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LWT 40 (2007) 737743 www.elsevier.com/locate/lwt

Research Note

Non-invasive through-package assessment of the microstructural quality of a model food emulsion by the NMR MOUSE
Adrian M. Haiduca, Elena E. Trezzaa, Dagmar van Dusschotenb, Aleksander A. Reszkac, John P.M. van Duynhovena,
a

Unilever Food and Health Research Institute, Unilever R&D, P.O. Box 120, 3133 AT Vlaardingen, The Netherlands b DSM Research, P.O. Box 18, 6160 MD Geleen, The Netherlands c SCC Global Technology Centre, Unilever R & D, P.O. Box 120, 3133 AT Vlaardingen, The Netherlands Received 4 August 2005; received in revised form 11 February 2006; accepted 16 February 2006

Abstract Recently, NMR has been demonstrated in a truly non-invasive through-package sensor mode, also denoted as the MObile Universal Surface Explorer (MOUSE). In this feasibility study, we present a rst example where we use the MOUSE sensor for assessment of the microstructural quality of a food material. We have taken model systems consisting of protein-stabilized oil-in-water emulsions, where an important microstructural quality parameter is water exudation (WE). In order to establish a sound relation between MOUSE signals and WE, it was necessary to deploy multivariate calibration techniques. It was found that the performance of the MOUSE is comparable to that of conventional benchtop NMR. Thus it was demonstrated that the NMR MOUSE presents a good option for non-invasive assessment of microstructural quality parameters, e.g. in manufacturing and in the supply chain. r 2006 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
Keywords: NMR MOUSE sensor emulsions

1. Introduction Within the foods industry we are witnessing a transition from the classical process and quality control taking place in an off-line laboratory, towards non-invasive, on-line and real-time measurement of product quality parameters. This has resulted in the tight integration of process and quality control, and is yielding signicant economical benets. Many on-line measurement technologies have become available that can be integrated with process and quality control in a versatile manner (Senorans, Ibanez, & Cifuentes, 2003). Most of these measurement tools are based on spectrophotometry (Williams & Norris, 2003) and provide only feedback on the chemical composition of the food system of interest, however. Although food quality is often perceived as compositional, i.e. the presence

Corresponding author. Tel.: 31 10 4605534; fax: 31 10 4605310.

E-mail address: john-van.duynhoven@unilever.com (J.P.M. van Duynhoven).

of molecules that can be tasted or smelled, the dominating factor is often the microstructure of the product. The microstructure of a product is particularly important for semi-solid foods, ranging from sauces and dressings to yoghurt, spreads and ice cream. Sofar primarily ultrasound technology has matured enough to allow the on-line measurement of solid-to-liquid ratios (Prakash & Ramana, 2003) and (for specic systems) rheological properties (Bamberger & Greenwood, 2004; Ross, Pyrak-Nolte, & Campanella, 2004). Despite these successes, one has to conclude that these techniques have their limitations with respect to versatility when it comes to assessment of a broad range of microstructural parameters. NMR is a good candidate to full such a role, and within the foods industry it is applied to assess a range of microstructural features on (relatively) low-cost benchtop spectrometers (Todt, Burk, Guthausen, Kamlowski, & Schmalbein, 2001). However, since these applications still require that a sample is taken and placed in the benchtop NMR spectrometer, this technology still belongs to domain of the classical process and quality control laboratory.

0023-6438/$30.00 r 2006 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2006.02.026

