical (pH, ionic strength, protein concentration) and processing (heating and cooling kinetics, flow, shear) conditions (Xiong, 1992; Hoffmann et al., 1996; Verheul et al., 1999; Foegeding et al., 2002; Simmons et al., 2007; De Wit, 2009). In particular, it has been recognised that the presence of divalent calcium ions enhances WP heat induced aggregation and strongly influenced WP aggregates size. The time/temperature/shear rate ⇑ Corresponding author. E-mail address: [email protected] (F.T. Ndoye). 0260-8774/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jfoodeng.2012.09.021
history also affects rheological properties (Ndoye et al., 2012), and aggregates size distribution (ASD). Therefore, ASD and viscosity monitoring and control are of major interest. Existing microparticulation processes consist on a heat treatment followed by a mechanical step of shearing (high pressure homogeneisation) to break aggregates for the desired size. Most of these processes have been developed through trials and errors, but an optimization of the current process is still possible, especially by in situ characterization of heat induced WP aggregation. Conventional granulometric and rheological methods need sampling which could damage the aggregates and introduce systematic errors. In situ measurements provide a key insight in the dominate process mechanisms and a quick assessment of the impact of process variables on ASD and viscosity. Furthermore, real time analytics make possible the following of the overall aggregation process with time and offer the possibility to rapidly act on the physicochemical and operating conditions. The objective of the work was to implement in situ sensors in a laboratory scale heat treatment device to monitor the aggregation of WP. In-line particles characterisation techniques and rheological characterisation methods have been selected. A feasibility study was performed to assess the potential of innovative in situ sensors to properly follow the WP aggregates properties (ASD, aggregates mean size, viscosity). Integration of the selected sensors in the heat treatment pilot allowed inline measurements of the WP aggregates
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Nomenclature A Lt dP Q R
absorbance (–) tube length (m) pressure drop (Pa) flow rate (m3 s1) tube radius (m)
l r
dynamic viscosity (Pa s) shear stress (Pa)
Indices w
tube wall
Greek letters c_ shear rate (s1)
size and of the viscosity of the suspension depending on various calcium concentrations and heating conditions. 2. Selection of in situ sensors for WP aggregation process characterization Techniques of characterization of WP aggregates are often based on various physical properties of aggregates. But the choice of the most suitable measurement technique must be made with consideration for the type of results desired and the practicalities of the situation. Multiple analytical techniques ensure that the true nature of the aggregates is determined and that the biases inherent in each measuring technique do not give a misleading representation. In this work, it was chosen to combine several techniques for the characterisation of heat induced b-lg aggregate: the Focused Beam Reflectance Measurement (FBRM) method and the tubular viscometry respectively for aggregates size and for rheological characterizations; a turbidity technique, by use of a spectrophotometer, to have global information about protein particles size and concentration of the suspension. 2.1. In situ aggregates size measurements Counting the number of particles and sizing them is the earliest method to follow aggregation and particles processes. An innovative in situ method of counting and sizing particles and aggregates in food technology is FBRM. Haddad Amamou et al. (2010) had given a detailed description of the FBRM measurement principle. The technology returns a chord length distribution (CLD) which depends on both the particle size distribution (PSD) and the shape of the particle. However, the relationship between chord length and particle size is not straightforward. This problem has been examined by many authors who proposed theoretical methods for the translation of a measured CLD into its PSD (Heath et al., 2002; Wynn, 2003; Li and Wilkinson, 2005; Kail et al., 2009). Several studies have been devoted to the comparison of FBRM response to conventional particle sizing techniques. Heath et al. (2002) used laser diffraction (Malvern Mastersizer) and electrical sensing zone technique (Coulter Multisizer) as alternative sizing techniques. They analysed the effects on the measured FBRM CLD of several factors such as the mode of weighting the distribution, the position of the focal point, the agitation speed, the particle properties and the suspension volume fraction. They concluded that the mean of square-weighted chord lengths compare well with the mean diameter obtained with the used conventional sizing techniques over the range from 50 to 400 lm. They also found that the square-weighted FBRM results were not affected by changes of the focal position, the suspension fluid flow velocity, or the solid fraction. Similar study achieved by Li et al. (2005); compared FBRM technique with laser diffraction (LD), ultrasonic attenuation spectroscopy (UAS) and image analysis (IA). They found a good agreement between PSDs obtained by LD, UAS and IA, but that particle shape strongly affects the result obtained by different techniques.