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Recently, NMR has also been presented in a truly noninvasive mode, by deploying one-sided magnets with builtin measurement coils (Blumich et al., 1998), also denoted as the MObile Universal Surface Explorer (MOUSE). The MOUSE consists of two separate magnets attached antiparallel wise to an iron yoke. Sandwiched between these magnets is a simple RF coil, which is used for transmitting RF pulses and receiving the NMR signal (Fig. 1). The rst applications of the NMR MOUSE were in non-invasive assessment of polymer quality (Guthausen et al., 2000), but also the rst applications in food technology have appeared. These food applications of the MOUSE focussed on the non-invasive assessment of chemical compositional parameters (Guthausen et al., 2004; Pedersen, Ablett, Martin, Mallet, & Engelsen, 2003). However, the aforementioned sensitivity of NMR to distribution and dynamic state of water implies that the MOUSE should also be a versatile sensor for the microstructural quality of foods (Martinez et al., 2003). In this study, we will present a rst example where we use the MOUSE sensor in this respect. For this feasibility study we have taken model systems consisting of oil-in-water emulsion gels, stabilized by proteins. These systems form complex structures (Haiduc & van Duynhoven, 2005; Haiduc, van Duynhoven, Heussen, Reszka, & Reiffers-Magnani, submitted; Pudney, Hancewicz, Cunningham, & Brown, 2004), built from fat droplets and a protein aggregate network in which water is dispersed in pores with a range of sizes (schematically depicted in Fig. 2). One important quality parameter of these materials is their water exudation (WE). In the classical approach, WE is determined by measuring the amount of water that is lost from the material when it is subjected to gravitational forces. As long as these materials are contained, e.g. in a tub, water cannot leave the

structure and water is distributed homogeneously. However, when the material is put in geometry where water can leave the structure, exudation takes place, which can take between 1 and 8 h. Recently, also a NMR method was described that could measure loss of water from a food microstructure (Hinrichs, Gotz, & Weisser, 2003), but also this method is intrinsically slow. When a more rapid alternative methodology would become available, in a truly non-invasive, through-package mode, this would open opportunities for process and quality control in manufacturing and the supply chain. In our study, we rst identied the underlying microstructural basis of WE by conventional benchtop NMR. Subsequently we assessed the microstructural quality of the model systems by non-invasive, through-package MOUSE measurements. We will also outline the requirement of multivariate calibration techniques for exploiting experimental data generated by the MOUSE sensor.

2. Materials and methods 2.1. Acquisition A Bruker Minispec MQ 20 NMR spectrometer (Bruker BioSpin, Rheinstetten, Germany), equipped with a variable temperature relaxation probe (dead time 7 ms) was used for the conventional benchtop NMR experiments. Samples were measured in a 10-mm diameter tube, lled to a height of 1 cm. Transverse relation decays, consisting of a free induction decay (FID) and a subsequent CarrPurcell MeiboomGill (CPMG) pulse train (Meiboom & Gill, 1958) were recorded, using an in-house pulse programme written in the Bruker Exspel programming language. Using this combined FID-CPMG method, a large range of transverse relaxation times can be assessed in a single experiment. The 901801 pulse spacing in the CPMG sequence was 0.3 ms. The CPMG decay was sampled by averaging 20 points at the top of each echo. The FID part of the sequence was sampled with a time interval of 1 ms. The decays were measured at 25 1C. All MOUSE NMR measurements were performed on a Bruker MQ MiniSpec spectrometer that was connected to a separate radio frequency (RF) unit and a portable magnet with a builtin RF coil (RWTH, Aachen, Germany). In order to get meaningful data, a CPMG train of very short RF pulses is required. In between these pulses, the NMR signal arises in the form of very sharp echoes that are each tted to a polynomial to determine the amplitude. The phase of the rst ve echoes can be determined and the whole echo train is then subjected to a so-called zero-order phase correction, using this phase information. Now, the imaginary part of the complex signal can be disregarded. This improves the signal to noise ratio and signicantly reduces the baseline. These improvements were implemented directly into the Bruker control software. Decay signals were recorded with the RF pulse spacing varying between 50 and 250 ms.

tub

N S

S N

Fig. 1. Schematic presentation of the NMR MOUSE.

A B C

Fig. 2. Schematic representation of oil-in-water emulsion gel stabilized by protein: (A) continuous protein gel phase, (B) oil droplets and (C) pores.