They also showed that the CLD measured by FBRM is complex, depending not only on the PSD, but also on particle optical properties and shape, especially for particles diameters <20 lm. 2.2. In situ rheological characterization Rheological data can be used as a quantitative indicator of product microstructure with associated links to quality attributes. Measurements of viscosity data from a single point, as it is often done, give no information about the flow behaviour. The viscosity of a fluid during processing can increase in linear or a non linear way with a transition from Newtonian to non-Newtonian behaviour as the total solids concentration is increased (Walstra and Jenness, 1984). WP aggregation phenomena induce a modification of the protein volume fraction and, consequently of the apparent viscosity of the fluid. Aggregate size seems to influence the apparent viscosity. Walkenström et al. (1999) pointed out the dependence of the WP aggregates suspension viscosity on the microstructure (various sizes and compactness) which is determined by process parameters. A decreased viscosity related to a decreased aggregate size is observed with an increasing shear rate. The challenge is to perform online rheological measurements and not only a viscosity measurement after aggregation treatment, in order to characterize the fluid flow behaviour and monitor changes occurring according to process conditions. Tube viscometers have been used by several authors to measure the flow behaviour of gelatinized starch dispersions at high temperature (Lagarrigue and Alvarez, 2001). Online rheometry system composed of a set of pipes of several diameters have been successfully used to follow a crystallization process (Cerecero-Enriquez, 2003). This type of viscometer is based on Rabinowitsch–Mooney equation (Eq. (1)) relating wall shear stress rw (Eq. (2)) to wall shear rate c_ w for laminar and steady flow (Steffe, 1996):
c_ w ¼ f ðrw Þ ¼
rw ¼
3Q
pR3
" # dðQ =ðpR3 ÞÞ þ rw drw
ðdPÞR 2Lt
ð1Þ
ð2Þ
The pressure drop dP is measured over a fixed length Lt of a horizontal tube of inside radius R for a given volumetric flow rate Q. Plotting the apparent wall shear rate versus the wall shear stress for different operating conditions and each diameter of tube allow to determine the rheological behaviour of the fluid. 2.3. Turbidity characterization The properties of suspended particles or aggregates can also be derived by measurement of turbidity. A light beam illuminating a suspension of particles is partially scattered. The intensity of light scattering depends on particles size, shape concentration and refractive index of the surrounding liquid, as well as on the light wavelength and the angle of detection. An increase in particle size
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makes a very large increase in scattered light intensity and an increase in the proportion of light scattered at low angles (Gregory, 2009). Scattering by particles causes a reduction of transmitted light intensity through the suspension. The change in turbidity might be a useful measure of the degree of WP aggregation. Turbidity is often measured as absorbance with a spectrophotometer at wavelengths where the attenuation of the light beam is essentially due to scattering and absorption is negligible (Ju and Kilara, 1998; Simons et al., 2002; Giroux et al., 2010). 3. Materials and methods 3.1. Experimental procedure The experimental procedure adopted in this work is as following: The first step of the work is a feasibility study devoted to the evaluation of FBRM as an innovative sensor technology to detect and measure WP aggregation, and of the ability of spectrophotometer to characterize WP aggregation. The second step of the study is the integration of in situ sensors (FBRM, and tube viscometer) in a heat treatment pilot for inline characterization of the b-lg heat induced aggregation process. Off-line measurements were performed with the spectrophotometer for correlation with FBRM and viscosity measurements, and with a rheometer to compare with in-line viscosity data. 3.2. Whey protein A commercial b-lg powder containing 88.9% of b-lg was used for experiments. b-lg is the major WP in milk and its aggregation kinetic is known to govern the one of the whole WPs. The protein was rehydrated at 6% w/w in distilled water at 40 °C. Calcium was added to the protein solution during the preparation using a 1 M CaCl2 solution. Concentrations used in this study were varied between 5.0 and 7.0 mM of CaCl2. The solution was kept at 40 °C at least 2 h to achieve a complete rehydration. Measurement of the pH solution gives a value of 6.8. 