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The number of echoes we acquired was always set to 512. The number of averages was set to 128. Due to slow T1relaxation, the delay between the individual averages was set to 4 s, which yields an experimental time of 8 min for one MOUSE measurement. 2.2. Samples A series of 9 protein-stabilized oil-in-water model emulsions was prepared, containing vegetable fat, predominantly native whey protein concentrate (Nutrilac QU7560, ex Arla foods), skimmed milk powder, potassium sorbate as a preservative, demineralized water and gelatine. An emulsion premix was prepared at 60 1C, subsequently heated to 85 1C and then homogenized. Heating and holding steps took 20 min. Homogenization was performed using an APV Lab1000 homogenizer, single stage at 300 bar by default, and is always preceded by turraxing the mixture at 8000 rpm for 2 min (Kiokias, Reiffers-Magnani, & Bot, 2004). Samples were acidied to a pH in the range 4.54.9 using a 50% citric acid solution in demineralized water, and homogenized again at 200 bar. Emulsions were lled in tubs, sealed, and stored at 5 1C until further analysis. The WE of these samples was characterized by means of a simple leakage test, and is expressed as percentage water lost. The repeatability and reproducibility of the leakage test is 1.6% and 2.8%, respectively. 2.3. NMR data analysis Partial least squares (PLS) and multilinear regression (MLR) are well known techniques and their theoretical background will not be discussed here (Massart, Vandeginste, Buydens, de Jong, & Smeyers-Verbeke, 1997). In this work PLS is used directly on the NMR decays. For MLR, the decays are rst transformed from the continuous domain to the discrete domain of T2 distributions and amplitudes using the Nonnegative Least Squares (NNLS) algorithm (Bro & De Jong, 1997). The decays are averaged, and the resultant data is tted without an initial guess of the number of components or T2 values. The basis matrix used in the NNLS regression is constructed from the NMR CPMG magnetization function: Mt M expt=T 2 0 (1)

sum of exponential decays tted with NNLS: X St M expt=T 2n ; n 1 to N, n

(3)

where Mn is the amplitude of the nth exponential, T2n is the corresponding relaxation time constant, t is the acquisition time axis and N is the number of exponential functions or components in the sample. The sum SMn is smaller than the normalized value, the difference is due to components with very short T2s for which the magnetization decays to zero in the FID part. For MLR the following calibration equation is used: X P B0 C Bn M n , (4) where P is the calibrated parameter, B0 is the MLR constant and the unit constant C the sum of all magnetizations. This equation can be rewritten as X X P B0 M 0 M n Bn M n B0 M 0 X B0 Bn M n . 5 In this work, the term B0 Bn is denoted as scaled coefcients. 2.4. Monte-Carlo analysis The value of the amplitudes obtained by a NNLS is inuenced by the noise on the experimental decays. This is a well-known problem in numerical inversion and the usual work-around is by the use of a regularization parameter (Butler, Reeds, & Dawson, 1981; Provencher, 1982). However, a regularized t leads to a continuous distribution of T2s over a wide range, which would impede the use MLR for performing a calibration. Furthermore, in regularized tting approaches small disturbances in the decays are known to introduce relatively large artefacts in the T2 distributions. Hence, in this work, it was chosen not to deploy regularization. As an alternative, the inuence of the noise on the NNLS result was tested using a MonteCarlo approach. For each recorded decay, the difference between the NNLS t and the experimental data (residuals) is assumed to be due to normally distributed noise. Simulated noise is constructed using a random number generator with the same variance as the residuals and then added to the NNLS t, thus simulating a new experiment on the same samples. The resultant new decays are then tted again. The process is repeated 100 times, resulting in a distribution of amplitude values which can be used to derive statistical parameters. 2.5. Software The commercial software package SIMCA-P (Umetrics, Umea, Sweden) was used for (PLS) model development. Orthogonal Signal Correction (Wold, Antti, Lindgren, & Ohman, 1998) was applied in order to yield a PLS model with a single principal component. Data preprocessing was performed in EXCEL and MATLAB

where M0 is the amplitude at time t 0, T2 is the corresponding relaxation time constant and t is the acquisition time axis. M0 is set to 1 and the T2 value is varied across the interval 0.23000 ms with an equidistant step of 30 ms. In matrix notation, the equation solved with NNLS is S A K, (2) subject to AX0, where S is the experimental NMR decay matrix, and A are the unknown amplitudes of the exponential basis function K. The CPMG part follows a

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3. Results and discussion


(B)