3.3. Heat treatments Two heat treatment methods have been used in this work: a static system for the feasibility study and a dynamic device consisting on a heat treatment laboratory scale plant for in situ measurements. For static heating, 6% b-lg solutions with 6.6 mM CaCl2 were placed in sealed glass tubes (1 cm internal diameter) fitted with thermocouple to allow the acquisition of temperature during heating. Samples were first heated in boiling water for 1 min before being held in a thermostatically controlled bath (Fluke 7340 – USA). Samples removed from the bath, were immediately placed in water at room temperature (20–25 °C) to stop aggregation process. Investigated conditions are temperatures of 75 °C, 80 °C, 85 °C and 90 °C for 1, 3 and 5 min of holding time. This system has been used to assess the applicability of FBRM and spectrophotometer to WP aggregation. For in situ characterisation of aggregates, experiments were performed using an ultra high temperature (UHT) laboratory scale plant (Armfield FT174X, Ringwood, UK) schematized on Fig. 1. The heat treatment system has a nominal capacity of 20–50 L/h where the product is fed by a volumetric pump. In situ sensors were implemented at the outlet of the cooler to characterize the process in real time. Experiments in the UHT device have been performed with 6% b-lg solutions with 5 different concentrations of CaCl2 (5.0, 6.0, 6.4, 6.6 and 7.0 mM) and at 3 different temperatures (75 °C,
75
87 °C and 95 °C) with a constant holding time of 60 s and at a constant flow rate of 20 L/h. 3.4. Instrumentation 3.4.1. FBRM instrument The LasentecÒ FBRM S400-8 (Mettler Toledo, USA) was used to measure CLDs in experiments. FBRM instrument is composed of a measurement probe, an electronic measurement unit and a computer for data acquisition and analysis. The probe, schematized on Fig. 2 is in stainless steel and has an external diameter of 9.5 mm and a length of 120 mm. The laser beam is projected through the sapphire window at a wavelength of 780 nm. For batch analysis, the probe is inserted into the agitated suspension with an inclination of 45° using a dedicated stand (LasentecÒ). The data analysis software gives the counts number per second for each selected size class of chord length. CLDs are given in different formats such as non weighted, square weighted and cubic weighted distribution in 100 predefined size classes. Thousands of chords can be measured per second, giving a CLD from 1 lm to more than 1 mm. Square-weighted mean chord length can be compared to the Sauter mean diameter defined as the diameter of monodispersed spheres that would have the same volume and the same surface area as the measured polydispersed particles. A dedicated device was designed in order to integrate FBRM probe on the UHT pilot for in-line measurements (Fig. 1). It comprises a derivation for the dilution of the aggregates suspensions outgoing the cooling section of the pilot with non-heated b-lg solution. A flowmeter is used in order to set the derivated flux. The probe is inserted in counter-current into the tube where circulates the diluted suspension, with an inclination of 45 °C. This inclination allows the renewal of the flux near the probe window. Arellano et al. (2012) has compared the FBRM probe used in this work with an IA method using Polyamid Seeding Particles (PSP) of known mean diameter. Very good agreement was found between the Sauter mean chord lengths obtained by FBRM measurements and those given by IA. This result validated the FBRM technique as a method of particle size measurement. 3.4.2. Tubular viscometer A tubular viscometer has been designed and manufactured in order to follow indirectly the protein aggregation process (Fig. 1). The system is formed of 4 PVC ducts of a length of 100 cm and of several diameters placed in series. In order to maintain a laminar flow regime inside the ducts, diameters of 5.8, 10, 13 and 16.7 mm have been retained. Each duct is equipped with two piezometric rings separated by 50 cm and at 25 cm of the extremity of the tube. These piezometric rings allow a homogeneous pressure and constitute the measurement point of pressure drop. Preliminary study with water has been performed in order to validate the system. A difference <3% was found between the measured water viscosity and the given values in the literature for temperature ranging from 4 °C to 25 °C. For online measurements with the UHT Armfield pilot, the tubular viscometer is placed at the outlet of the cooling exchanger. Aggregates suspension flowed inside the tubes at room temperature. Variation on temperature was continuously recorded using temperature sensors (Pt100) placed at the inlet and at the outlet of the set of pipes. Raw data of this online rheological measurement unit are pressure drop and flow rate continuously recorded respectively with a differential pressure drop transmitter and a flowmeter transmitter. These data allow calculating the wall shear stress and the apparent wall shear rate in order to represent the fluid behaviour using the Rabinowitsch–Mooney equation (Eq. (1). For Newtonian behaviour, the viscosity of the aggregates suspension can be obtained using the Poiseuille–Hagen law (Eq. (3)):
l¼
pdPR4 8Lt Q
ð3Þ
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Fig. 1. UHT pilot plant and in-line measurements instrumentation.
Off-line viscosity measurements were achieved systematically after each experiment in the pilot with a rheometer (ReoRox G2, Medirox AB, Sweden) based on the patented FOR (Free Oscillation Rheometry) technology. This device, calibrated for low viscosity (0.5–4 mPa s) measurements, uses a torsion wire system to set a sample into oscillation. The viscosity of the sample is calculated by analysing the frequency change and the amplitude damping from free oscillation. The technology is especially suitable for low viscous material. The FOR principle is widely used to study the kinetic of milk coagulation (Hennessy, 2011). Assays were done at 20 °C and in triplicate. The goal was to compare these measurements with the in situ ones and to validate the tubular viscometer. 3.4.3. Turbidimetric technique A spectrophotometer (Beckman Coulter DU 730, Fullerton, USA) was used for turbidity measurements. Spectrophotometer is employed to measure the amount of light that a sample absorbs or
scatters at a given wavelength, this amount of light being linked with the suspended particles sizes and concentration. The instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching the photometer. Measurements were carried out by wavelength scan with 1 nmwavelength intervals in the range of 190 and 1100 nm, giving absorbance spectra. 4. Results and discussion 4.1. Offline whey protein aggregation characterisation 4.1.1. Offline FBRM analyzes The ability of FBRM technique to characterize WP aggregation process has been assessed by investigating the effect of heating temperatures and times on b-lg solutions. Fig. 3-a represents the aggregates square weighted cumulative CLD obtained at heating
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Square wieghted cumulative frequency (%)
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100 94%
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Fig. 3. Effect of heating temperature on a 6% b-lg solution aggregation (6.6 mM CaCl2 – 5 min of holding time); (a) square weighted cumulative CLD; (b) sauter mean chord length and (c) total counts number.
temperatures of 77 °C, 80 °C, 85 °C and 90 °C during 5 min of holding. Investigations at 75 °C are not reported because the FBRM measurements did not give significant values (counts number <300 s1). It is shown a remarkable difference between the four distributions. Large aggregates are obtained with increased heating temperature. Measurements have underlined that 94% of aggregates formed at 77 °C have a chord length between 1 and 10 lm while this rate decreases at 24% at 90 °C in favour of larger aggregates. This tendency is confirmed by the calculation of aggregates Sauter mean chord length as shown in Fig. 3-b, on which it can be seen an increasing in mean aggregates size with temperature. Total counts number of aggregates also increased with increasing temperature as can be seen in Fig. 3-c. These results are in agreement with the previous findings that heat induced WP aggregates size increase with heating temperature and time (Simmons et al., 2007). Results are also inline with earlier studies which reported that irreversible WP aggregation only occurs at temperatures above 75 °C (De Wit and Klarenbeek, 1984; Hoffmann and van Mil, 1999; Tolkach and Kulozik, 2007). FBRM measurements also showed the effect of heating time. A slight increase of the aggregate mean chord length was found as the holding time is extended. These preliminary measurements showed that FBRM is a valuable tool for characterizing aggregation of WPs in solution. The effect of aggregates fraction on FBRM measurements has been investigated. Aggregates concentration has been varied by diluting pure aggregated suspensions with the raw solution (non aggregated solution