3.1. Inspection of conventional and MOUSE NMR decays Raw NMR decays obtained by conventional benchtop NMR and the MOUSE are presented in Fig. 3. Whereas both types of signal were recorded within comparable measurement times (ca. 8 min), they differ signicantly with respect to signal-to-noise ratio. Furthermore, the MOUSE signals decay much more rapidly. In order to extract common T2 populations in the NMR decays, a NNLS transformation was carried out (Bro & De Jong, 1997). The NNLS procedure was performed on decays recorded by conventional (benchtop) means, and by the MOUSE. The resulting T2 distributions are presented in Fig. 4. In the NMR decays that were measured by conventional means, three T2 components can be discerned, which can be assigned (Haiduc & van Duynhoven, 2005) to (I) oil (E170 ms), (II) oil and protein-associated water (E200 ms) and (III) water in pores (4500 ms). For the NMR MOUSE, two T2 population distributions are
1.0 0.8 0.6 0.4 0.2 0 0.1 (B) 1 0.8 0.6 0.4 0.2 0 0.1 (A)

Amplitude

(The MathWorks, Natick, MS, USA), using in house developed routines. Transformation of the NMR decays to discrete distributions of transverse relaxation times (T2) by means of NNLS and MLR of the resulting amplitudes versus functional material parameters was achieved using Matlab routines developed in house.

II Amplitude I III 0
(A)

150

300

450

600 750 900 1050 1200 1350 1500 time [ms]

Fig. 4. T2 distribution obtained by NNLS tting of CPMG data obtained by conventional benchtop NMR (A) and the NMR-MOUSE (B). The assignments of T2 populations IIII in (A) are outlined in the text.

obtained, centred around 10 and 70 ms. The T2 populations as measured by the MOUSE cannot be related to the ones assessed by regular benchtop NMR in a straightforward manner. The strong magnetic eld inhomogeneities that are inherent to the one-sided MOUSE magnets lead to strong diffusional contributions to the transverse relaxation time. This is particularly effective for rapidly diffusing water components and less for slowly diffusion oil. From this, we may assume that the component at 10 and 70 ms can (roughly) be assigned to water and oil, respectively. 3.2. Prediction of WE from NMR decays recorded by conventional NMR and the MOUSE

10 Time [ms]

100

1000

10 Time [ms]

100

1000

Fig. 3. Raw NMR decays obtained by (A) conventional benchtop timedomain NMR and (B) the MOUSE. Decays were normalized with respect to intensity at 0.1 ms and time scale (horizontal) is plotted logarithmically.

Since conventional benchtop NMR decays allow for physical interpretation, these were rst used for explorative multivariate modelling. The most straightforward method to build such a model is by means of PLS. PLS has the advantage that it can predict WE directly from the continuous NMR exponential decays. As is shown in Table 1A, this results in a good predicting model for WE. The PLS model also generates a so-called loading which represents the signal in the NMR decays that is responsible for the relation between decays and WE. This signal is presented in Fig. 5A, but is difcult to be interpreted in a physical manner. This is due to use of centred and scaled data in the PLS model. This procedure has the advantage that it avoids extracting a very strong component which is just an average NMR decay and has no relation with WE. The disadvantage, however, is that when dealing with continuous NMR decays, a loading is obtained with both positive and negative amplitudes. This complicates the

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straightforward explanation of WE in terms of microstructural features, i.e. T2 populations. In order to obtain calibration models that allow for physical interpretation, another approach was explored where MLR is applied to NNLS signal amplitudes extracted from decays measured by conventional means. The MLR model is built using the NNLS amplitudes as responses, and the functional parameters as predictors. The quality of the NNLSMLR models is illustrated by the plot

Table 1 Goodness of t (R2) and root mean square errors (RMSE) of calibration (Cal) and cross validation (xVal) for PLS and MLR models constructed from (A) conventional benchtop and (B) MOUSE decays A Conventional PLS R2 RMSE-Cal RMSE-xVal 0.93 1.17 1.57 MLR 0.96 1.13 1.71 B MOUSE PLS 0.78 3.05 3.42 MLR 0.97 0.99 1.99

Values in bold indicate strong models.