of 6% b-lg – 6.6 mM CaCl2) at protein mass fraction ranging from 0.15% w/w to 6% w/w. Experiments were performed with two aggregated samples produced at the UHT pilot at different conditions (87 °C and 18 s of holding time and 87 °C and 60 s of holding time). Fig. 4-a and b represent respectively the unweighted and the square weighted cumulative CLDs obtained with a b-lg aggregates suspension produced on the UHT pilot at a heating temperature of 87 °C for 18 s of holding time. Plots in Fig. 4-a reveal important differences between the pure suspension (6% w/w) CLD and the diluted suspensions CLDs. It seems that the sensitivity of FBRM to fine particles is increased in concentrated solutions. Results show that FBRM oversizes small particles (<10 lm) and undersizes larger ones (>10 lm) in the pure sample of b-lg aggregates. This is probably due to the overlapping of aggregates at high concentration or to fines overcrowding in the measurement zone near the window. Ruf et al. (2000) reported a masking effect of fines particles yielding to their overestimation by FBRM. However, by applying a normalized chord square weighting to the distributions, it can be seen in Fig. 4-b a progressive shift of the CLDs to the large sizes zone, with the concentration
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Chord length (µm) Fig. 4. Effect of the aggregates mass fraction on the (a) unweighted and the (b) square weighted cumulative CLD (6% b-lg; 6.6 mM CaCl2; 87 °C/18 s; 20 L/h).
decreasing. For the most diluted solutions (0.15% w/w, 0.3% w/w and 0.6% w/w, respectively a dilution rate of 0.025, 0.05 and 0.1), the three distributions are tightening with a constant Sauter mean chord lengths of about 57 lm, while the 6% w/w solution had a Sauter mean chord lengths of 21 lm. Comparative results were found with aggregated suspension produced at 87 °C and 60 s of holding time. Heath et al. (2002) have also reported similar results for aluminum particles showing that the solid volume fraction largely affects the CLD. On the basis of these results, it seems that a dilution rate of 0.1 is a threshold value to overcome the effect of particles volume fraction. This result suggests the use of a derivative system in the UHT pilot for the installation of FBRM sensor for in situ measurements, in order to make dilution, as shown on Fig. 1. 4.1.2. Offline turbidimetric measurements To assess spectrophotometry as a technique of characterization of WP aggregates, turbidity measurements were carried out with WP aggregated samples produced by static heat treatment at four different temperatures (75 °C, 80 °C, 85 °C and 90 °C) during 5 min. Fig. 5-a shows the influence of heating temperature on absorbance spectra of b-lg aggregates suspensions. Between 190 and 500 nm, there is overlap between spectra obtained at 85 °C and 90 °C, while spectra obtained at 75 °C and 80 °C showed noticeable difference. Above the overlapping region, absorbance increases with heating temperature. The difference is the highest at about 900 nm. It is known that the transmitted light is reduced when the particle size increases, then increasing the turbidity of the analyzed suspension (Gregory, 2009). So variations obtained on turbidity data may be explained by the aggregates size increasing with heating temperature as seen above with FBRM measurements. These results have proven the ability of the turbidity technique to characterize WP aggregation process. The dependence of turbidity on aggregates fraction was studied. Fig. 5-b shows an effect of the protein mass fraction on the
Fig. 5. Absorbance spectra of aggregates suspension; (a) effect of the heating temperature (6% b-lg; 6.6 mM CaCl2), (b) effect of the aggregate mass fraction (87 °C/18 s; 20 L/h).
absorbance of the WP suspension obtained with a b-lg aggregates suspension produced on the UHT pilot at a heating temperature of 87 °C for 18 s of holding time. Aggregates solutions at 6% w/w, 2% w/w and 1.2% w/w showed spectrum with absorbencies essentially constant with the wavelength. Important absorbance variations with wavelength were found for the most diluted solution (0.6% w/w, 0.3% w/w and 0.15% w/w). Turbidity decreased with the particles mass fraction decreasing. It may be attributed to a difference of sensitivity according to the wavelength (Ryan et al., 2012). It was chosen to use absorbance at 900 nm (A900) in order to give exhaustive comparisons in further sections and to be sure that measured absorbance data are only due to light scattering, in other words, due to turbidity (negligible protein light absorption at 900 nm). It was also chosen to work at a dilution rate of 0.1 for turbidity off-line measurements, in coherence with FBRM measurements. 4.2. In-line whey protein aggregation characterization Aggregates size and viscosity measurements were performed in-line on the UHT pilot plant. Off-line turbidity and viscosity measurements were systematically carried out for each experiment, respectively with the spectrophotometer and the rheometer in order to compare with in situ measurements. Two variables: the heating temperature and the CaCl2 concentration were investigated to measure the in-line sensors sensibility on WP aggregation process. 4.2.1. Aggregates size FBRM measurements were continuously performed during the aggregation process and allow the observation of real-time changes in aggregates counts and size. Fig. 6-a showed the evolution of Sauter aggregates mean chord size with temperature and calcium concentration. It can be seen an increasing of aggregates mean size with both heating temperature and concentration in
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CaCl2. These results suggest that aggregation is promoted by temperature on the one hand and by calcium ions on the other hand, or by their combination, as it was widely recognized in the literature (Morr and Josephson, 1968; Donovan and Mulvihill, 1987; Mulvihill and Donovan, 1987; Galani and Owusu Apenten, 1999; Simons et al., 2002; Mounsey and O’Kennedy, 2007; Simmons et al., 2007; Tolkach and Kulozik, 2007; Mercadé-Prieto et al., 2009). Upon heating above 70 °C near neutral pH, b-lg native molecules undergo conformational changes exposing buried thiol groups and hydrophobic residues (Verheul et al., 1998; Schokker et al., 1999; Sava et al., 2005). The so denaturated molecules aggregate according to a two-consecutive-steps mechanism: (1) formation of b-lg oligomers through the establishment of intermolecular thiol-disulphide interactions and thiol–thiol oxidation, (2) formation of b-lg polymers via non-covalent interactions between hydrophobic residues (Sawyer, 1968; Roefs and Kruif, 1994; Schokker et al., 1999). Increasing temperature increases the accessibility of the thiol groups and then the rate of aggregation via thiol-disulphide exchange reaction resulting in larger aggregates (De Wit, 2009). Results of this work are inline with findings of Simmons et al. (2007) who reported that small and weakly bonded aggregates of WP are formed at temperature below 75 °C while large and strong aggregates are obtained at higher temperatures. Furthermore, at elevated temperature, the presence of salts like CaCl2 result in more stable hydrophobic interactions inducing aggregates size increasing (Xiong et al., 1993; Sherwin and Foegeding, 1997; Simons et al., 2002; Mounsey and O’Kennedy, 2007; O’Kennedy and Mounsey, 2009). However, investigating the role of calcium in b-lg aggregation, more recent studies of Simons et al. (2002) and Mounsey and O’Kennedy (2007) have questioned the order of the two-steps aggregation mechanism
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described above. They rather suggested that non-covalent physical interactions were mainly responsible for the heat-induced primary aggregation in presence of calcium at neutral pH. Secondary formation of covalent bonds through thiol-disulphide interchange reactions would then proceed leading to irreversible aggregate formation. Simons et al. (2002) reported that calcium acts in the screening of surface charges and promotes aggregation by binding to carboxylates with a threshold affinity. This result have been confirmed by Mounsey and O’Kennedy (2007) who also found that a stoichiometric ratio of calcium to protein was required to initiate aggregate formation. This result is consistent with that showed on Fig. 6-b representing the evolution of the aggregates counts number/s with heating temperature and CaCl2 concentration. It can be seen on Fig. 6-b that the number of particles increases with temperature but decreases slightly with calcium concentration. The decrease in aggregates concentration when the calcium concentration increases may be explain by the promoting effect of calcium in the aggregation of the denaturated molecules, resulting in larger aggregates and reducing the total number of aggregates in suspension. Giroux et al. (2010) have calculated WP aggregates voluminosity and reported that the protein concentration in aggregates (which is the reciprocal of voluminosity) increased with calcium chloride concentration, giving more compact and less porous aggregates. An increase of protein concentration in aggregates results on a decrease in aggregates concentration in the suspension. Reduction in particles number is more frank above 6 mM of CaCl2, corresponding to an excess of calcium. The increase in aggregates concentration with temperature can be linked to the denaturation level which increase fastly with heating temperature as mentioned in previous works (Petit et al., 2011). 