in Fig. 6A. MLR is performing similar, or better, as PLS as is illustrated in Table 1A. The MLR model has the additional advantage that these can be interpreted in a physical manner (Haiduc & van Duynhoven, 2005). The coefcients of the MLR establish a direct linear relation between the amplitudes of the three T2 populations and the calibrated parameter. The relaxation times T2 for these three populations relate to different microstructural features of the emulsion, thus enabling physical interpretation. The scaled MLR coefcients in Fig. 5B represent the physical relation between microstructural features (in the form of T2 populations) and the calibrating (functional) parameters P. In Fig. 5B one can observe that WE is dependent on population I and (mostly) on pore population III. The large T2 value associated with Population III point towards water conned in relatively large pores in the continuous phase of the emulsion. The water in these pores is more prone to drain from the emulsion than other populations. The results of both PLS and MLR demonstrate that a microstructure related quality property can be predicted from decays recorded on benchtop NMR equipment. The MLR model is not simply a multivariate black box, it also allows for building understanding of WE in

0.1 250 0.05 200

Amplitude

Amplitude
0 500 1000 1500 2000 2500 3000

150 100 50 0

-0.05

-0.1

-50 0 150 300 450 600 750 900 1050 1200 1350 1500

(A)

Time [s]

(B)

Time [ms]

Fig. 5. (A) PLS loading (coefcients) for calibration of conventional benchtop NMR decays with Water Exudation. (B) Scaled coefcients for an MLR model that predicts Water Exudation based on conventional benchtop decays.

25 Measured Water Exudation [%] Measured Water Exudation [%] 0


(A)

25

20

20

15

15

10

10

0 5 10 15 20 Predicted Water Exudation [%] 25


(B)

0 0 5 10 15 20 Predicted Water Exudation [%] 25

Fig. 6. Goodness of ts of NNLS-MLR models based on (A) conventional benchtop NMR decays and (B) MOUSE decays.

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742 A.M. Haiduc et al. / LWT 40 (2007) 737743 Table 2 Monte-Carlo simulation results on the MLR regression coefcient (R2) for water exudation R2 original R2 average from MC simulation R2 95% condence 0.970 0.971 0.9680.973

Acknowledgements We acknowledge the European Union (Marie Curie Fellowship, HPMI-CT-2002-00167, Benchtop NMR techniques for the structural characterization of foods) for the funding of A.H. and E.T. References
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microstructural terms. The aforementioned PLS and MLR approaches were also applied to the NMR decays recorded with the MOUSE. There is a small difference between the calibration and cross-validation errors of the MOUSE PLS model (Table 1B), indicating that the model is not overtted. Compared to the model based on conventional benchtop NMR (Table 1A), the MOUSE PLS model is less favourable. This is because PLS needed only one statistically relevant component in the MOUSE model, whereas three components were needed to describe the conventional benchtop NMR data. Much more promising are the MLR results obtained after the NMR MOUSE decays were transformed with NNLS into sets of amplitudes and observed T* values (Fig. 4B). The quality of the MOUSE 2 MLR model for WE (Fig. 6B) is comparable to the results from the conventional benchtop NMR experiments (Fig. 6A). We note that only two NNLS amplitudes were used in the MLR model, hence there is a minimal risk of overtting the data. This is also illustrated by the relative small difference between calibration and crossvalidation errors. MLR model errors for the MOUSE data are larger than for conventional benchtop NMR (Table 1B), which can be attributed to the poorer signal to noise ratio of the MOUSE data. This will have an impact on the value of the amplitudes obtained by a NNLS t to the exponential decays. The inuence of the noise on the amplitudes was tested using a Monte-Carlo approach. The result for the WE model shows that this parameter is independent of the noise (Table 2), which on average has a variance (square of standard deviation) of 7 105 for the NMR-MOUSE and 2 106 for a conventional benchtop NMR experiment. Simulated data showed that the NNLS t becomes unstable when the noise variance reaches 5 104, almost an order of magnitude higher than in our experiments. Hence, we conclude that MOUSE decays can be used to make reliable predictions of WE. This work demonstrates that low eld NMR relaxometry can be deployed to assess the microstructural quality of food products, by both conventional benchtop NMR as well as the NMR MOUSE. The models constructed from conventional benchtop NMR data, have the advantage that they allow for the understanding of WE in microstructural terms. The NMR MOUSE data are more difcult to be interpreted, but allow for assessment of WE in a truly non-invasive (through package) mode. This may offer promising options for non-invasive assessment of other microstructural quality parameters, i.e. in manufacturing and in the supply chain.

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