4.2.2. Viscosity In situ viscosity characterization has been carried out by direct measurement of differential pressure drop with an in-house developed tubular viscometer. Results showed a Newtonian behaviour in the investigated conditions, and the viscosity of the aggregates suspension was derived using the Poiseuille–Hagen law (Eq. (3)). Evolution of the viscosity of the aggregates suspension as a function of heating temperature and CaCl2 concentration is shown in Fig. 7-a. The doted line represents the viscosity of the native b-lg solution (no thermal treatment). As expected, increased heating temperature resulted in increased viscosity. At a given heating temperature, the b-lg aggregates solution became more viscous as the CaCl2 concentration increased. Maximum viscosity was reached at 95 °C and 6.6 mM of CaCl2, increasing the viscosity almost by a factor 2. The increase in aggregates size induces an increase particles volume fraction resulting in a more viscous solution (Vardhanabhuti and Foegeding, 1999). In-line viscosity measurements were compared to the off-line ones in Fig. 7-b. The parity plot showed that all data points are distributed below and above the parity line without significant deviation, indicating a good agreement between off-line and in-line viscosity measurements. These results showed that the in situ viscosity measurements gave reliable data of the small variations with the operating conditions. In Fig. 8, viscosity data measured at the three heating temperatures and the five CaCl2 concentrations are plotted against the corresponding Sauter mean chord length measured by FBRM. The graph shows a good correlation (R2 = 0.9593) between in situ size and viscosity measurements, with a non-linear evolution of the aggregates mean size in function of the viscosity of the aggregates solution. This correlation confirms the close relation between the WP aggregates size and the rheology of the WP aggregates solution.
[CaCl2] (mM)
Fig. 6. Dependence of the aggregates size on the heating temperature and the CaCl2 concentration; (a) evolution of aggregates Sauter mean chord length; (b) evolution of FBRM aggregates counts number.
4.2.3. Correlations with turbidity measurements In situ turbidity measurements were considered at the beginning of the work, but due to technical difficulties related to its
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Fig. 7. Dependence of the aggregates solutions viscosity on the heating temperature and the CaCl2 concentration; (a) evolution of viscosity data; (b) parity plot of in-line and off-line viscosity data.
implementation, it was decided to still carry out offline measurements. WP aggregated samples were collected at the outlet of the cooling exchanger. Before performing turbidity measurements, collected sample was diluted with the 6% b-lg native solution added with the corresponding calcium concentration, at a dilution rate of 0.1. Evolution of A900 with heating temperature and calcium concentration is plotted in Fig. 9. A clear increase in the turbidity is found with heating temperature and calcium concentration. At 95 °C, turbidity quickly increased from 0.8 to 2.13 between 5 and 6 mM of CaCl2 and seems to reach a saturation level for the highest calcium concentrations. The turbidity increasing may be related to the larger aggregates produced with increasing heating temperatures and calcium concentration. Simons et al. (2002) reported a turbidity increase of a 20 mg/mL b-lg solution heated at 70 °C for 2 h when calcium concentration was increased from 0 to 15 mM,
while the turbidity decreased at higher calcium concentrations (50 and 100 mM). This decrease may be explained by the precipitation of very large aggregates as observed by other authors (Ju and Kilara, 1998). Using a microheater able to mimick industrial time temperature profiles as in the UHT pilot used in this work, Purwanti et al., (2009) studied the effect of heating temperature (50–95 °C) and NaCl concentration (0, 20 and 50 mM) on the turbidity of a WP solution containing 3.2 g/L of b-lg. The authors reported an increase of the turbidity with the temperature of treatment and mentioned that this increase was more pronounced at temperatures above 75 °C. The turbidity also increased quickly with the NaCl concentration, inline with the findings of this work with the CaCl2 concentration. Like calcium ions, sodium ions have been found to promote heat induced aggregation of WP (Xiong, 1992; Majhi et al., 2006; Abhyankar et al., 2010), albeit to a lesser extent. 35
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Fig. 10. Correlation of aggregates (a) Sauter mean chord length and (b) viscosity with the absorbance at 900 nm.
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Turbidity data can be correlated to aggregates mean size and viscosity. Fig. 10-a and b represented, respectively Sauter mean aggregates size and viscosity data against A900. Both aggregates mean size and viscosity increased non-linearly with turbidity. In the turbidity range between 0 and about 1.5, FBRM Sauter mean chord length and viscosity varied very few. Above a turbidity value of 1.5 (data corresponding to high heating temperatures and high calcium concentrations), aggregates Sauter mean chord length and viscosity increased significantly with turbidity. This result may be linked to the presence of a majority of small particles (<1–2 lm) generated by the protein denaturation and few aggregation at low heating temperatures and low calcium concentrations. These fine aggregates can modify the turbidity (interaction with light at 900 nm) without significantly change the viscosity at the same time. Moreover, particles of this size cannot be detected by the FBRM, so the aggregates Sauter mean size did not change with turbidity. 5. Conclusion The study showed that FBRM and tubular viscometer are promising sensors for in situ characterization of WP aggregation processes. Pronounced sensitivity to heating temperature and calcium concentration has been found for both sensors. Furthermore, aggregates size data obtained with FBRM correlated well with viscosity measurements. Turbidity measurements at 900 nm were also correlated to FBRM and viscosity data and could be use as an indicator of protein aggregation. Results obtained in this work show that in-line characterization of WP aggregation processes changes with short response time is possible. Real time command-control based on such measurements can then permit the development of optimized and more efficient WP aggregation processes. Acknowledgement The authors would like to acknowledge financial support from ANR (Agence Nationale de la Recherche) within the framework of the GLOBULE Project (ANR–08–ALIA–08). References Abhyankar, A.R., Mulvihill, D.M., Fenelon, M.A., Auty, M.A.E., 2010. Microstructural characterization of b-lactoglobulin–konjac glucomannan systems: effect of NaCl concentration and heating conditions. Food Hydrocolloids 24 (1), 18–26. Arellano, M., Benkhelifa, H., Flick, D., Alvarez, G., 2012. Online ice crystal size measurements during sorbet freezing by means of the focused beam reflectance measurement (FBRM) technology. Influence of operating conditions. Journal of Food Engineering 113 (2), 351–359. Cayot, P., Lorient, D., 1998. Effets des traitements thermiques sur la structure des proteines. Structures et Technofonctions des Proteines du Lait. T. Doc. Paris, Lavoisier, pp. 107–129. Cerecero-Enriquez, R., 2003. Etude des Transferts Thermiques et du Comportement à l’écoulement lors de la production d’un sorbet au sein d’un échangeur à surface raclée, Ph.D. INAPG – CEMAGREF, Paris. De Wit, J.N., 2009. Thermal behaviour of bovine beta-lactoglobulin at temperatures up to 150 °C. A review. Trends in Food Science & Technology 20 (1), 27–34. De Wit, J.N., Klarenbeek, G., 1984. Effects of various heat treatments on structure and solubility of whey proteins. Journal of Dairy Science 67 (11), 2701–2710. Donovan, M., Mulvihill, D.M., 1987. Thermal denaturation and aggregation of whey proteins. Irish Journal of Food Science and Technology 11, 87–100. Foegeding, E.A., Davis, J.P., Doucet, D., McGuffey, M.K., 2002. Advances in modifying and understanding whey protein functionality. Trends in Food Science & Technology 13 (5), 151–159. Galani, D., Owusu Apenten, R.K., 1999. Heat-induced denaturation and aggregation of beta-lactoglobulin: kinetics of formation of hydrophobic and disulphidelinked aggregates. International Journal of Food Science & Technology 34 (5–6), 467–476. Giroux, H.J., Houde, J., Britten, M., 2010. Preparation of nanoparticles from denatured whey protein by pH-cycling treatment. Food Hydrocolloids 24 (4), 341–346. Gregory, J., 2009. Monitoring particle aggregation processes. Advances in Colloid and Interface Science 147–148, 109–123.
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